Some members of the omptins family of outer membrane proteases present in Gram-negative bacteria are reported to mediate and contribute to resistance to AMPs. The PgtE protease of *S. enterica* Serovar *typhimurium* and CroP protease of *Citrobacter rodention* promote resistance to α-helical peptides (Guina et al., 2000; Le Sage et al., 2009). OmpT has an important role in the resistance of *E. coli* to protamine and urinary cationic peptides (Stumpe et al., 1998; Hui et al., 2010). In enterohemorrhagic *E. coli* (EHEC), OmpT contributes to the degradation of LL-37 more efficiently than in enteropathogenic *E. coli* (EPEC). The difference between EHEC OmpT and EPEC OmpT in providing defense against AMPs is due to the differences in the regulation of *ompT* genes in a promotor dependent manner, resulting in high levels of *ompT* transcripts and OmpT protein in EHEC (Thomassin et al., 2012). A posterior study with uropathogenic *E. coli* (UPEC) demonstrated that UPEC OmpT can cleave and protect the membrane from LL-37, but only when is expressed in high levels (Brannon et al., 2013), similar to those reported by Thomassin et al. (2012) for EPEC. Therefore, UPEC OmpT is not essential to LL-37 resistance. These data suggest that the role of OmpT in resistance to AMPs is specific to each pathogen.

In order to avoid killing by AMPs, pathogens may form complexes that retain their own proteases near the bacterial surface (Nyberg et al., 2004). This study shows that the host proteinase inhibitor α2-macroglobulin (α2−M) binds with high affinity to GRAB, a protein attached to *S. pyogenes* cell wall. The α2−M, on the other hand, traps the SpeB protease secreted by the bacteria, but SpeB retains the ability to degrade LL-37 and, more interestingly, its proteolytic activity against that AMP molecule is enhanced.

#### **EFFLUX PUMPS**

Efflux pumps are energy-dependent transporters that extrude toxic compounds, including antibiotics, being one of the major mechanisms by which microbial pathogens resist to different classes of antibiotics (Piddock, 2006; Poole, 2007). Kupferwasser et al. (1999) demonstrated that a naturally occurring plasmid containing *qacA* gene, which encodes a proton motive forcedependent multidrug efflux pump, confers resistance to tPMP-1 in *S. aureus*, while constructions with deletions that compromise the *qacA* gene abolish tPMP-1 resistance. The *qacA-*mediated resistance seems to be specific to tPMP-1, because the expression of *qacA* did not confer resistance to other AMPs. It is possible that the efflux function of QacA protein itself is not responsible for the resistance to tPMP-1, but the alterations on the cytoplasmic membrane of strains expressing *qacA*. On the other hand, in *Neisseria gonorrhoeae* and *N. meningitidis*, the energy-dependent Mrt efflux pump mediates the resistance to antibacterial peptides PG-1, PC-8, LL-37, and TP-1 (Shafer et al., 1998; Tzeng et al., 2005). Notably, this pump recognizes structurally unrelated AMPs, such as PG-1, which folds into a β-sheet conformation, and LL-37, that assumes n α-helical structure. The importance of Mrt efflux pump system to avoid AMPs-killing during *in vivo* infections by *N. gonorrhoeae* was demonstrated by Jerse et al. (2003) where Mtr-CDE mutants were more susceptible to the host immune system. Warner et al. also showed that mutations upstream of *mrtC,* on the promoter of the *mtrR* gene encoding a repressor for MtrCDE, or in the binding site of MrtR, confer more resistance to LL-37 and CRAMP-8, a murine analogous to LL-37, by increasing the levels of MrtE and *mrtC*RNA stability (Warner et al., 2008). These mutants were also more fitted *in vivo* than wild-type strains, highlighting the importance of this pump in evasion of the immune system.

Resistance to polymyxin B in *Y. enterocolitica* and *K. pneumonia* involves a system composed of two proteins, RosA/RosB and AcrAB, respectively (Bengoechea and Skurnik, 2000; Padilla et al., 2010). The dissipation of the proton-motive force by the addition of Carbonyl cyanide *m*-chlorophenyl hydrazone CCCP reduced the resistance to polymyxin B, highlighting the importance of the energy-driven efflux pump of AMPs in resistance to these molecules. In *Y. enterocolitica,* RosA functions as a CAMP proton motive force-dependent efflux pump, while RosB has a potassium antiporter activity, involved in the regulation of intracellular pH, which is linked to CAMPs resistance by acidification of the cytoplasm. Both, RosA and RosB, are required for resistance to CAMPs and the presence of these defense molecules increases the expression of the *ros locus*, as observed in *S. pneumoniae* LL-37 resistance, mediated by MefE/Mel efflux pump (Zahner et al., 2010). Similarly, the protein TrkA of the potassium transport complex Trk is required for CAMP resistance in *Vibrio vulnificus* (Chen et al., 2004). It is hypothesized that protamine forms channels through which K+leaks from the cell; then, the high-rate K+uptake systems pump potassium inside the cell, preventing bacterial death until the protamin is degraded by extracellular proteases. Likewise,*sapG* mutants, a *S. typhimurium* gene that encodes a protein involved in potassium transport higlhy homologous to TrkA, exhibited hypersensitivity to protamin (Parra-Lopez et al., 1994). On the other hand, the overexpression of AcrAB, MexAB and NorA pumps in *E. coli*, *P. aeruginosa*, and *S. aureus*, respectively, did not increase resistance to CAMPs such HNP-1, HNP-3, HD-5, HBD-2, HBD-3, and LL-37, when compared to the wild-type strains and inactivated mutants. These results indicate that specific efflux pumps extrude specific types of AMPs (Rieg et al., 2009).

#### **TRAPPING**

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Some secreted molecules can bind to and neutralize the microbicidal activity of AMPs. For example, SIC proteins produced by *S. pyogenes* M1 bind to several AMPs, blocking their antimicrobial activity in the early stages of infection (Frick et al., 2003; Fernie-King et al., 2004; Pence et al., 2010). SIC binds more efficiently to LL-37 than to the defensing human neutrophil peptide HNP-1 and SIC deficient mutants are more sensitive to LL-37 than wildtype strains. Other SIC variants from other serotypes also bind and interfere with the antimicrobial activity from AMPs, but SIC from M1 strain is more potent (Frick et al., 2003). In addition to the plasminogen binding activity, Staphylokinase (SAK) from *S. aureus* can also binds to HNP-1 and HNP-2. SAK induces HNPs release from neutrophils, and the formation of a complex of SAK and the α-defensin results in the loss of antimicrobicidal activity of those defense molecules. This complex abrogates bactericidal activity of HNPs in other species, when grown together.

Other trapping molecules can be found attached to the bacterial surface, like M1 protein from *S. pyogenes.* M1 mutant strains are killed more efficiently by cathelicidins, LL-37 or mCRAMP than the wild-type strain. It seems that M1 acts as a shield, binding, and trapping the AMPs before they can reach the cell membrane and kill the pathogen (Lauth et al., 2009). Pili also may be a trap to AMPs. Maisey et al. (2008) reported that PilB from Group B *S.* sequesters LL-37 and mCRAMP, increasing the resistance to those peptides. GRABs also mediate an important binding mechanism in resistance to LL-37 by *S. pyogenes* (Nyberg et al., 2004).

It is also described that the shedding of host molecules by proteases from pathogens are involved in sequestration and resistance to several AMPs. For example, extracellular proteases secreted by *P. aeruginosa*, and *E. faecalis* degrade decorin proteoglycan, producing dermatan sulphate that protects those bacteria from killing mediated by HPN-1 through directly binding to the α-defensin (Schmidtchen et al., 2001).

#### **CONCLUSION**

The increasing emergence of antibiotic resistance among human pathogens has led to the search for alternatives to overcome this alarming problem. Despite their ancient origin, only in the past decades the study of AMPs have gained increased attention as a promising therapeutic alternative against pathogenic microorganisms due their broad spectrum of activity against several species of bacteria, fungi, protozoa, and enveloped virus. Although the main target of AMPs was previously thought to be bacterial membrane several new and frequently, combined strategies of killing have been described in the past decades. Resistance to these molecules seems to be less common than to conventional antibiotics but was selected in several pathogens during the co-evolution of host and pathogens. A better comprehension of AMPs mode of action and counterpart resistance mechanisms is fundamental for the design of optimized AMPS that could be efficiently used as therapeutic drugs.

#### **ACKNOWLEDGMENTS**

We thank Hugo Costa Paes for writing assistance. We also thank CAPES, CNPq, FAP/DF for financial support.

#### **REFERENCES**

*sanguinea* induces apoptosis in *Candida albicans*. *Front. Microbiol.* 2:47. doi: 10.3389/fmicb.2011.00047

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by a naturally occurring antibacterial peptide. *J. Clin. Invest.* 106, 439–448. doi: 10.1172/JCI9826

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The editor and authors declare that while the editor shares the same institution and department as the authors, there has been no conflict of interest during the review and handling of this manuscript.

*Received: 10 October 2013; accepted: 06 November 2013; published online: 09 December 2013.*

*Citation: Guilhelmelli F, Vilela N, Albuquerque P, Derengowski LS, Silva-Pereira I and Kyaw CM (2013) Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance. Front. Microbiol. 4:353. doi: 10.3389/fmicb.2013.00353*

*This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology.*

*Copyright © 2013 Guilhelmelli, Vilela, Albuquerque, Derengowski, Silva-Pereira and Kyaw. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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**REVIEW ARTICLE** published: 20 March 2014 doi: 10.3389/fmicb.2014.00097

# Defensins: antifungal lessons from eukaryotes

# *Patrícia M. Silva, Sónia Gonçalves and Nuno C. Santos\**

*Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal*

#### *Edited by:*

*Octavio Luiz Franco, Universidade Católica de Brasília, Brazil*

#### *Reviewed by:*

*Valdirene Moreira Gomes, Universidade Estadual do Norte Fluminense, Brazil Françoise Gosti, Centre National de la Recherche Scientifique, France*

#### *\*Correspondence:*

*Nuno C. Santos, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisbon, Portugal e-mail<EMAIL_ADDRESS>
Over the last years, antimicrobial peptides (AMPs) have been the focus of intense research toward the finding of a viable alternative to current antifungal drugs. Defensins are one of the major families of AMPs and the most represented among all eukaryotic groups, providing an important first line of host defense against pathogenic microorganisms. Several of these cysteine-stabilized peptides present a relevant effect against fungi. Defensins are the AMPs with the broader distribution across all eukaryotic kingdoms, namely, Fungi, Plantae, and Animalia, and were recently shown to have an ancestor in a bacterial organism. As a part of the host defense, defensins act as an important vehicle of information between innate and adaptive immune system and have a role in immunomodulation. This multidimensionality represents a powerful host shield, hard for microorganisms to overcome using single approach resistance strategies. Pathogenic fungi resistance to conventional antimycotic drugs is becoming a major problem. Defensins, as other AMPs, have shown to be an effective alternative to the current antimycotic therapies, demonstrating potential as novel therapeutic agents or drug leads. In this review, we summarize the current knowledge on some eukaryotic defensins with antifungal action. An overview of the main targets in the fungal cell and the mechanism of action of these AMPs (namely, the selectivity for some fungal membrane components) are presented. Additionally, recent works on antifungal defensins structure, activity, and cytotoxicity are also reviewed.

**Keywords: antimicrobial peptides, defensins, antifungal, resistance, host defense peptides**

#### **INTRODUCTION**

Naturally occurring antimicrobial peptides (AMPs) probably represent one of the first successful forms of chemical defense of eukaryotic cells against bacteria, protozoa, fungi, and viruses (Ganz and Lehrer, 1998; Lehrer and Ganz, 1999; Zasloff, 2002; Mookherjee and Hancock, 2007; Lai and Gallo, 2009; Guo et al., 2012; Domingues et al., 2014), being also active against cancer cells (Hoskin and Ramamoorthy, 2008; Gaspar et al., 2013). Currently commercialized antibiotics are mostly of microbial origin or synthesized from those. These antibiotics are losing efficacy as a result of high selection pressure, leading to rapid emergence of resistance in many important human pathogens, thus threatening to put an end to the golden age of antibiotics (Clardy et al., 2009; Fisher et al., 2012). The use of antifungal treatments has increased as a consequence of the increase of immunocompromised patients, mostly due to improvements in oncology and transplant fields (Mehra et al., 2012), leading to more frequent resistances to the drugs used. A strategy to overcome this problem can be found in AMPs, which are part of the innate immune system of different living organisms (Hegedus and Marx, 2013), such as plants (Thomma et al., 2002; Lay and Anderson, 2005; Gonçalves et al., 2012b), fungi (Mygind et al., 2005; Schneider et al., 2010; Oeemig et al., 2012), bacteria (Zhu, 2007; Gao et al., 2009), invertebrates (Bulet and Stocklin, 2005; Ayroza et al., 2012), and vertebrates (Ganz, 2004; Sahl et al., 2005; van Dijk et al., 2008; Gonçalves et al., 2012a).

Although some AMPs have had their target unveiled, many are still unclear. Some mechanisms of action of antifungal peptides have been reported, such as binding to the cell wall, membrane permeabilization, receptor-mediated internalization inducing signaling cascades, and interaction with intracellular targets, inducing the formation of reactive oxygen species (ROS), leading ultimately to apoptosis (Hancock and Rozek, 2002; Oberparleiter et al., 2003; Thevissen et al., 2003a, 2004; de Coninck et al., 2013; van der Weerden et al., 2013). Apoptosis is a type of programed cell death, which is regulated by a complex network of proteins and metabolic pathways. The central core of this process is regulated by a family of proteins named caspases. Yeasts have at least one ortholog of mammalian caspases: the metacaspase YCA1 (yeast caspase 1; Madeo et al., 2002). Routinely used assays aiming at the detection of these apoptotic features are used for the identification of fungal cells undergoing apoptosis after treatment with antifungal agents.

Antimicrobial peptides have variable amino acid composition and size (ranging from less than 10 to more than 100 amino acid residues), commonly being cationic and amphipathic molecules (Brogden et al., 1996; Yang et al., 2003; Fontana et al., 2004; Glaser et al., 2005; Domingues et al., 2014). To date, more than 2200 natural or synthetic AMPs have been identified, as listed by the Antimicrobial Peptide Database (APD1), of which over 1900 have antibacterial activity and 800 have antifungal activity.

1http://aps.unmc.edu/AP/main.php

This discrepancy, however, may be redundant as antibacterial AMPs may also have antifungal activity, but this property is not systematically assessed.

Antimicrobial peptides may have linear structures, like indolicidin (Ladokhin et al., 1999), or they may have tertiary structures stabilized by disulfide bonds, with β-sheet (e.g., protegrin; Aumelas et al., 1996; and the defensin human neutrophil peptide 1, HNP-1; Zhang et al., 2011), α-helix (e.g., dermaseptin; Mor et al., 1991) or αβ-motif secondary structure (e.g., drosomycin; Landon et al., 1997; and *Pisum sativum* defensin 1, *Ps*d1; Almeida et al., 2002).

The most studied families of AMPs are cathelicidins, dermaseptins, magainins, cecropins, and defensins. Cathelicidins are found in the innate immune system of mammals, amphibians, and reptiles (Wang et al., 2008; Tsai et al., 2011; Hao et al., 2012); dermaseptins and magainins are found in amphibians (Morton et al., 2007); cecropins are found in insects (YiZeng et al., 1989); and defensins, which are the largest family of AMPs, have also the broader distribution across the majority of eukaryotic organisms (Wang et al., 2013). Besides having antimicrobial activity, defensins also have immunomodulatory functions in the organisms that produce these peptides. Defensins with antifungal properties are present in all eukaryotic kingdoms, pointing out to a common ancestor. This review is focused on defensins (as well as some defensin-like peptides) with antifungal activity. However, it is impossible to describe here all the defensins with this activity. Therefore, we highlight some of the most recent research made on this field. The chosen peptides are described taking into consideration their specific properties, evolutionary background, organism of origin, and antifungal mode of action.

Some databases have been created in order to provide useful information for the study of AMPs. Among the AMP databases, PhytAMP is a database dedicated to antimicrobial plant peptides2 (Hammami et al., 2009). This resource contains valuable information on these AMPs, including peptide sequences, taxonomic, microbiological, and physicochemical data. Another database, Collection of Anti-Microbial Peptides (CAMP3), holds

2http://phytamp.pfba-lab-tun.org/main.php

experimentally validated and predicted AMP sequences and structures of AMPs. These databases include several tools for AMPs analysis and prediction, helping in the design of new therapeutic peptides based on specific structure and functional features.

# **DEFENSINS**

Defensins are the largest groups of AMPs. These peptides are cysteine-rich and have diverse sequences and structures, stabilized into compact shapes by three or four conserved cysteine disulfide bridges. They have at least two positive charges (lysine or arginine residues) and are small, ranging approximately from 12 to 50 amino acid residues (2–6 kDa; Ganz, 2003; Ren et al., 2011; Gao and Zhu, 2012).

Vertebrates' defensins are divided into three subfamilies: α-, β-, and θ-defensins. α-Defensins are present in mammals such as humans, monkeys, and several rodent species, being particularly abundant in neutrophils, certain macrophage subpopulations and Paneth cells of the small intestine (Ouellette and Selsted, 1996; Ganz and Lehrer, 1998; Lehrer and Ganz, 1999). β-defensins are found in a wide range of vertebrates, presenting a cysteinestabilized αβ-motif composed of an antiparallel β-sheet and an α-helix. As an example, on bovine neutrophils, as many as 13 β-defensins have been identified (Yang et al., 2002a). However, in other species, β-defensins are mostly produced by epithelial cells lining different organs (e.g., epidermis, bronchial tree, and genitourinary tract). θ-Defensins, present only in OldWorld monkeys, are cyclic and derived from α-defensins (Lehrer, 2004; Lehrer and Lu, 2011; Semple and Dorin, 2012). In **Figure 1**, conserved cysteine residues among defensins from different kingdoms are shown.

Plants, fungi, and many invertebrates produce defensin-like peptides structurally similar to the β-defensins from vertebrates (Thomma et al., 2002; Bulet and Stocklin, 2005; Mygind et al., 2005; Sahl et al., 2005; Yount and Yeaman, 2006; van Dijk et al., 2008; Ayroza et al., 2012; Oeemig et al., 2012). These observations allowed to assume that defensins and defensin-like peptides all evolved from a common precursor. The relatively recent identification of three defensins in lower eukaryotes, plectasin from *Pseudoplectania nigrella* (Mygind et al., 2005), eurocin from *Eurotium amstelodami* (Oeemig et al., 2012), and bubble protein

residues.

**and** *Penicillium brevicompactum* **Dierckx bubble protein (BP),**

3http://www.camp.bicnirrh.res.in/

(BP) from *Penicillium brevicompactum* Dierckx (Seibold et al., 2011), is important to demonstrate the wide distribution of these peptides over diverse eukaryotic lineages, which suggests that ancestral defensin genes existed over 1500 million years ago, before Fungi, Plantae, and Animalia kingdoms diverged (Wang et al., 1999). The wide distribution of these peptides in the Eukarya domain could suggest their uniqueness to eukaryotic cells, but it was possible to determine that these peptides may have had their ancestor in a prokaryotic organism after the discovery of the first defensin-like peptide in the bacteria *Anaeromyxobacter dehalogenans* (AdDLP; Zhu, 2007; Gao et al., 2009). This defensin-like peptide is proposed as an ancestor of eukaryotic defensins and defensin-like peptides due to the similarity of their structures, namely at the level of the cysteine-stabilized αβ-motif (Zhu, 2007). These findings further support the concept that AMPs may have been fundamental to the evolution of multicellular organisms within microbial-exposed environments.

Although defensins were initially identified only as AMPs, recent studies have demonstrated that they have a much broader range of action, including immunomodulatory function (issue further developed in the text, in Section "Immunomodulatory Function"; Ulm et al., 2012).

# **RECENTLY STUDIED ANTIFUNGAL DEFENSINS**

When a new AMP is described, the most usual properties to assess are structure and peptide sequence, antimicrobial activity, expressed mainly in terms of minimal inhibitory concentration (MIC) or half maximal inhibitory concentration (IC50), cytotoxicity and lytic activity against human cells (whenever the AMP origin is not mammalian), target, and mode of action toward the pathogen tested. The following AMPs were classified as defensins or defensin-like peptides due to their structural and sequence homologies with other defensins. They are examples of some of the recently studied antifungal defensins from fungal, plant, and animal origin that fulfill most of the properties expressed above. Further details and a list of some of these defensins can be found in **Table 1**.

#### **FUNGAL SOURCES**

Defensin-like antifungal peptides secreted by filamentous fungi have a low molecular mass (5.8–6.6 kDa), a basic character, presence of 4–10 cysteine residues and several disulfide bonds (providing resistance against temperature stress or adverse solvent conditions), and a β-barrel conformation (Hagen et al., 2007; Seibold et al., 2011). Proteins with such properties with antifungal activity have been isolated and investigated from several Ascomycota fungal species, such as *Penicillium chrysogenum*, *Penicillium nalgiovense*, *Penicillium brevicompactum* Dierckx, *Aspergillus giganteus*, and *Aspergillus niger* (Galgóczy et al., 2010; Seibold et al., 2011). Among these fungus-derived antifungal peptides, the most intensively studied are *Penicillium chrysogenum* antifungal protein (PAF) and *Aspergillus giganteus* antifungal protein (AFP), with six and eight cysteines, respectively. *Penicillium brevicompactum* Dierckx bubble protein structure is considerably similar to PAF and AFP.

# *Penicillium chrysogenum* **antifungal protein**

PAF acts through a G protein-coupled signal transduction pathway, although this mechanism is not entirely understood (Marx et al., 2008). The G protein-coupled activity of PAF was confirmed by the study of the *fadA* mutant of *Aspergillus nidulans* which proved to be less sensitive to PAF treatment compared to the wild-type. The *fadA* gene encodes the heterotrimeric G protein α subunit, which dissociation from the Gβγ complex is inhibited in the *fadA* mutant *Aspergillus nidulans* (Leiter et al., 2005). These results indicate that PAF toxicity requires active heterotrimeric G protein signaling (Leiter et al., 2005; Marx et al., 2008). Based on these observations, Marx et al. (2008) hypothesized that PAF interacts directly or indirectly with G protein signal transduction pathways. These authors also hypothesized that the cell wall could be a selective barrier for PAF, but *in vivo* chitin-binding activity of PAF has not been demonstrated yet. In the sensitive organisms, PAF exerts multiple detrimental effects: induction of plasma membrane polarization, increased exposure of phosphatidylserine (PS) on the cell surface, DNA fragmentation, membrane blebbing, cell shrinking, intracellular ROS production, and apoptosis-like phenotype (Kaiserer et al., 2003; Leiter et al., 2005; Marx et al., 2008).

# *Aspergillus giganteus* **antifungal protein**

In susceptible organisms, AFP disturbs the polarized growth of hyphae by interfering directly or indirectly with the cell wall biosynthesis (Theis et al., 2003). Hagen et al. (2007) demonstrated that AFP can bind to chitin *in vitro*, and inhibits the cell wall chitin biosynthesis by the specific inhibition of chitin synthase III andV. These enzymes are unique among fungi and essential for the maintenance of the polarized growth and virulence of pathogenic fungi. Presence of chitin synthase III and V is confirmed in the AFP-sensitive fungi, while the insensitive fungal species do not have these enzyme classes. Sphingolipid membrane components in the sensitive fungi might serve as secondary receptors for AFP. This was further confirmed by the discovery that the depletion of cellular glucosylceramide (GlcCer) levels in AFP-sensitive fungal species (*Aspergillus fumigatus* and *Aspergillus niger*) resulted in reduced AFP susceptibility (Meyer, 2008). This, together with the observation that the sphingolipids are necessary to maintain the polarized hyphal growth, elucidates the mechanism of polarized growth degeneration effect of AFP (Li et al., 2006; Meyer, 2008). The species specificity of AFP may be related with the sphingolipid profile of the sensitive fungi (Meyer, 2008).

# *Penicillium brevicompactum* **Dierckx bubble protein**

This fungal defensin was first described in 2003 (Olsen et al., 2004). It is found in the bright yellow–green fluorescent exudate bubbles of the ascomycete fungus *Penicillium brevicompactum* Dierckx. Similarly to other ascomycetes, BP produces a small antimicrobial molecule, mycophenolic acid, which gives the bubbles their yellow–green fluorescence. This combined production suggests a possible synergistic action between defensins and other antibiotic agents produced by this class of fungi (Seibold et al., 2011). BP has 64 amino acid residues, with high content of basic amino acids, β-barrel conformation (Olsen et al., 2004), and a cage-like pattern of four very stable disulfide bridges. In addition, it was discovered

 **1 |**

**Recently studied antifungal**

 **defensins from fungal, plant, and animal sources.**

*(Continued)*

*n.d., not determined.*

**Table 1 |**

**Continued** that the closely related fungus *Penicillium chrysogenum* encodes a BP homolog (in addition to PAF), indicating that fungi may have more than one defensin (Seibold et al., 2011).

#### **PLANT SOURCES**

Many fungi are phytopathogenic, with species such as *Fusarium* spp., *Cladosporium* spp., *Pythium* spp., *Curvularia* spp.,*Aspergillus flavus*, and *Puccinia pittieriana* affecting potato, rice, corn, wheat, tobacco, and cotton crops by causing wilt, mold, crown rot, mildew, and rust, just to name a few plant diseases (The American Phytopathological Society, APS4). These diseases can deplete entire crops, bearing enormous costs for agriculture due to the difficulty in eliminating fungal infections from plants, once they appear. Soils harbor plants for most of their life cycle, but also a considerable amount of bacteria, fungi, and parasites, many of which can be phytopathogenic. For this reason, plants need to have good defenses against these microorganisms; thus, it is easy conceivable that plants are major AMPs producers, often with antifungal activity, but also antibacterial activity (Moreno et al., 1994; Segura et al., 1998; Thomma et al., 2002; Mayer et al., 2013).

In fact, a major research effort has been put forward on the screening for these molecules in plants. Besides defensins, other AMPs are also produced by plants, being exclusive to them. Examples of these plant exclusive AMPs are thionins, lipid transfer proteins and snakins, which were also demonstrated to have antifungal activity (Segura et al., 1999; Silverstein et al., 2007; Sun et al., 2008; Asano et al., 2013). Plant defensins with antifungal activity have been purified from several plants, such as *Pisum sativum* (Almeida et al., 2000; Lobo et al., 2007; de Medeiros, 2009; de Medeiros et al., 2010; Gonçalves et al., 2012b), *Raphanus sativus* (Aerts et al., 2007, 2009; Tavares et al., 2008; Thevissen et al., 2012), and *Heuchera sanguinea* (Thevissen et al., 1997; Aerts et al., 2011), which will be addressed below. Several other plant defensins with antifungal activity have also been studied.

# *Ps***d1**

This garden pea (*Pisum sativum*) seed defensin, firstly characterized in 2000 (Almeida et al., 2000), has 46 amino acid residues. Its secondary structure comprises a globular fold composed of β-sheets and an α-helix stabilized by four disulfide bridges, i.e., a cysteine-stabilized αβ-motif (Almeida et al., 2002). As demonstrated by a yeast two-hybrid screening system, *Ps*d1 has affinity to a *Neurospora crassa* protein related to the cell cycle control, cyclin F (Lobo et al., 2007). Using a developing retinal tissue of neonatal

rats as a model to study this interaction, it was proven that *Ps*d1 impairs the correct cell cycle progression, by blocking cyclin F role in the transition of S to G2 phases of the cell cycle, promoting endoreduplication and disturbing nuclear migration. Recently, it has been demonstrated through partition studies that *Ps*d1 has a high affinity with high specificity to model membranes enriched with ergosterol, the main sterol present in fungal membranes, and GlcCer (Gonçalves et al., 2012b). On the contrary, there is no interaction between the defensin and model membranes enriched in cholesterol (a characteristic of mammalian cells), reducing *Ps*d1 toxicity to human cells.

#### *Rs***AFP2**

This defensin, isolated from radish (*Raphanus sativus*) seeds in 1992 (Terras et al., 1992), has 51 amino acid residues and is highly cationic. It has eight cysteine residues, forming four disulfide bridges that stabilize its αβ-motif structure. *Rs*AFP2 has fungal GlcCer as its target, as observed in experiments performed with wild-type *Candida albicans* and a mutant lacking GlcCer in the membrane (*gcs*; Aerts et al., 2007). It does not need to be internalized to have its antifungal effect. After the initial contact, a signaling cascade is activated inside the cell and ROS are formed, leading to membrane permeabilization and consequent cell death (Aerts et al., 2007). Other effects of *Rs*AFP2 comprise the induction of apoptosis in *C. albicans* by triggering caspases activation, but not of metacaspases, implying that different apoptotic pathways can be induced in *C. albicans* (Aerts et al., 2009). *Rs*AFP2 also promotes an accumulation of ceramides in *C. albicans*, which can be lethal to the cell, and blocks the yeast-to-hypha transition (Thevissen et al., 2012). *In vivo* experiments were performed in murine models, proving that *Rs*AFP2 considerably reduces the fungal burden in kidneys of mice infected with *C. albicans*. This defensin has low susceptibility to serum peptidases, meaning that upon entering the bloodstream it will not be degraded, maintaining its antifungal activity. Lactate dehydrogenase (LDH) release levels are indicative of cell damage and tissue breakdown. Human brain endothelial cells incubated with *Rs*AFP2 show no release of LDH, hence supporting the conclusion that this defensin has limited toxicity to mammalian cells.

# *Hs***AFP1**

Firstly identified in 1995, this defensin found in the seeds of coral bells plant *H. sanguinea* (Osborn et al., 1995) was shown to have high affinity to specific sites in fungal membranes and to permeabilize cells of susceptible fungi (Thevissen et al., 1997). Unlike *Rs*AFP2, which relies on an interaction with GlcCer to exert its antifungal effect, *Hs*AFP1 has antifungal activity against *C. albicans gcs* and its wild-type counterpart (Aerts et al., 2011). It was proposed that *Hs*AFP1 may interact with essential components of the fungal membrane, resulting in a low occurrence of resistance *in vitro*, an advantage for the use of *Hs*AFP1 as a novel antifungal agent (Aerts et al., 2011). Using sodium azide, a respiratory inhibitor, mitochondrial function is impaired and *Hs*AFP1 antifungal activity is affected, indicating that the defensin requires a properly working respiratory chain. This defensin induces ROS formation and apoptosis in yeast. It was also proposed that mitogen-activated protein kinase (MAPK) signaling pathways may

4http://www.apsnet.org/

be a possible strategy for yeast tolerance to *Hs*AFP1 (Aerts et al., 2011).

# **ANIMAL SOURCES**

#### **Mammal defensins**

Antimicrobial peptides from animal sources have shown antifungal and immunomodulatory activities, being mammals major producers of defensins (Yang et al., 2002a,b; Sahl et al., 2005). θ-Defensins are the less studied defensin family, at least partially due to their source. To date, no antifungal activity was attributed to θ-defensins; as such, these defensins will not be further discussed in the present review. Being vertebrates, mammals possess an adaptive immune system, hence having a more complex network of signaling pathways, diverse responses against pathogens invading the organism and an array of AMPs produced in different organs and tissues, each with its particular function and mode of action (Ganz and Lehrer, 1998; Ganz, 2004; Lai and Gallo, 2009; Pasupuleti et al., 2012). Human β-defensins 1 and 2 are chemotactic for memory T cells and immature dendritic cells (Pazgier et al.,2006). Mammal defensins differ substantially in their antimicrobial specificities. For example, HNP-1, HNP-5 and human beta-defensins 1 and 3 (HBD1 and HBD3, respectively) have broad antimicrobial activities against Gram-negative and Gram-positive bacteria and yeasts (Ganz et al., 1985; Bensch et al., 1995; Porter et al.,1997; Harder et al.,2001; Hoover et al.,2003; Joly et al.,2004). HBD1 and HBD3 have been shown to be effective against *C. albicans* (Krishnakumari et al., 2009), while HBD2 has been shown to possess significant microbicidal activity against Gram-negative bacteria and *C. albicans* (Schroder and Harder, 1999). Recombinant human intestinal defensin 5 (rHD-5) exhibits microbicidal activity against *Listeria monocytogenes*, *Escherichia coli*, and *C. albicans*. Opposed to cryptdins, the mouse intestinal defensins, rHD-5 is active against both mouse-virulent wild-type *Salmonella typhimurium* and its isogenic, mouse-avirulent *pho*P mutant (Porter et al., 1997).

Mouse β-defensin 3 (MBD3), a HBD2 homolog, is an AMP expressed in the mouse epithelial and mucosal tissues (Jiang et al., 2010). The fungicidal properties of recombinant MBD3 suggest that similar peptide formulations can be used in the treatment of fungal and/or bacterial infections. MBD3 is expressed in footpads, skin, and mucosal membranes (tongue) of normal mice. Potent antifungal activity was observed against filamentous fungi, such as *Aspergillus fumaricus*, *Microsporum canis*, *Trichophyton rubrum*, *Trichophyton tonsurans*, and *Trichophyton violaceum* (all these species are primary human pathogens, meaning they cause infection whether or not the immune defenses are weakened, as opposed to opportunistic fungal infections), as well as yeast strains like *C. albicans* and *Cryptococcus neoformans*. This peptide also presents bactericidal activity against Gram-positive and Gram-negative bacteria, such as *Staphylococcus aureus*, *E. coli*, and *Salmonella typhi* (Jiang et al., 2010).

# **Arthropods defensins**

*Coprisin.* This 43 amino acid residues beetle defensin-like peptide was described in 2009 as an antibacterial peptide (Hwang et al., 2009). Its structure comprises an αβ-motif, stabilized by three cysteine disulfide bridges (Lee et al., 2013). In 2012, the same authors investigated its antifungal activity against *C. albicans*, revealing that coprisin enters the fungal cell and localizes in the nucleus, which indicates that coprisin penetrated the membrane without disrupting the fungal plasma membrane, as confirmed with 1,6-diphenyl-1,3,5-hexatriene (DPH) analysis, calcein-leakage, and giant unilamellar vesicle assays (Lee et al., 2012). Using H2O2 as a positive control for apoptotic induction, coprisin proved to have the same effects in inducing early and late apoptosis, features shown by the annexin V conjugated with fluorescein and propidium iodide co-staining method. Apoptosis induced by this AMP is metacaspase-dependent. Concomitantly, coprisin compromises mitochondrial membrane potential and ROS production, in addition to the release of cytochrome *c* from the mitochondria to the cytosol. No hemolytic activity was observed for this peptide in human erythrocytes (Lee et al., 2012).

*Juruin.* This defensin-like peptide was discovered in 2012 by screening the venom on the theraposid Amazonian pink toe spider *Avicularia juruensis* (Ayroza et al., 2012). It has 38 amino acid residues, three disulfide bonds and, like neurotoxins reported to have antimicrobial activity, it has a putative inhibitory cysteine knot (ICK) motif, i.e., a fold common to venom peptides from spiders, scorpions, and aquatic cone snails (Smith et al., 2011). ICK-containing peptides of spider venom are likely to have evolved from β-defensins (Fry et al., 2009). Based on amino acid sequence and structure similarities with insecticidal peptides of other spiders, this peptide is likely to belong to a group of conserved toxins with voltage-gated ion channels inhibitory action. Juruin showed a fungicidal rather than fungistatic effect against *C. albicans* and *C. tropicalis*, without hemolytic activity (Fry et al., 2009).

# **Reptile defensins**

*Crotamine.* This highly basic peptide, isolated from the venom of a South-American rattlesnake, was discovered in 1947 (Gonçalves and Polson, 1947). It shares structural similarity to β-defensins due to an identical disulfide bridge pattern (Fadel et al., 2005; Yamane et al., 2013). Crotamine structure comprises an antiparallel β-sheet and an α-helix stabilized by three disulfide bridges (Nicastro et al., 2003; Fadel et al., 2005). Recombinant crotamine displayed a more potent antimicrobial activity than native and synthesized crotamine (Yamane et al., 2013). This peptide induces extensive ultrastructural modifications in *C. albicans*. TEM studies showed deformed cell shape, irregular layering structure of cell wall and cytoplasmic contents coagulation, but without detectable hemolytic effects and low toxicity to mammalian cells (Yamane et al., 2013).

# **IMMUNOMODULATORY FUNCTION**

Defensins may be produced constitutively or have their expression triggered when there is an inflammatory process, by the recognition of microbial conserved structures, such as lipopolysaccharide (LPS) and lipoteichoic acid, or inflammatory effectors, like cytokines. These AMPs are expressed differentially depending on the peptide itself and on the tissue or cell type (Ulm et al., 2012). Defensins, besides their antimicrobial action, can also be immunomodulatory and inhibitors of virulence factors. This ability is not exclusive of defensins, as other AMPs also

share this property. Thus, they can enhance the host's immune system, with this multifunctional character rendering these peptides lower probability of becoming tolerated by microorganisms (Mehra et al., 2012; Jarczak et al., 2013).

Pro-inflammatory mediation has been recognized in some of these molecules, as they can bind to chemokine receptors, being able to recruit immune cells, thus enhancing the immune response (Mookherjee and Hancock, 2007; Lai and Gallo, 2009; Alba et al., 2012; Semple and Dorin, 2012; Ulm et al., 2012; Zhu and Gao, 2013). β-Defensins were demonstrated to have the capability to induce chemoattraction of CD4+ memory T cells, macrophages, and immature dendritic cells, by binding to receptors in the membrane (Yang et al., 1999; Wu et al., 2003; Taylor et al., 2008). This binding favors the attraction and migration of inflammatory cells to the inflammation site, in order to improve and speed up the inflammatory response. α and β-Defensins have also been shown to inhibit neutrophil apoptosis (Nagaoka et al., 2008). These authors showed that HBD3 binds to CCR6 at the neutrophil cell surface, initiating an increase in the levels of the antiapoptotic protein Bcl-xL and inhibiting caspase activity. This increases neutrophils life span and is an inflammatory event that is beneficial to eradicate invading microorganisms (Nagaoka et al., 2008), thus promoting the production of proinflammatory cytokines and chemokines, which in turn, amplifies the immune system response.  Treatment of these cells with HBD2 HBD3 or HBD4 leads to the increase of the expression of proinflammatory mediators, like monocyte chemoattractant protein-1, macrophage inflammatory protein-3, and some interleukins (Niyonsaba et al., 2007).

Surprisingly, some defensins are also able to attenuate proinflammatory responses whenever these can be harmful to the organism (Lande et al., 2007; Yamasaki et al., 2007). These antagonistic effects depend on the level of expression, disease state, and pathogen exposure. It has been previously described for α-defensins that mice having a matrilysin deficiency (hence without mature α-defensins in the intestine) are more susceptible to chemically induced colitis than wild-type controls. Interleukin-1β (IL-1β), a cytokine with an important role in mediation of inflammation, reaches level significantly increased in the deficient mice and it was ultimately shown that α-defensins are able to inhibit the production of IL-1β (Shi et al., 2007).

It has been demonstrated that HBD3 (mainly expressed in epithelial cells), when in basal concentration, has an immunosuppressive effect in the presence of LPS, contributing to the maintenance of a non-inflammatory environment over continual low-level exposure to microorganisms, commensal or pathogenic (Semple et al., 2010). Concentrations of HBD3 ranging from 0.5 to 1 μM are able to suppress the induction of tumor necrosis factor α (TNFα), a proinflammatory effector of the immune system, and IL-6, an interleukin that acts both as pro and anti-inflammatory. At these concentrations, proinflammatory proteins are not induced and there is no proinflammatory gene expression (Semple et al., 2010). The proinflammatory effects of β-defensins were observed at slightly higher concentrations of the defensin, in the 4–6 μM range (Funderburg et al., 2007; Niyonsaba et al., 2010). This was not the first case observed of opposite effects in immunomodulating AMPs. Cathelicidin LL-37 has been shown to have also a duality in inflammatory effects, being proinflammatory at concentrations above 20 μg/ml but anti-inflammatory at 1–5 μg/ml (Scott et al., 2002). Defensins were also shown to have a role in other biological processes, namely wound healing (Hirsch et al., 2009), dog coat color determination (Candille et al., 2007), fertility (Li et al., 2001), plant development (Stotz et al., 2009), and carcinogenesis regulation (Donald et al., 2003; Gambichler et al., 2006; Joly et al., 2009).

It is clear that defensins have many functions that are determined by the level of expression. Whereas higher expression of defensins takes place at the pathogen's site of entry, with a proinflammatory response and the chemoattraction of macrophages and other immune cells, defensins expressed at lower levels may be involved in the resolution of the immune response. When the danger is neutralized and defensins and other proinflammatory molecules decrease in the inflammation site, defensins may then have a role in resolving inflammation (Semple and Dorin, 2012).

Due to this multifunctionality, AMPs have also been referred to as host defense peptides (HDPs; Steinstraesser et al., 2010; Ulm et al., 2012).

# **RESISTANCE**

Like other antibiotics resistance, it is easily conceivable that AMPs resistance is a key characteristic for increased virulence of pathogenic strains. Despite this fact, and contrary to antibiotics that act through a single approach (meaning that microbes can evade them through a single resistance system), AMPs follow a multidimensional strategy against microbial invasion (Lai and Gallo, 2009). Therefore, selective pressures on microbes are avoided, reducing the development of resistant strains (Zasloff, 2002).

A synergistic effect between different host AMPs is also possible, as evidenced by the fact that the MIC of AMPs *in vitro* are usually higher than the physiological concentrations of those AMPs*in vivo* (Lai and Gallo, 2009). Two distinct AMPs may have their combined MIC much lower than when acting isolated, strongly suggesting heterologous HDP interactions (Westerhoff et al., 1995).

Microorganisms have evolved their own strategies for evading the antimicrobial action of the compounds used against them. AMPs frequently have the ability to disrupt microbial membranes and to inhibit the synthesis of some of their components; thus, strategies to escape the action of those AMPs follow the redesign of cell membranes, as described for both Gram-negative and Grampositive bacteria (Gunn et al., 2000; Li et al., 2007). Other evasion mechanisms include affecting the correct function of the AMP by turning off its expression, releasing plasmid DNA in epithelial cells, a strategy adopted by highly contagious bacteria from the *Shigella* genus that cause dysentery (Islam et al., 2001). As AMPs frequently rely on transmembrane potential to interact with microbial pathogens and exert their mechanism of action against them, it is probable that another microbe strategy for evading AMPs could be to change their transmembrane potential status (Yeaman and Yount, 2003).

*Candida albicans* resistance to some AMPs is regulated by the protein Ssd1, combined with the transcription factor Bcr1 (biofilm and cell wall regulator; Nobile and Mitchell, 2005; Gank et al., 2008; Jung et al., 2013). Ssd1 is an RNA-binding protein and a component of the regulation of morphogenesis pathway (Saputo et al., 2012). In *C. albicans*, this pathway governs multiple processes, including filamentation and cell wall integrity (Song et al., 2008; Bharucha et al., 2011). This combination yields resistance to protamine, RP-1 and HBD2 by maintaining mitochondrial energetics and reducing membrane permeabilization, thus allowing the fungus to counteract the negative effects of these AMPs (Jung et al., 2013). Protamine is an α-helical cationic polypeptide, frequently used to screen for AMP susceptibility (Yeaman et al., 1996), and RP-1 is a synthetic AMP modeled upon the C-terminal α-helical domain existent in the human platelet factor-4 kinocidins; this domain is responsible for RP-1 microbicidal activity (Bourbigot et al., 2009). *C. albicans* mutant strains in Bcr1 and Ssd1 proteins are more susceptible to the AMPs described above; thus, Jung et al. (2013) were able to conclude that these proteins are necessary for the resistance to protamine, RP-1 and HBD2. Further studies are necessary to clarify the roles of Bcr1 and Ssd1 in early *versus* late mechanisms of resistance to AMPs.

The Hog1 (high osmolarity glycerol) MAPK pathway, which provides a response to osmotic, oxidative, and heavy-metal exposure stresses in fungal cells, was shown to be activated in the presence of AMPs, such as *Na*D1, HBD2, HBD3, and histatin-5 (a salivary cationic AMP that has a role in keeping *C. albicans* in its commensal state;Yeaman et al.,1996;Vylkova et al.,2007;Argimon et al., 2011; Hayes et al., 2013). The injuries imposed on *C. albicans* by these defensins seem to share common features with osmotic and/or oxidative stress (Argimon et al., 2011). Upon exposure to these defensins, the Hog1 MAP kinase is activated, triggering a transcriptional response aimed to rescue the cells from the source of injury, i.e., the core and osmotic-stress transcriptional responses (Enjalbert et al., 2006; Argimon et al., 2011).

Another strategy for evading AMP function is to enzymatically degrade these peptides before they exert their effects. This is possible by producing proteases and peptidases involved in tissue degradation, as described for *C. albicans* secreted aspartic proteases (Saps). Namely, histatin-5, present in human saliva, is a host-specific substrate of Sap9, enabling the transition of the fungus from commensal to pathogenic in HIV+ individuals. These patients, who have lower levels of this isoenzyme in the saliva, have an increased incidence of oral candidiasis (Meiller et al., 2009; Khan et al., 2013). Also regarding histatin-5, a transport mechanism of efflux mediated by the flu-1 transporter has been described for *C. albicans*, rendering the pathogen the ability to reduce the isoenzyme cytosolic concentration and fungicidal activity (Li et al., 2013). The LL-37 cathelicidin and histatins bind to cell wall carbohydrates, preventing adhesion of *C. albicans* to host cells; thus, the release of AMP-binding proteins acts as a decoy for these AMPs, diverting them from binding to fungal cell surface (den Hertog et al., 2005, 2006; Mochon and Liu, 2008). For example, Msb2 (multicopy suppressor of a budding defect) is a *C. albicans* surface protein (a mucin) highly soluble and proteolytically stable, which is shed to the extracellular environment, acting as a basal AMP-resistance decoy by binding to LL-37 and histatin-5, avoiding the antimicrobial action of these AMPs (Szafranski-Schneider et al., 2012).

The characteristics described above are associated with a decrease in microbes' susceptibility to AMPs, indicating that microbial pathogens have developed some structure-specific and energy-dependent mechanisms to subvert the action of these host defense systems.

# **FUNGAL CELL MEMBRANE**

Fungi possess a unique cell wall and cell membrane that can serve as specific targets for antifungal agents. The fungal cell membrane is similar to those of other eukaryotic cells, composed of a lipid bilayer with proteins embedded within it (Katzung et al., 2011). Sterols (absent in prokaryotes) are major components of fungal membranes. The sterol present in higher eukaryotic membranes is cholesterol, but in fungal membranes the main sterol present is ergosterol, providing stability and flexibility to the cell membrane (Thevissen et al., 1999, 2003a).

Glycosphingolipids (GSLs) are a family of lipids that act as key components of biological membranes. They exist in animals, plants, and fungi (Leipelt et al., 2001; Halter et al., 2007; Daniotti and Iglesias-Bartolome, 2011). GSLs were initially described as components of the architecture of cell membranes, straightly connected with fluidity and stability (Feinstein et al., 1975; Aaronson and Martin, 1983; Campanella, 1992). Recently, however, it was demonstrated that their role goes clearly beyond the initial concept, since these molecules are major components of specialized membrane domains called lipid rafts (Bagnat et al., 2001; Hakomori, 2003, 2008). GSLs have been characterized as important structures in cell–cell interaction, cell signaling, and protein sorting (Bagnat et al., 2000; Bagnat and Simons, 2002; Nimrichter et al., 2008; Staubach and Hanisch, 2011). Lipid rafts are more ordered and tightly packed than the surrounding bilayer, serving as organizing centers for the assembly of signaling molecules, influencing membrane fluidity and membrane protein trafficking (Chiantia and London, 2013).

The most common GSL found in fungi is GlcCer, present in the membranes of most fungi, such as *Pichia pastoris*, *C. albicans*, *Cryptococcus neoformans*, *Aspergillus fumigatus*, *Sporothrix schenckii* and *Neurospora crassa* (Saito et al., 2006). Large amounts of this GSL have also been found in the fungal cell wall (Nimrichter and Rodrigues, 2011). GlcCer has been identified as a fungal component decades ago. Its functions during fungal growth/dimorphism, lipid raft formation, and correlation with virulence have been reported (Rittershaus et al., 2006). In fact, it was recently shown to be required for virulence in *C. albicans* (de Medeiros et al., 2010; Noble et al., 2010; de Coninck et al., 2013).

Work published by Thevissen and colleagues strongly suggested that fungal GlcCer targeting by the AMPs *Rs*AFP2 and *Hs*AFP1 could initiate a cell signaling response in fungi, with formation of ROS and subsequent cell death by apoptosis (Thevissen et al., 2004; Aerts et al., 2007, 2009, 2011). The use of anti-GlcCer antibodies was shown to block germ tube formation in *C. albicans*, *Colletotrichum gloeosporioides*, and *Pseudallescheria boydii* (Pinto et al., 2002; da Silva et al., 2004), and also to protect mice upon

the potentially lethal infection by *C. neoformans* (Rodrigues et al., 2007).

The crescent knowledge of GlcCer functions in eukaryotes (may these be related to virulence, growth or morphological transitions), together with the findings described above, can be connected to specific and essential structural features and particular biosynthetic steps to validate this GSL, as well as other fungal specific membrane lipids and sterols, as potential targets on the development and discovery of new antifungal drugs (Nimrichter and Rodrigues, 2011; Gonçalves et al., 2012b). Besides GlcCer, fungal membranes are also rich in phosphomannans and in the negatively charged phospholipids PS, phosphatidylinositol (PI) and diphosphatidylglycerol (DPG), which confer a highly negative surface charge to these membranes (Pasupuleti et al., 2012).

# **MODELS OF MEMBRANE ACTIVITY – MECHANISM OF ACTION**

The biological activity of AMPs is strongly influenced by peptide– membrane interactions. To explain how some AMPs show differential membrane affinity, their biological activities, and modes of action have been assessed on studies of defensins interaction with fungal membrane model systems, which showed a strong dependence on membrane lipid composition and on the concentration of specific components (de Medeiros et al., 2010; Gonçalves et al., 2012a,b). As with other AMPs, the mechanisms of action of some plant defensins with antifungal activity involve membrane binding, binding to the cell wall, interaction with intracellular targets leading to apoptosis, membrane permeabilization, and receptor-mediated internalization (van der Weerden et al., 2013).

The mechanisms of action of some defensins have been studied by using synthetic lipid vesicles mimicking the lipid composition of fungal, bacterial and mammal membranes (de Medeiros et al., 2010; Wimley and Hristova, 2011; Gonçalves et al., 2012a,b). The permeabilization models used to explain the mode of action of defensins could be classified into two main groups: transmembrane poreformation, such as the barrel-stave and toroidal models, and non-pore formation, such as the carpet, aggregate channel, Shai–Matsuzaki–Huang, lipid clustering, and interfacial activity models (Alba et al., 2012). The carpet model can evolve to disrupt the membrane through pore formation models or through a detergent-like mechanism, with partial micellization of the membrane (Bechinger and Lohner, 2006; Chang et al., 2008; Hoskin and Ramamoorthy, 2008). There are currently at least three different commonly accepted models describing possible AMPs mode of action: the barrel-stave pore model, the toroidal pore model, and the carpet model (Shai, 2002; Chang et al., 2008; Hoskin and Ramamoorthy, 2008; Alba et al., 2012).

Most defensins are amphipathic molecules with clusters of positively charged amino acid residues side chains and hydrophobic amino acid side chains (Lehrer and Lu, 2011). This structural behavior allows them to interact with microbial membranes both at the level of the negatively charged phospholipid head groups and of the hydrophobic fatty acid chains. The orientation of the peptide on the membrane surface depends on the specific peptide– lipid system, but it is commonfor theAMP to stay at the membrane interface until a threshold peptide concentration is reached (Yang

et al., 2000; Yount and Yeaman, 2005; Pasupuleti et al., 2012). In the barrel-stave model (**Figure 2**), once the critical threshold concentration of peptide is reached, peptides self-aggregate in the membrane resulting in the formation of a transmembrane pore lined by peptide, which dissipates proton and ionic gradients (Ehrenstein and Lecar, 1977; Reddy et al., 2004), but the membrane thickness and homogeneity do not change (Chang et al., 2008). The toroidal pore model is a variant of the barrel-stave model, claiming that, at some critical peptide concentration, curvature strain induces membranes to curve inward, resulting in the formation of a pore that is lined by both peptides and lipid headgroups (**Figure 2**). Toroidal pores seem to have varying lifetimes and longer-lived pores may have a lethal effect similar to barrel-stave pores, with dissipation of proton and ion gradients. This type of mechanism of AMPs action also causes a decrease in membrane thickness and a slightly decreased surface homogeneity (Chang et al., 2008). In the carpet model (**Figure 2**), peptides bind to phospholipid head groups by electrostatic interactions and align themselves parallel to the membrane surface in a carpet-like fashion until a critical threshold concentration is reached. When a detergent-like membrane micellization takes place, a strong decrease of membrane homogeneity occurs (Chang et al., 2008; Hoskin and Ramamoorthy, 2008; Epand et al., 2010; Hazlett and Wu, 2011; Li et al., 2012; Pasupuleti et al., 2012).

Besides targeting fungal membranes' specific components, defensins may also have other mechanisms of action, as previously referred. These mechanisms comprise binding to the cell wall, membrane permeabilization, receptor-mediated internalization inducing signaling cascades and interaction with intracellular targets, which would cause the formation of ROS, leading ultimately to programed cell death. To address these mechanisms the reader is directed to some relevant references on this topic (Hancock and Rozek, 2002; Oberparleiter et al., 2003; Thevissen et al., 2003a, 2004; Schroeder et al., 2011; de Coninck et al., 2013; De

Initially, the peptide (magenta) is adsorbed at the membrane surface. After an initial recognition of the surface, a conformational change of the peptide occurs. Once a threshold concentration of peptide on the membrane is reached, it is followed by membrane disruption by one of these three mechanisms.

Paula et al., 2013; Jaeger et al., 2013; van der Weerden et al., 2013; Zhang et al., 2013).

#### **CONCLUSION**

The knowledge on AMPs has been increasing considerably during the last 20 years. This increased knowledge shows that AMPs have much more than only antimicrobial activity, presenting a broad spectrum of physiological functions. Defensins are the most represented AMPs across the eukaryotic domain, and in all types of eukaryotic organisms we can find defensins not only with antifungal activity but also with other potential applications.

Despite this relevance, defensins may have limitations in terms of new drug development, due to their cationic, amphiphilic, and protease labile nature, leading to a low serum half-life that limits their systemic administration (Maisetta et al., 2008). This limitation can be overcome by the use of peptidomimetics, like the substitution of natural occurring L-amino acid residues by Damino acid residues or unusual amino acids (Oren et al., 1997; McPhee et al., 2005). Defensins bare a favorable characteristic against this problem, as their disulfide-stabilized structure confers increased protease-resistance (Wu et al., 2003). Nonetheless, defensins combine targeted antimicrobial activity with the capacity to positively modulate the immune system, and have proven to be effective across life evolution, making these peptides highly appealing as an anti-infective strategy.

Defensins have evolved as successful barrier of defense not only against bacteria, but also pathogenic fungi, present among plants, animals, and fungi. This ability may serve as a "lesson" on how selective pressures that shape organisms and their components served and continue to serve as a lever for the evolution of better defenses. Most antibiotics used nowadays are from bacterial origin or synthetic (Clardy et al., 2009; Fisher et al., 2012). The molecular design and synthesis of new molecules inspired on defensins or on other AMP structures and sequences seem to be a promising approach to develop a new and extensive field of applications, ranging from antimicrobial therapy, to their possible use as vaccine adjuvants. Therefore, a better understanding of function and mechanism of action of HDPs, specially defensins, is highly relevant for the development of new anti-infective and immunomodulatory therapeutics (Guaní-Guerra et al., 2013).

#### **ACKNOWLEDGMENTS**

This work was partially supported by Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCT-MEC, Portugal) and by the European Union FP7-IRSES project MEM-PEPACROSS.

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Zhu, S. (2007). Evidence for myxobacterial origin of eukaryotic defensins. *Immunogenetics* 59, 949–954. doi: 10.1007/s00251-007-0259-x

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 August 2013; accepted: 21 February 2014; published online: 20 March 2014.*

*Citation: Silva PM, Gonçalves S and Santos NC (2014) Defensins: antifungal lessons from eukaryotes. Front. Microbiol. 5:97. doi: 10.3389/fmicb.2014.00097*

*This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology.*

*Copyright © 2014 Silva, Gonçalves and Santos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Antifungal defensins and their role in plant defense

# *Ariane F. Lacerda1,2 , Érico A. R. Vasconcelos 2,3 , Patrícia Barbosa Pelegrini 2 and Maria F. Grossi de Sa2,3 \**

*1 Department of Biochemistry and Molecular Biology, Federal University of Rio Grande do Norte, Natal, Brazil*

*2 Plant-Pest Interaction Laboratory, Embrapa – Genetic Resources and Biotechnology, Brasília, Brazil*

*3 Catholic University of Brasilia, Brasília, Brazil*

#### *Edited by:*

*Tzi Bun Ng, The Chinese University of Hong Kong, China*

#### *Reviewed by:*

*Noton Kumar Dutta, Johns Hopkins University, USA Dmitri Debabov, NovaBay Pharmaceuticals, USA*

#### *\*Correspondence:*

*Maria F. Grossi de Sa, Plant-Pest Interaction Laboratory, Embrapa – Genetic Resources and Biotechnology, PqEB Avenue W5 Norte (final), P. O. BOX 02372, Brasília, DF 70770-917, Brazil e-mail<EMAIL_ADDRESS>
Since the beginning of the 90s lots of cationic plant, cysteine-rich antimicrobial peptides (AMP) have been studied. However, Broekaert et al. (1995) only coined the term "plant defensin," after comparison of a new class of plant antifungal peptides with known insect defensins. From there, many plant defensins have been reported and studies on this class of peptides encompass its activity toward microorganisms and molecular features of the mechanism of action against bacteria and fungi. Plant defensins also have been tested as biotechnological tools to improve crop production through fungi resistance generation in organisms genetically modified (OGM). Its low effective concentration towards fungi, ranging from 0.1 to 10 μM and its safety to mammals and birds makes them a better choice, in place of chemicals, to control fungi infection on crop fields. Herein, is a review of the history of plant defensins since their discovery at the beginning of 90s, following the advances on its structure conformation and mechanism of action towards microorganisms is reported. This review also points out some important topics, including: (i) the most studied plant defensins and their fungal targets; (ii) the molecular features of plant defensins and their relation with antifungal activity; (iii) the possibility of using plant defensin(s) genes to generate fungi resistant GM crops and biofungicides; and (iv) a brief discussion about the absence of products in the market containing plant antifungal defensins.

**Keywords: plant defensins, antifungal, phytopathogens, peptide structure, peptide function, transgeny**

#### **INTRODUCTION**

Plants are constantly exposed to several pests and pathogens in nature. They have developed complex defense mechanisms to protect themselves against the attack of pathogens (Gachomo et al., 2003, 2010).

Defensins are small cationic peptides of 45–54 amino acid residues with a conserved signature of cysteines, which can form three to four disulfide bridges. Plant defensins exhibit a conserved tertiary structure that consists of a triple-stranded antiparallel βsheet and one α-helix that are stabilized into a compact shape by the disulfide bridges. These bridges form a cysteine-stabilized α-helix β-sheet motif (CSα/β) (Kobayashi et al., 1991; Zhu et al., 2005). In addition to the CSα/β motif, two additional conserved motives, named α-core, encompassing the loop connecting the first β-strand to the α-helix, and the γ-core containing the hairpin loop that links β-strands 2 and 3 (Lβ2β3) were also present in the defensin structure (Yount and Yeaman, 2004; Yount et al., 2007). Despite the low level of amino acid sequence identity between defensins, their three dimensional structures are remarkably similar between different plant defensins (Pelegrini and Franco, 2005). Variations in the amino acids are reflected by small conformational changes in the tertiary structure that contribute to the broad range

of biological activities in these proteins. Only one amino acid substitution can change the spectrum of activity exhibited by these peptides (Carvalho and Gomes, 2011).

Since the beginning of 1990s, lots of cationic plant cysteine-rich antimicrobial peptides (AMP) have been studied. Plant defensins were first described in the seeds of wheat (*Triticum turgidum*) and barley (*Hordeum vulgare*) (Colilla et al., 1990; Mendez et al., 1990). They were characterized as a new member of the thionine family due to their similarity in molecular mass, amino acid sequence and number of cysteines. However, subsequent studies performed by Bruix et al. (1995) revealed the existence of differences in the pattern of the disulfide bridges, demonstrating that these two peptide families are unrelated. Broekaert et al. (1995) renamed these peptides as "plant defensins," after comparing their structural and functional resemblance to previously characterized AMPs found in insects and mammals.

#### **DEFENSINS AND THEIR CONTRIBUTION TO PLANT DEFENSE HOW CAN DEFENSINS HAVE A ROLE IN PLANT DEFENSE?**

The role of defensins in the preformed defense of plants is well reported. Several reports show that defensins are an integral part of the plant innate immune system. Most plant defensins already characterized show a constitutive pattern of expression with up regulation in response to pathogen attack, injury and some abiotic stresses (de Beer and Vivier, 2011).

Several features make clear that defensin peptides are involved in plant defense (Selitrennikoff, 2001). Their distribution is consistent with their putative defense role. They have been identified in leaves, tubers, flowers, pods and seeds, playing an important role in the protection of germinating seeds and developing seedlings (Garcia-Olmedo et al., 1998). In addition, plant defensins are also localized in the xylem, stomata, and stomata cells, parenchyma cells, and other peripheral areas (Kragh et al., 1995; Segura et al., 1998; Chen et al., 2002). The presence in the different tissues is consistent with a defensive role of such peptides, once it is believed that such sites are the place of the first contact with a potential pathogen (Carvalho and Gomes, 2011).

Moreover, plant defensins have a broad spectrum of *in vitro* antimicrobial activity and, currently, there are several reports describing the production of transgenic plants constitutively expressing foreigner defensins. Hence, they possess an enormous multiplicity of biological activities, such as antimicrobial, insecticidal, inhibiting protein synthesis, mediating abiotic stress, and Zn tolerance, and as inhibitors of digestive enzymes (Carvalho and Gomes, 2009, 2011). According to Franco (2011), these defense peptides are classified as promiscuous proteins, as they show numerous biological activities. As an example, there is the family of defensins isolated from *Vigna unguiculata*, in which different homologous forms may act as antifungal, antibacterial, and enzyme inhibitors (Franco, 2011). Although they present multiple functions, the antimicrobial activity of plant defensins is mainly observed against fungi.

Therefore, the present review explores the current knowledge about the structure and mechanism of action of plant defensins with emphasis on its activity against phytopathogenic fungi. Furthermore, we describe the current use of these peptides as biotechnological tools in the production of transgenic plants that could result in the future release of agronomically important crops resistant to various diseases.

# **STRUCTURAL CONFORMATION AND MECHANISM OF ACTION**

Plant defensins present a well-conserved three-dimensional structure composed by a cysteine-stabilized α/β (CSαβ) motif, which forms one α-helix followed by three anti-parallel β-sheets. The amino acid sequence is also quite conserved, especially due to the presence of six to eight cysteine residues, which form three to four disulfide bridges in the sequence of Cys1-Cys8, Cys2-Cys5, Cys3-Cys6, and Cys4-Cys7 (Lay and Anderson, 2005). Nevertheless, plant defensins with five disulfide bonds have been described, such as the peptide from *Petunia hybrida* (PhD1), whose cysteine residues interact in the following order: Cys1-Cys10, Cys2-Cys5, Cys3-Cys7, Cys4-Cys8, and Cys6-Cys9 (Janssen et al., 2003). The additional disulfide bond does not affect the typical three-dimensional structure of the defensin, which is located after the α-helix and the first β-sheet (Janssen et al., 2003).

Furthermore, plant defensins with alternative structures have been identified in the literature, including defensins from *Nicotiana alata* (NaD1), *Petunia hybrida* (PhD1 and PhD2), and ZmESR6 isolated from developing maize kernels. These defensins contain an extra acidic C-terminal prodomain whose function is still unknown, although it has been suggested that it is involved in vacuolar targeting or in eliminating potential detrimental effects caused by the basic nature of the defensin (De Coninck et al., 2013).

As they are peptides consisting of 45–54 amino acid residues, structural studies on crystallography and nuclear magnetic resonance (NMR) have been widely extended during the last few years. Among the peptides with antifungal activity, whose structures have been elucidated, are included the defensins from *Nicotiana alata* (NaD1), *Pachyrrhizus erosus* (SPE10), *Petunia hybrida* (PhD1), *Pisum sativum* (Psd1), *Raphanus sativus* (Rs-AFP1), and *Saccharum officinarum* (Sd5) (Fant et al., 1998; Almeida et al., 2002; Janssen et al., 2003; Lay et al., 2003; de Paula et al., 2011; Song et al., 2011; Van der Weerden and Anderson, 2013; **Figure 1**). An amino acid sequence alignment of antifungal defensins from plants shows that they do not present conservative amino acid sequences, except the cysteine residues and a glycine residue positioned in the second β-sheet (Pelegrini and Franco, 2005; Van der Weerden and Anderson, 2013). According to their structural features, plant defensins show a conserved γ-core signature classified as the dextromeric isoform, which is related to the amino acid sequence conservation of the region NH2...[X1−3]-[GXC] = [X3−9]-[C]...COOH (**Figures 1** and **2**). This preservation in the primary sequence gives them a threedimensional conformation denominated γ-core motif, consisting of two antiparallel β-sheets, with an interpolated turn region. Earlier studies classified plant defensins as belonging to the β-γ-α Group, according to their relative structural γ-core (Yount and Yeaman, 2004). It has been described that the γ-core motif is important for antimicrobial activity in disulfide-stabilized peptides (Yount andYeaman, 2004), not only for their cysteine content, but especially due to the presence of positively charged residues at the second β-turn of their structure (Fant et al., 1998). This characteristic was first observed when the structure of *R. sativus* defensin 1 (Rs-AFP1) was determined by 1HNMR, and mutation analyzes was also performed using the peptide isoform Rs-AFP2 (De Samblanx et al., 1997; Fant et al., 1998). In both cases, it was demonstrated that positively-charged amino acids located at the γ-core motif were essential for the antifungal activity of theses peptides, and the substitution of neutral residues inside this γcore by other positively-charged amino acid residues increased their activity towards pathogenic fungi. Spelbrink et al. (2004), while studying defensins from *Medicago trunculata*, verified that the antifungal activity of MtDef1 was due to the presence of four positively-charged amino acids, also located in the γ-core region, which was lacking in the structure of the non-antifungal peptide MtDef2. Moreover, *in vitro* assays revealed that this region might be involved in the ability of MtDef1 to block L-type Ca++ channels in mammalian cells.

There are two major hypothesis that tries to explain the mechanism of action of antimicrobial defensins: (i) the carpet model and (ii) the pore model. In both models, defensins are described to interact with the negatively charged molecules present at the cell membrane of pathogens, causing an increase of its permeabilization, leading to cell leakage and death by necrosis. While the carpet model emphasizes the pore formation of several peptides into the membrane, the pore model shows that peptides form oligomers that, then, form multiple pores into the cell membrane. However, there is an alternative hypothesis, where defensins do not damage

**FIGURE 1 | Alignment of the amino acid sequence of antifungal plant defensins.**  Asterisk indicates conserved cysteine amino acid residues among antifungal defensins (gray boxes). Gray lines represents the disulfide bridges between cysteine amino acid residues. Pink box and blue amino acid residues correspond to the γ-core region. Green arrows indicate β-sheet region and green cylinder indicate α-helix region. Alignment was done using ClustalW2 Tool.

**plants.** Pink region highlight the γ-core motif of each peptide. β1: β-sheet 1; β2: β-sheet 2; β3: β-sheet3; L1: Loop1; L2; Loop 2: NaD1: *Nicotiana alata* defensin 1 (Accession: 4ABO\_A); Rs-AFP1: *Raphanus sativus* antifungal peptide 1 (Accession: 1AYJ\_A); SPE10: *Pachyrrihizus erosu* peptide

(Accession: 3PSM\_A); PhD1: *Petunia hybrida* defensin 1 (Accession: 1N4N\_A); Sd5: *Saccharum officinarum* defensin 5 (Accession: 2KSK\_A); VrD2: *Vigna radiata* defensin 2 (Accession: 2GL1\_A). All figures were designed using PyMol Molecular Graphic System Version 1.2r3pre, Schrödinger, LLC.

the cell membrane, but interact with the phospholipids, leading to an increase of ion permeability, or even to the transportation of these peptides to the intracellular environment (Wilmes et al., 2011; Hegedus and Marx, 2013). Hence, they can also enhance reactive oxygen species (ROS) and activate programmed cell death (PCD; Wilmes et al., 2011; Hegedus and Marx, 2013).

Moreover, positively charged amino acid residues were described to be important for antifungal activity, when located at loops and β-sheet regions. Hence, it was observed that the concave side of theVI β-turnfrom Rs-AFP1 was positively-charged, leading to the suggestion that the contact of this peptide with pathogenic fungi may occur through electrostatic interactions (De Samblanx et al., 1997; Fant et al., 1998). Other studies on the structural analyses of plant defensins, such as NaD1, described the importance of positively-charged amino acid residues at the loop region between β2 and β3, not only for antifungal activity, but for also functioning as a specificity factor towards different pathogens (Lay et al., 2003). Recently, it was reported that the amino acid residues located in the γ-core motif of MtDef4 are key tools for its antifungal activity and its specificity towards pathogenic fungi (Sagaram et al., 2011). First, *in vitro* assays using only the γ-core sequence of Mtdef4 and MsDef1 (alfAFP) showed that the high content of positively charged residues with the core of MtDef4 could, alone, provide antifungal activity, in contrary to the core of alfAFP, which was inactive against filamentous fungi (Sagaram et al., 2011). Later, mutagenesis studies on the region RGFRRR from MtDef4 showed that the substitution of the hydrophobic and positively-charged residues, Phe and Arg, at positions 3 and 4, respectively, by Ala residues decreased intensely its activity against fungi. Furthermore, it was shown that both defensins present differences on their kinetics of permeabilization, when assayed against *Fusarium graminearum*, as MtDef4 was able to induce a more potent antifungal activity and could take up the molecular probe SYTOX Green (SG) at a dependent concentration, indicating physical damage of cell membranes. In comparison, alfAFP induced a less effective membrane permeabilization, and did not induce a concentration dependent SG uptake (Sagaram et al., 2011).

Further reports displayed a comparison between the electrostatic potential surfaces of different defensins with their potential antimicrobial activities (Almeida et al., 2002). However, although there was no pattern of charge distribution among defensins, there was a high indication that plant defensins may act as potassium channel inhibitors, due to their similarities with neurotoxins, which contains residues for such activity (Almeida et al., 2002). **Figure 3** shows the electrostatic surface area of three antifungal plant defensins (Phd1, Rs-AFP1, and VrD2), in which the site related to the second loop of the defensins that contains the γ-core region is described as the most important site for their antifungal activity. This is highly positively-charged in the cartoons where electrostatic surfaces were designed in vacuum. Therefore, it corroborates with the antifungal assays and the *in silico* studies performed by many researchers over the last 20 years.

A structural study on sugarcane defensin, Sd5, provided new information about the mechanism of action for those antifungal peptides. It was described that the hydrophobic core at the C-terminal of the defensin is also important for membrane interaction and permeabilization (de Paula et al., 2011). In

addition, evaluations on the backbone conformational dynamics of Sd5 suggest that the mechanisms of its structural exchange is related to modifications in the hydrogen bond distances of the β-sheet and α-helix of the peptide, giving it the ability to bind to membranes. Hence, membrane permeabilization and vesicle leakage induced by Sd5 may occur through the interaction of the side chains of residues of three serines and the glycosyl part of the membrane model with glucosylceramide extracted from the hyphae of *F. solani* (de Paula et al., 2011). Recent studies on dynamics of Sd5 structure revealed that this peptide displays many dynamic properties. It was able to interact with a sphingolipid glycosylceramide (CMH) membrane in a conformational selection process, which involved a specific binding, while other flexible regions of Sd5 showed to interact with the interface in a nonspecific manner (Valente et al., 2013).

Recent reports described the structural conformation of dimeric defensins being highly significant for its antifungal activity (Song et al., 2011; **Figure 4**). In this way, analyses of the defensin from *Pachyrrihizus erosus*, SPE10, provided the selection of the binding pattern Arg36-Trp42-Arg40 as essential for dimer formation. Moreover, it was demonstrated that Trp42 is fundamental for antifungal activity of plant defensins, as it is absent in nonantifungal peptides Therefore, dimers of SPE10 are arranged in a side-by-side manner with the α-helix of one monomer interacting with the β-sheet of the second monomer, leading to a stretched

and twisted molecular surface. Conformational changes on Arg36 and Trp42 would alter the dimeric interface of SPE10, destabilizing the dimer (Song et al., 2011). In addition, the dimerization of the defensin NaD1 was performed in order to evaluate the relation between structural conformation and antifungal activity. In contrary to what was observed by SPE10 dimer, monomers of NaD1 were connected by a β-sheet/β-sheet configuration, although the antifungal activity was maintained (Lay et al., 2012; **Figure 4**). Hence, plant defensins that form dimers coupled with their positively charged surface area become highly efficient molecules against pathogenic fungi, as they can strongly interact with the negatively charged glycoproteins located at the fungal cell walls (Lay et al., 2012).

#### **TARGETED FUNGI AND EFFECTIVE CONCENTRATIONS**

One of the first studies that attempted to highlight this class of plant antimicrobial defensins was carried out with two peptides isolated from Radish seeds, Rs-AFP1 and Rs-AFP2. Both peptides were assayed against 20 different plant pathogenic fungi and the lower protein concentration required for 50% inhibition of fungal growth (IC50) was obtained by Rs-AFP2, when assayed against *Pyricularia oryzae.* Its IC50 ranged from 0.08 to 5 μM. Since that lots of defensins were reporter to show high biological activity in the range of micromolar to nanomolar as will soon be shown. Terras et al. (1992) were the first ones to report the importance of disulfide bonds to defensins stabilization and the role of inorganic ions in its antifungal activity. They also showed how thermostable defensins are, once they found that heating Rs-AFP1 and 2 at 100◦C for 10 min did not affect antifungal properties of such molecules. The stability of such molecules is an important feature which allows wondering a wide range of biotechnological applications to plant defensins.

Few years after Terras report, Osborn et al. (1995) increased the knowledge about plant defensins and their effects under fungi. They assayed four AMP isolated from *Aesculus hippocastanum* (Ah-AMP1)*, Clitoria ternatea* (Ct-AMP1), *Dahlia merckii* (Dm-AMP1), *Heuchera sanguinea* (Hs-AFP1) against eight different fungi in the presence, or absence, of inorganic ions. The lower IC50, around 0.1 μM, was acquired when Ah-AMP1 was tested towards *Cladosporium sphaerospermum*, *Leptosphaeria maculans*, and *Septoria tritici*. Hs-AFP1 presents the same antifungal activity when assayed against *Septoria tritici*. In all the studies, inorganic ions decreased IC50. When visualized under a microscope, it was possible to see that such antifungal peptides caused distinct morphological changes during germ tube elongation and hyphae development, like multiple hyphae buds or the diminished of the rate of germ tube elongation (Osborn et al., 1995; **Table 1**).

A great number of the earlier studies about the mechanism of action of plant defensins agree on the membrane permeabilization outcome (Thevissen et al.,1996,1999). More recently, two peptides similar to plant defensins were reported to show such disruption power. The first one, from *Phaseolus vulgaris*, permeabilizes *Mycosphaerella arachidicola* membrane, among other fungi (Wong et al., 2012). The second, from *Picea glauca*, was reported to act on permeabilization of *Verticillium dahlia* membranes (Picart et al., 2012). Membrane permeabilization seems to be just one of a huge variety of mechanism of action for such molecules. While some results point to cellular membranes as the point of action, others suggest intracellular targets (Thevissen et al., 2000).

The use of antifungal peptide genes to generate important agronomical traits resistant to fungal disease have been seen with some skepticism by the biotechnological thinkers. Plant defensins proved to be useful for biotechnological purposes in the year of 2000, when Gao et al. (2000) showed that AlfAFP, an antifungal peptide from *Medica sativa* and active towards*Verticillium dahliae*, was expressed in a transgenic potato, increasing resistance against such filamentous fungus. The IC50 of AlfAFP towards *Verticillium dahlia* was determined at 1 μM, around ten times higher than the previous AMP described here (Gao et al., 2000). However, the resistance of transgenic potato expressing AlfAFP towards*Verticillium dahliae* showed to be more effective in greenhouse conditions and in the field than the chemical methods, what make of it a useful choice to plant transformation aiming resistance to phytopathogenic fungi, which will be discussed in detail later in this review.

Almeida et al. (2001) reported the heterologous expression of a *Pisum sativum* defensin (Psd1) in a eukaryotic expression, system based on the methilotrophic yeast *Pichia pastoris*. The high amount of Psd1 produced by *P. pastoris* expression system (13.8 mg/L), allowed investigations about the conformational features between wide type and recombinant form of Psd1 (rPsd1). Besides being active towards filamentous fungi, such as *Neurospora crassa*, Psd1 did not demonstrate any activity against yeasts, even at high (20 μM) concentrations (Almeida et al., 2000). According

**Table 1 | Short sample of plant defensins and its IC50 concentration against its fungal targets.**

to the report, the heterologous expression in *Pichia pastoris* did not significantly affect the defensin conformational features, and all post-translational modifications needed to its activity had been done. One of the small differences between Psd1 and rPsd1 was their N-terminal sequences. rPsd1 kept four amino acids residues from the recombinant signal peptide, and this seemed to be related to the 5-fold decrease on its activity towards *F. solani* and *Aspergillus niger*, in comparison to the wide type peptide. rPsd1 activity towards *N. crassa* was not affected, which suggests distinct modes of action of Psd1 against fungi belonging to different classes (Almeida et al., 2001). Furthermore, the *Pichia pastoris* system was also used to produce the recombinant *Nicotiana megalosiphon* defensin (NmDef02) active against *F. oxysporum* (Portieles et al., 2010).

Plant defensins have also been expressed in prokaryotic system and tested against fungi. TvD1, a defensin from *Tephrosia villosa*, was expressed in *Escherichia coli* and assayed towards *Pheaoisariopsis personata* (Vijayan et al., 2008; **Table 1**). A comparison between the expression of Pdc1, a corn defensin, in yeast and *E. coli* was done and in both cases the peptide kept its antimicrobial activity, however, Pdc1 expressed in yeast (IC50 7.5 μM) was more efficient than when expressed in *E. coli* (IC50 30 μM) in arrest *F. graminearum* growth. The presence or absence of a His-tag also influences its activity, suggesting that defensins are sensible to covalent modifications on its terminal ends (Kant et al., 2009).

Different from some results, which suggest the importance of N-terminus in defensin activity (Almeida et al., 2001), Spelbrink et al. (2004) demonstrated that the major determinant of antifungal activity of a defensin from *Medicago sativa* (MsDef1) resides in the carboxy-terminal region. They evaluated six different defensin chimeras obtained from molecular combinations of MsDef1, active towards *F. graminearum*. They also analyzed MtDef2, a defensin from *Medicago truncatula*, which did not have any activity towards *F. graminearum*. Among the six chimeras, only the ones harboring the MsDef1 portion on the C-terminal displayed some activity against *F. graminearum* (Spelbrink et al., 2004). The divergence among results pointing to C-terminus and to N-terminus as essentials to plant defensin activity, expose the uncertainty about the relation between structure and function of such molecules and even more on its modulation mechanism of activity.

Three years after Spelbrink findings,Ramamoorthy et al. (2007) tried to go a little deeper into the cellular mechanisms of activity modulation using Medicago defensins against *F. graminearum*. They have demonstrated that mutants of *F. graminearum* can react differently to Medicago defensins MsDef1 and MtDef4. *F. graminearum* mutant, whose MAP Kinase cascades were disrupted, were hypersensitive to MsDef1. However, it did not show any difference on its sensitivity to MtDef4. MAP kinase signaling cascades seemed to provide protection towards MsDef1, but not to MtDef4, which suggests that these plant defensins utilize specific signaling pathways to alter fungal growth (Ramamoorthy et al., 2007).

Besides antimicrobial activity, a plant defensin from *Phaseolus limensis* named Limyin and active against *F. solani*, were also reported to show antiproliferative activity towards human tumor cells (Wang et al., 2009), suggesting there are lots of things to be discovered about the cellular targets and mechanisms of action of plant defensins.

Plant defensins encompass a class of biomolecules with the potential to be explored as biotechnological tools towards phytopathogenic fungi, which nowadays, are controlled only by chemicals. The wide natural sources of these molecules and the heterogeneity of their action on different targets allow hundreds of possible biotechnological approaches that, together with their low effective concentration, as shown in **Table 1**, could lead to phytopathogeninc fungi control with less environmental impact.

#### **BIOTECHNOLOGICAL APPLICATIONS AND TRANSGENY**

Although there are many transformed plants in the market with additional genes coding to proteins that confer resistance towards herbicides and insect-pests, there is still no transgenic plant available against phytopathogenic fungi, nor even containing

**Table 2 | Antifungal defensins from plant sources used for transformation into foreign species.**

plant defensins as the resistant factor. Nevertheless, several studies describe the efficient activity of antifungal defensins when transformed into different host plants (**Table 2**). Therefore, plant defensins with antifungal activity have become the first molecule for the development of transgenic crops resistant to phytopathogens.

The first attempt to evaluate of transgenic plants containing foreigner antifungal defensin genes was done in tobacco plants expressing Rs-AFP2, a peptide from radish. High levels of peptide expression were observed in the transformed tobacco plants, as well as an increasing resistance towards the phytopathogenic fungus *Alternatia longipes* (Terras et al., 1995). Four years later, the same defensin was used for studies with transgenic apple plants and evaluation against pathogenic fungi species (De Bondt et al., 1999). Hence, after transformation through *Agrobacterium tumefaciens*, the transgenic plants were selected and the expressed peptide was isolated and quantified. *In vitro* assays showed that the recombinant peptide was able to inhibit the germination of *Fusarium culmorum* spores (De Bondt et al., 1999). Tomato lines have also been transformed with Rs-AFP2, generating the increase in their antifungal activity. In this study, leaves of tomato plants overexpressing the radish defensin were extracted and tested against some phytopathogenic fungi, including *Alternatia solani*, *F. oxysporum*, *Phytophthora infestans*, and *Rhizoctonia solani* (Parashina et al., 2000). It was demonstrated that the crude extract of tomato leaves containing the radish defensin could decrease the activity of all the fungi cited above.

Furthermore, in 2002, Rs-AFP2 was again evaluated in transgenic plants, this time using two pear cultivars – Burakovka and Pamyat' Yakoyleva. After transformation, leaves of pear plants were collected for PCR and Western Blot Hybridization analyses. The presence of the foreigner gene and recombinant peptide were detected through the respective techniques, confirming the success of plant transformation (Lebedev et al., 2002). Nevertheless, *in vitro* and *in vivo* assays against pathogen fungi are still to be done in order to check the antifungal activity of transgenic pear plants expressing Rs-AFP2. The most recent work on Rs-AFP2 was published in 2010, when Jha and Chattoo (2010) transformed this peptide into rice (*Oryza sativa* L. cv. *Pusa* basmati 1). The transgenic plants were tested *in vitro* and *in vitro* against *Magnaporthe oryzae* and *Rhizoctonia solani*, the main causes of rice losses in agriculture, revealing that overexpression of Rs-AFP2 can control the rice blast and sheath blight diseases (Jha and Chattoo, 2010).

In addition, other works on transgenic plants expressing an antifungal defensin were published. Hence, it was demonstrated that pea defensins transformed into *Brassica napus* cultivars enhanced their resistance against *Leptosphaeria maculans*, which causes blackleg diseases in plants (Wang et al., 1999). Tobacco plants transformed via *Agrobacterium tumefasciens* and containing a synthetic antifungal gene was also performed. The expressed peptide, named D4E1, provided an increasing resistance of tobacco against *Aspergillus flavus* and *Verticillium dahlia* (Cary et al., 2000). Tobacco was also used for transformation of the stamen

defensin BSD1, where the expressed peptide provided higher tolerance to the plant against the attack of *Phytophtora parasitica* (Park et al., 2002). Transformation of tobacco with the mustard defensin – BjD – once more validated the potential of these peptide-family members as excellent antifungal agents, as transgenic plants displayed improved resistance towards *F. moniliforme* and *Phytophtora parasitica* (Anuradha et al., 2008). More recently, a defensin purified from maize, ZmDEF1, when transformed into tobacco plants, showed increased tolerance against *Phytophtora parasitica* (Wang et al., 2011). Transgenic peanut plants, expressing the same mustard defensin, also provided an enhancement of tolerance against *Cercospora arachidicola* and *Pheaoisariopsis personata*, which mutually cause the late leaf spot disease (Anuradha et al., 2008).

Kanzaki et al. (2002) performed a successful attempt of expressing a defensin from*Wasabia japonica* into rice plants, as an effort to increase the plant resistance against the phytopathogenic fungus *Magnaporthe grisea*. Moreover, they showed that T3-generation transformed rice plants could still overexpress the wasabi defensin and maintain its ability to control *Magnaporthe grisea in vivo*. Earlier, it was demonstrated that transgenic potato expressing an antifungal defensin from alfalfa (alfAFP) was more resistant to the attack of *Verticillium dahliae*, when compared to nontransformed plants (Gao et al., 2000). A summary of information of expressed plant defensins into plant cultivars can be seen at **Table 2**.

An attempt at transforming two different genes at the same time in tomato plants was performed using genetic material of a defensin and a glucanase from alfalfa, in order to analyze their efficiency towards phytopathogenic fungi. Therefore, T1-generation transgenic plants revealed enhanced tolerance to *R. solanacearum*, when compared to non-transformed plants, indicating the existence of a synergic effect of both proteins as antifungal molecules in tomato cultivars (Chen et al., 2006). Further efforts using other plant defensins into transformed tomato plants were carried out. In this way, Abdallah et al. (2010) inserted the *Medicago sativa* defensin gene into *Licopersicum esculentum* cultivar CastleRock and evaluated the transformed plants against the pathogenic fungus *F. oxysporum* f. sp. *Lycopersici. In vivo* assays demonstrated that T1- and T2-generations of transgenic tomato plants presented increased resistance against the fungal pathogen, when compared to non-transformed plants.

Plant defensins have also displayed indirect responses towards phytopathogenic fungi in transgenic plants, when other foreigner genes are being overexpressed (Murad et al., 2007). Hence, earlier reports showed that a peptide from *Arabidopsis thaliana*, named AtPep1 stimulated the transcription activation of the defensin gene *pdf1.2* (Huffaker et al., 2006). When AtPep1 precursor gene PROPEP1 was expressed into transgenic *Arabidopsis* plants, the transcription of PDF1.2 was also observed. Moreover, the expressed defensin stimulated root development, which, consequently, improved plant resistance against the filamentous fungus *Pythium irregular* (Huffaker et al., 2006).

Similar results were obtained when an ionotropic glutamate receptor (RsGluR) was transformed into *Arabidopsis* plants. The expression of RsGluR led to an up-regulation of defensins, causing an increase of the plant resistance towards *Botrytis cinerea* (Kang et al., 2006). Microarray analyses later confirmed that up-regulated defensins and jasmonic acid-responsive genes were produced after overexpression of RsGluR in *Arabidopsis*. Furthermore, the same plant species was transformed with a cotton nonsymbiotic hemoglobin for tolerance against fungal pathogens. However, the foreigner gene could also induce a constitutive expression of the PR protein K (PR-1) as well as the defensin PDF1.2, providing an enhanced resistance to *Verticillium dahliae* (Qu et al., 2006).

# **CONCLUSION AND PERSPECTIVES**

Plant defensins correspond to a world of possibilities for defense mechanisms, and new peptides with different activities are still to be discovered, as well, studies with thousands of plant species need to be performed. Nowadays, several peptides already show satisfactory efficacy against such pathogens with strong potential to be applied for the production of a commercial fungicide or application into transgenic plants. But, the question remains. Why is there still no product containing antifungal plant defensins – in its natural form or in nanocapsules – already available in the market?

It is interesting that plant defensins with antifungal peptides are mostly studied for pathogens located in tropical areas, including Latin American, African, and some Asian countries. Moreover, the loss of commercially important crops due to the attack of phytopathogenic fungi is considered worldwide, until now, less detrimental than the losses caused by drought stress and insectpests. Therefore, the efforts focused on the release of novel plant varieties resistant to drought stress and insect-pests are more significant, as there is mounting pressure to control these adversities in order to provide an increase in crop production. However, the development of transgenic plants expressing antifungal defensins or the production of defensin-based biofungicide depends, mainly, on the determination of regional research teams focusing on specific fungal targets, so these products can reach the market.

Furthermore, there is a long process required for analyzing the efficiency, environmental risks, safety towards animal and human consumption, and reproducibility of transformed plants expressing certain molecules, as well as the need for having an extremely stable, effective, and easy-to-produce peptide to be used in the fabrication of a biofungicide. Therefore, it is possible that there are already plant defensin-based products on the horizon that will soon be released on to the market.

Also, it is expected that, in the near future, antifungal defensinbased commercial agro-products be targeted as essential for the increase of crop production. This will stimulate and accelerate the transition between biotechnological research and field application of bioproducts.

#### **ACKNOWLEDGMENTS**

The authors are thankful for the financial support of CAPES, CNPq, and Embrapa.

# **REFERENCES**

Abdallah, N. A., Shah, D., Abbas, D., and Madkour, M. (2010). Stable integration and expression of a plant defensin in tomato confers resistance to Fusarium wilt. *GM Crops* 1, 344–350. doi: 10.4161/gmcr.1.5.15091

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 10 August 2013; accepted: 07 March 2014; published online: 02 April 2014. Citation: Lacerda AF, Vasconcelos ÉAR, Pelegrini PB and Grossi de Sa MF (2014) Antifungal defensins and their role in plant defense. Front. Microbiol. 5:116. doi: 10.3389/fmicb.2014.00116*

*This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology.*

*Copyright © 2014 Lacerda, Vasconcelos, Pelegrini and Grossi de Sa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Peptide array based discovery of synthetic antimicrobial peptides

# *Chris W. Diehnelt\**

*Center for Innovations in Medicine, The Biodesign Institute at Arizona State University, Tempe, AZ, USA \*Correspondence<EMAIL_ADDRESS>
*Edited by:*

*Octavio L. Franco, Universidade Catolica de Brasilia, Brazil*

#### *Reviewed by:*

*Márcia R. Mortari, University of Brasília, Brazil Marciane Magnani, Federal University of Paraíba, Brazil*

**Keywords: peptide arrays, antimicrobial peptides, targeted antibiotic, antimicrobial resistance, drug discovery**

# **ANTIMICROBIAL PEPTIDES AS A MEANS TO COMBAT ANTIBIOTIC RESISTANCE**

The rise of antibiotic resistance has emphasized the shortcomings in antibiotic drug development (Boucher et al., 2013). The move from biological based discovery methods to chemical approaches to identify candidates has left the antibiotic pipeline painfully dry (Lewis, 2013). The paucity of compounds that are effective against antibiotic resistant pathogens has led to great interest in antimicrobial peptides (AMPs) as potential solutions to the rise of resistant organisms (Hancock and Sahl, 2006; Fox, 2013). AMPs are short (5–50 amino acid) peptides that are produced by virtually all organisms as part of an innate immune system. There are 2,398 AMPs that have been reported (Antimicrobial Peptide Database—September 2013) and over 80% are cationic AMPs (CAMPs). Most positively charged AMPs interact with anionic bacterial membranes (Schmidtchen and Malmsten, 2013) which leads to a rapid breakdown in membrane function and subsequent cell death (Wimley, 2010). It is this mechanism of action that is of interest as it should be difficult for bacteria to develop resistance against lethal concentrations of CAMPs.

However, many AMPs have poor druglike properties and questions remain about that their ultimate utility as antibiotics (Brogden and Brogden, 2011). Great strides have been made in improving the protease stability; pharmacokinetics and therapeutic profile of peptide drugs and these methods have been used to improve the drug-like properties of AMPs. Despite the significant developments that have been made to advance AMPs through the clinical pipeline there has yet to be an approved AMP therapeutic (Vila-Farres et al., 2012). Clearly there is an ongoing need for additional AMP candidates as a tool in the fight against antibiotic resistant bacteria.

# **DISCOVERY OF SYNTHETIC AMPs**

Many groups are turning to non-natural sources to discover the next generation of AMPs. These efforts are focused on computational design of AMPs or by screening large libraries of peptides for new candidates. There have been significant advances in computational design of AMPs and progress continues in this field, illustrated by the recent work of Deslouches et al. (2013). However, these studies are guided by rules learned from natural AMPs and could be limiting in terms of designing peptides that function like natural AMPs with all of their inherent strengths and weaknesses. Other groups have used peptide discovery systems to screen large libraries of peptides with the aim of identifying synthetic AMPs and potentially novel classes of AMPs. Display technologies, such as phage display, are capable of producing large libraries of peptides (∼107 peptides) that can be used to discover AMPs (Huang et al., 2012). However, display techniques can be difficult to adapt to whole bacteria screening and require multiple rounds of selection to identify peptides with activities similar to natural AMPs.

A promising approach to discover antibacterial candidates is to screen a target bacterium against a peptide library arrayed on a solid surface. This approach uses two different types of peptide libraries: *in situ* synthesized peptide arrays and libraries of peptides prepared as spotted peptide microarrays. Seminal work in the use of *in situ* peptide microarrays for AMP development was demonstrated for small libraries of variants of natural AMPs (Hilpert et al., 2005, 2007, 2009; Hilpert and Hancock, 2007). In this method, hundreds to thousands of peptides are synthesized on a nitrocellulose membrane, then chemically cleaved into micro wellplates where there are then tested for activity. In contrast to this approach, spotted peptide microarrays are prepared by the synthesis of thousands to tens of thousands of peptides, which are printed on glass slides using standard microarray printing technology. Peptide microarrays have been used for ligand discovery by many groups and assays have been developed to screen whole cells against immobilized ligands (Papp et al., 2012).

# **DISCOVERY OF ANTIBACTERIAL PEPTIDES DIRECTLY ON PEPTIDE MICROARRAYS**

We have recently introduced an activity based assay that enables the selection of peptides with antibacterial activity directly on peptide microarrays (Domenyuk et al., 2013). In this assay, the target bacteria is labeled with an internal dye that fluoresces while the cell is metabolically active (e.g., Cell-Tracker Orange) and the exterior of the bacteria (outer membrane for Gram-negative or peptidoglycan layer for Gram-positive bacteria) is labeled with an amine-reactive dye, such as AlexaFluor-555 (**Figure 1A**). Fluorescently labeled

bacteria are then screened against a spotted peptide microarray of 10,000, 20 amino acid long peptides to identify peptides that bind the bacteria and those that kill the bacteria. Peptides that bind but do not kill the bacteria produce two fluorescent colors, while those that bind the bacteria and disrupt the membrane produce one signal. Using this system, we were able to identify peptides that inhibited growth of both Gram-positive and Gram-negative bacteria with minimum inhibitory concentrations (MIC's) in the 20μM range.

This approach has several important advantages as a source of antibacterial peptides. First, the system can be used to screen a wide variety of bacteria. Laboratory strains or clinical isolates can be easily labeled and do not require genetic modification to express a fluorescent or colorimetric indicator. The labeling procedures are robust and effective for Gram-positive and Gram-negative bacteria. Second, the method is rapid; bacteria can be labeled, screened, and analyzed in the same day. Additionally, the convenience of solid phase synthesis enables the incorporation of non-natural amino acids, such as D-amino acids or β-amino acids, into peptide libraries, enabling the direct screening of protease stabilized peptides. The *in vitro* assay format is very flexible in terms of the screening conditions, buffers, sera or media that can be used. Finally, peptide libraries can be designed without the inherent biases present in natural AMPs, potentially enabling the discovery of active peptides that function with novel mechanisms of action.

# **FUTURE DIRECTIONS FOR PEPTIDE ARRAY BASED DISCOVERY—SELECTION OF PEPTIDES THAT SPECIFICALLY TARGET AMP RESISTANT BACTERIA**

In our opinion, the future of array based AMP discovery lays in the selective targeting of antibiotic and AMP resistant bacteria over normal flora. This could be possible due to the convergence of several parallel avenues of technical and scientific development. Recently, robust methods for the *in situ* synthesis of peptide microarrays with medium densities, *>*103–104peptides (Loeffler et al., 2012; Price et al., 2012), and those with much higher densities, 105–106 peptides per array (Legutki, submitted) have been reported. This significantly expands the peptide sequence space that can be explored in a single experiment. This could be an especially important development in the search for peptides that are active against either intrinsically AMP resistant bacteria, such as *Burkholderia cepacia* complex (Loutet and Valvano, 2011), or those that acquire AMP resistance after treatment with CAMPs (Anaya-López et al., 2013; Fernández et al., 2013; Napier et al., 2013; Pelletier et al., 2013; Shireen et al., 2013). Acquired AMP resistance generally involves membrane modifications that increase the charge of the surface of the bacteria that prevents binding of CAMPs to the cell surface. This potentially is a troubling development as the vast majority of AMPs (*>*80%) discovered to date are cationic and there is some evidence that acquired resistance for one CAMP can extend to host AMPs (Napier et al., 2013). The latter report argues for the selection of AMPs that are not related to natural ones. For these pathogens, it is possible that neutrally charged or anionic AMPs will be effective against AMP resistant bacteria that have membranes modified with cationic groups.

It is here that the recent advances in *in situ* peptide array synthesis could become important. As peptide libraries can be designed with a more even distribution of charged peptides, large numbers of neutral and anionic peptides can be screened against a resistant pathogen. This is illustrated in **Figure 1B**, where the number of anionic, neutral, and cationic peptides is plotted for: the peptides in the AMP database, the 30,000 peptides we have used in our spotted peptide libraries, and the 330,000 peptides of our recent highdensity *in situ* peptide arrays. As can be seen, peptide arrays offer the opportunity to screen thousands to over 130,000 neutral and anionic peptides in a single experiment. It is possible that by screening both the AMP resistant and sensitive phenotype of a pathogen against this array, one could identify reduced charge peptides that inhibit the resistant phenotype with little effect on the sensitive phenotype. It is also possible that a peptide that selectively targets the AMP resistant bacteria would have a much narrower spectrum of activity toward normal flora. The selective targeting of the pathogen should help limit the spread of resistance to other species in the microbiome and maintain normal flora. It is likely that as the understanding of the host microbiome increases, the importance of targeted therapeutics will be even more evident. The use of molecular methods to quickly identify bacteria from clinical specimens is rapidly being adopted and should enable physicians to match a targeted antibiotic with the correct pathogen. Advances in peptide array discovery assays could provide a system to develop pathogen-specific antibiotics (Casadevall, 2009; Lemon et al., 2012) and lead to the discovery of the first generation of targeted antibiotics.

# **ACKNOWLEDGMENTS**

The author is grateful for the contributions of Stephen Albert Johnston, V. Domenyuk, A. Loskotov, J. Lainson, and P. Stafford to this work. This work was supported by a grant from DARPA to Stephen Albert Johnston under the 7-Day Biodefense Program and unrestricted funds to Chris W. Diehnelt and Stephen Albert Johnston.

#### **REFERENCES**

Characterization of the polymyxin B resistome of pseudomonas aeruginosa. *Antimicrob. Agents Chemother.* 57, 110–119. doi: 10.1128/AAC.015 83-12

antimicrobials in *acinetobacter baumannii*. *MBio* 4:e00021-13. doi: 10.1128/mBio.00021-13

*Received: 10 October 2013; accepted: 06 December 2013; published online: December 2013. 25*

*Citation: Diehnelt CW (2013) Peptide array based discovery of synthetic antimicrobial peptides. Front. Microbiol. 4:402. doi: 10.3389/fmicb.2013.00402*

*This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology.*

*Copyright © 2013 Diehnelt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Potential of known and short prokaryotic protein motifs as a basis for novel peptide-based antibacterial therapeutics: a computational survey

*Heini Ruhanen1,2,3, Daniel Hurley1,2,3, Ambarnil Ghosh4, Kevin T. O'Brien1,2,3, Catrióna R. Johnston5 and Denis C. Shields 1,2,3\**

*1 Complex and Adaptive Systems Laboratory, University College Dublin, Dublin, Ireland*

*2 Conway Institute of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland*

*3 School of Medicine and Medical Science, University College Dublin, Dublin, Ireland*

*4 Crystallography and Molecular Biology Department, Saha Institute of Nuclear Physics, Kolkata, India*

*5 Translational Research Institute, Woolloongabba, QLD, Australia*

#### *Edited by:*

*Nádia S. Parachin, Universidade de Brasília, Brazil*

#### *Reviewed by:*

*M. Pilar Francino, Center for Public Health Research, Spain Luis C. N. Da Silva, Universidade Federal de Pernambuco, Brazil Viji Sarojini, University of Auckland, New Zealand Mrinal Bhave, Swinburne University of Technology, Australia*

#### *\*Correspondence:*

*Denis C. Shields, Complex and Adaptive Systems Laboratory, University College Dublin, Belfield Office Park-8, Dublin 4, Ireland e-mail<EMAIL_ADDRESS>
Short linear motifs (SLiMs) are functional stretches of protein sequence that are of crucial importance for numerous biological processes by mediating protein–protein interactions. These motifs often comprise peptides of less than 10 amino acids that modulate protein–protein interactions. While well-characterized in eukaryotic intracellular signaling, their role in prokaryotic signaling is less well-understood. We surveyed the distribution of known motifs in prokaryotic extracellular and virulence proteins across a range of bacterial species and conducted searches for novel motifs in virulence proteins. Many known motifs in virulence effector proteins mimic eukaryotic motifs and enable the pathogen to control the intracellular processes of their hosts. Novel motifs were detected by finding those that had evolved independently in three or more unrelated virulence proteins. The search returned several significantly over-represented linear motifs of which some were known motifs and others are novel candidates with potential roles in bacterial pathogenesis. A putative C-terminal G[AG].\$ motif found in type IV secretion system proteins was among the most significant detected. A KK\$ motif that has been previously identified in a plasminogen-binding protein, was demonstrated to be enriched across a number of adhesion and lipoproteins. While there is some potential to develop peptide drugs against bacterial infection based on bacterial peptides that mimic host components, this could have unwanted effects on host signaling. Thus, novel SLiMs in virulence factors that do not mimic host components but are crucial for bacterial pathogenesis, such as the type IV secretion system, may be more useful to develop as leads for anti-microbial peptides or drugs.

**Keywords: short linear motifs (SLiMs), virulence factor, motif mimicry, antibacterial, bioinformatics, pathogen**

#### **INTRODUCTION**

Short linear motifs (SLiMs) are functional microdomains in proteins that play a critical role in many distinct biological processes such as cell signaling and regulation, post-translational modifications, proteolytic cleavage, and protein trafficking (Davey et al., 2011b; Mooney et al., 2012). These motifs are typically found in eukaryotic disordered protein regions and vary in size from 3 to 12 amino acids (Fuxreiter et al., 2007). In general, SLiMs have less than five defined amino acid positions and frequently these positions have some degree of flexibility in amino acid composition. Their shortness makes them evolutionarily plastic, allowing them to evolve convergently in unrelated proteins. This can allow proteins to rapidly acquire new protein interaction functions (Neduva and Russell, 2005; Diella et al., 2008; Davey et al., 2010, 2012b). Their short length also presents a challenge for SLiM discovery both experimentally and computationally, since there may be many false positive findings using both methods.

The presence of SLiMs in eukaryotes and viruses has been wellestablished. Several pioneering viral studies were crucial for the original characterization of SLiMs (Davey et al., 2011b). Viruses use SLiMs as a principal mechanism of hijacking cells by binding to host proteins and recruiting them to process viral proteins. A viral genome can contain various short motifs, many of which are necessary for the viral life cycle, providing a plethora of ways for the virus to take over the molecular machinery of the host cell (Kadaveru et al., 2008; Davey et al., 2011b). Like viruses, pathogenic bacteria are extremely proficient in intercepting host cell functions and in many cases it is still poorly understood how bacteria carry out the manipulation of the host cells. SLiMs have been documented in a number of cases to play a role in bacterial pathogenicity. However, bacterial linear motifs are not as well-characterized as in eukaryotes.

Most of the known instances of bacterial motifs are involved in pathogenicity including signals in effector proteins or host motif mimicry (Cornelis and Van Gijsegem, 2000; Alto et al., 2006). The tripeptide RGD motif is a known host extracellular matrix adhesion factor that is also used by bacteria to attach onto host cells (Tegtmeyer et al., 2010; Zimmermann et al., 2010; Zhang et al., 2012). RGD based anticancer and antithrombotic drugs are currently being developed but their direct impact on limiting bacterial adhesion and infectivity has not been investigated. A second example of a bacterial motif is the EPIYA motif found in several bacterial type III or IV secretion system effector proteins, which mimics SH2 binding peptides of the host (Hayashi et al., 2013). A third example of a bacterial motif has evolved to antagonize host proteins, but does this using a motif for which there is no eukaryotic equivalent. This W. . . E motif (where "." indicates any amino acid) in bacterial effector proteins has been proposed to mimic host G-proteins (Alto et al., 2006; Jackson et al., 2008; Ham et al., 2009). Other motifs found in prokaryotes which are not simply mimicking known eukaryotic motifs play roles in transport, modification and proteolysis of the bacterial proteins (**Table 1**).

Since SLiMs are used in a plethora of cellular processes in eukaryotes and are utilized by both pathogenic bacteria and viruses, discovering and characterizing new linear motifs is of great importance. As well as shedding light on the mechanisms of fundamental cellular processes they also hold promise as future therapeutic targets. There is an urgent need for new classes of antimicrobial therapeutics that are effective against multidrug resistant bacteria. Conventional antibiotics are becoming increasingly ineffective against pathogenic bacteria, such as methicillin resistant *Staphylococcus aureus* (MRSA) which presents a severe threat to public health.

We were interested in whether SLiMs may be valuable when developing new antimicrobial peptides or drugs. Compared with recombinant proteins, the smaller size of peptides makes them easier to manufacture and deliver. The use of chemically synthesized peptides in pharmacological and clinical applications is relatively limited by their low systemic stability and high clearance, poor membrane permeability, negligible activity when administered orally and their high cost of manufacture in comparison to small chemical compounds. However, to date more than 100 peptide-based drugs have already reached the market and of these, the majority are at the smaller end of the size spectrum at 8–10 amino acids (Craik et al., 2013).

Here, we conducted a study to discover SLiMs computationally in bacterial virulence factor datasets. We surveyed the distribution of these novel motifs, and compared their distribution with that of known motifs observed in prokaryotic proteins. The list of motifs given here represents a useful resource for experimental scientists interested in targeting SLiMs that may be important for the pathogenicity of bacteria.

#### **MATERIALS AND METHODS**

We utilized data from a virulence factor database MvirDB (Lawrence Livermore National Laboratory), which integrates DNA and protein sequence information from Tox-Prot, SCORPION, the PRINTS database of virulence factors, VFDB, TVFac, Islander, ARGO, CONUS, KNOTTIN, a subset of VIDA and sequences derived by means of literature searches (Zhou et al., 2007). MvirDB can be accessed at http://mvirdb.llnl.gov. The MvirDB browser tool was used to search the database to retrieve virulence factors by functional categories (**Table 2**) and to download sequences of interest. Protein sequence identifiers for the downloaded sequences for each functional category are available in **Table S1**.

The recovered protein sequences in each functional category thought to be associated with pathogenicity were searched for SLiMs using SLiMFinder (Davey et al., 2010) both locally, and on a webserver that is available at http://bioware*.*ucd*.*ie*.* The default settings provided in SLiMFinder without any extra masking were used in the analysis. This method finds sets of three or more unrelated proteins in a dataset of proteins that share a motif. Chemotaxis and enzyme protein sequence datasets were filtered to contain only sequences longer than 20 amino acids and lipoprotein and Exotoxin datasets sequences longer than 40 amino acids prior to the analysis.

The motifs identified by the SLiMFinder analysis were further examined for similarity to known SLiMs from literature motifs using CompariMotif, which takes two lists of protein motifs and compares them to each other, identifying and scoring similarities between short motifs in the sets (Edwards et al., 2008).

Motifs were visualized using the MEME Suite (Bailey et al., 2009), by taking a stretch of 10 amino acid residues containing the motif of interest from each protein sequence where the motif was found. MEME represents motifs as position dependent letter probability matrices which describe the probability of each possible letter at each position in the pattern. These are displayed as "sequence LOGOS," containing stacks of letters at each position in the motif. The total height of the stack is the "information content" of that position in the motif in bits. The height of the individual letters in a stack is the probability of the letter at that position multiplied by the total information content of the stack.