For proteomic studies sessile cells were cultured on polypropylene beads contained in column bioreactors as was previously described (Serra et al., 2008) with minor modifications. Briefly, *B. pertussis* Tohama I strain or the clinical isolate *B. pertussis* 2723 were grown in 200-mL SS broth for 24 h and then used to inoculate column reactors. After 4 h of static incubation to allow cell attachment, the suspension was drained to remove non-adhered cells and 200-mL of fresh SS broth were added to each column. Bioreactors were incubated with a constant air supply (0.1 vvm) at 37◦C for 72 h (mature biofilm stage). Every 24 h the broth was replaced by fresh medium. Afterward, polypropylene beads were washed three times with PBS prior to harvest the cells. The biofilm was detached from the surface by lightly agitation on PBS, subsequently, cells were harvested (15◦C, 8000 × *g*, 20 min) and immediately frozen in liquid Nitrogen and stored at –80◦C before being freeze-dried.

# Fourier Transform Infrared Spectroscopy (FT-IR)

For infrared analysis of biofilms each strain was grown in 6 well plates. After 72 h incubation, the biofilms were washed three times with distilled water and the biomass attached to the wells were suspended in bi-distilled water, adjusting the OD650 to 10. Samples were prepared from three independent assays by triplicate in each case. One hundred microliters of each bacterial suspension were transferred to an optical cell of zinc selenide (ZnSe) and vacuum dried (3.6 kPa) to obtain transparent films on the cell. FT-IR absorption spectra from 4,000 to 600 cm−1 were acquired with a FT-IR spectrometer (Spectrum One, Perkin Elmer, USA) as was reported (Serra et al., 2007). Infrared analysis was carried out as by means of OPUS 7.0 software (Bruker Optics, USA).

# Quantification of Biofilm Biomass

Biofilm biomass was quantified using the crystal violet assay described by O'Toole and Kolter (1998) adapted to the system. The absorbance of the solubilized dye was measured at 590 nm (OD590). Triplicates were made for each strain and *t*-Student's test was used to compare absorbance against *B. pertussis* Tohama I's biofilm. Samples were considered significantly different when *p* ≤ 0.05. CLSM was used to study the architecture and quantitative information of 72 h biofilms. An inverted confocal microscope Carl Zeiss LSM510-Axiovert 100M (Germany) was used as previously reported (Serra et al., 2007). Briefly, biofilms coming from semi-continuous culture, adhered to glass cover slips were first washed very carefully in PBS, and fixed with 4% paraformaldheyde. Then, adhered cells were rinsed in PBS, stained for 20 min with Acridine Orange and washed three times. In order to obtain quantitative information of mature biofilm structure, images were analyzed by COMSTAT software (Heydorn et al., 2000).

# Preparation of Soluble Protein Fraction

Cytosolic proteins were obtained following the protocol described by Ehling-Schulz et al. (2002) with minor modifications. Planktonic and sessile freeze-dried bacteria were suspended in detergent buffer containing 7 M urea, 2 M thiourea, 4% CHAPS and 30 mM TRIS (Sigma, St. Louis, MO, USA), cooled and then passed through a precooled French pressure cell (SLM AMINCO) working at 140 mPa two times. Cellular debris was harvested by centrifugation (4◦C, 10000 × *g*, 15 min). The supernatant was transferred to ultracentrifuge tubes (Beckman, USA) and centrifuged at 30000 × *g* for 40 min at 15◦C. The supernatant containing cytosolic proteins was stored in aliquots at –80◦C. Protein concentration was estimated using the 2-D Quant kit following the manufacture's protocol (GE Healthcare, Amersham Biosciences).

# 2D-DIGE

For two-dimensional difference gel electrophoresis (2D-DIGE) protein samples were minimally labeled as previously described (Radwan et al., 2008) with minor modifications. CyDye DIGETM fluorescent dyes (GE Healthcare Life Science, Munich, Germany) were used to label 33 μg of proteins per sample using 8 nmol dye/mg proteins. For each mode of growth three biological replicates were used. Biofilm and planktonic samples from each strain were labeled with Cye3 and Cye5. The internal standard comprising a pool of equal amounts from all samples was labeled with Cye2. Isoelectric focusing was carried out on an IPGphor III (GE Healthcare, Amersham Biosciences) system using 18 cm IPG Dry strips with linear pH gradients of 4–7 and 6–9 (all GE Healthcare, Amersham Biosciences). The IPG strips were rehydrated over night with rehydration buffer [7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.4% (w/v) DTT, 0.5% carrier ampholytes] at room temperature. DTT 0.4% (w/v) and carrier ampholytes 0.5% (v/v) were added to the mixed proteins samples in detergent buffer and the final volume was adjusted to 50 μL with rehydration buffer. Protein samples were then loaded onto the strips via loading cups. pH 4–7 strips were focused for a total of 36 KVh and pH 6–9 strips were focused for a total of 27 KVh. The IPG strips were reduced with 1% w/v DTT for 15 min and alkylated using 4% (w/v) iodoacetamide for 15 min in equilibration buffer (6 M urea, 30% (v/v) glycerin, 2% (w/v) SDS) and SDS-PAGE (12.5% T) was subsequently performed overnight (13 mA per gel) using an Ettan Dalt Six Electrophoresis Chamber (GE Healthcare, Amersham, Biosciences).

# Imaging and Data Processing

Fluorescence images of the gels were acquired using a Typhoon 9400 scanner (GE Healthcare). Data analysis was performed with the DeCyder software version 7.0 (GE Healthcare). Spot detection, matching, and normalization were performed using a standard algorithm of the software. Spot matching as well as spot quality of proteins of interest were manually checked eliminating false positives. To assess the reproducibility of gel replicates, principal component analysis (PCA) was carried out employing the DeCyder analysis module. For PCA all spots within the ANOVA 95th confidence interval were included. Spots showing more than threefold changes (*p* ≤ 0.05) in abundance between the strains growing as biofilm or in planktonic mode were considered as significant differences, manually excised from silvers stained gels (Blum et al., 1989; Miller and Gemeiner, 1992) and subjected to mass-spectrometry for protein identification.

# In-gel Trypsin Digestion and MS-Based Protein Identification

Protein identification was carried out using a matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometer (Bruker Daltonics, Ultraflex I) in MS and MS/MS modes. Spot distaining, in gel digestion and sample purification using Zip-Tipμ-C18 (Millipore) pipette tips were performed as reported previously (Blum et al., 1989). Samples were applied on a disposable target plate (Bruker Daltonics, PAC target) prespotted with α-cyano-4-hydroxycinnamic acid as matrix. Spectral pre-processing and peak annotation were carried out using FlexAnalysis 3.0 and Biotools 3.2 (Bruker Daltonics). Processed MS and MS/MS spectra were submitted to MASCOT server (Matrix Science) searching the database NCBInr restricting to *B. pertussis* Tohama I strain. Peptide mass fingerprint (PMF) search parameters were set for mass accuracy: <150 ppm, fixed modification: carbamidomethylation, variable modifications: methionine oxidation and acetylation at the protein N-terminal end, and missed cleavages: one. Based on the measured PMF at least one peptide was selected for MS/MS experiments. Search parameters were identical to PMF experiments, except for product ion tolerance (±1.0 Da). A protein was considered as identified, if the scores of database searches clearly exceeded the algorithm's significance threshold (*p* < 0.05) for PMF data and for sequence tag ion analyses of at least one peptide.

# RNA Isolation, cDNA Synthesis, and Quantitative Real-time PCR (qRT-PCR)

Total RNA was isolated from planktonic and biofilm bacteria using Trizol reagent (Life Technologies, Invitrogen) following manufacturer's instructions. The RNA was treated with DNase I (Promega, Madison, WI, USA) to remove contaminating DNA and cDNA synthesis was performed using hexamers primers (Promega, Madison, WI, USA) and M-MLV retrotranscriptase enzyme (Invitrogen, Carlsbad, CA, USA) following supplier's protocol. Specific primers were used to determine transcript levels of the selected genes (**Table 2**). SYBR premix (Thermo Scientific) was used for qPCR assays following manufacturer's instructions. Reactions were carried out on triplicate samples, including technical duplicates. Relative mRNA expression ratios of selected genes were normalized to the expression of 16S rRNA. Calculations for comparison between samples were performed using the --*CT* (where *CT* is threshold cycle) method as described by Conover et al. (2012). In the case of differences in primers efficiency a modification was done following the method described by Pfaffl (2001).

# DNA Sequencing and Data Analysis

The *bvgS* gene was sequenced for all clinical isolates used in this study. In addition, the following genes and their promoter regions were sequenced for clinical isolate Bp2723: *fhaB*, *ptxS1*, *fim3*, *prn*, *bsp22*, *bcrH2*, *vag8*, *brkA*, and *bvgA* using the primers listed in **Table 2**. For chromosomal PCR amplification the procedure described by Van Loo and Mooi (2002) was employed. Briefly, 1 μL of DNA was added to 19 μL of buffer comprising 50% Hotstar Taq Master mix kit (Qiagen), 1 μM concentration of each primer, and 5–10% dimethyl sulfoxide. Amplification of genes was performed in a Hybaid Omnigene incubator. The PCR fragments were purified with QIAquick PCR purification kit (Qiagen) and sequenced with the primers used for amplification and internal primers (not shown). Sequence reactions were performed with an ABI Prism Big Dye terminator reaction Kit, and the reactions were analyzed using a 377 or 3700 ABI DNA Sequencer (Perkin–Elmer, Applied Biosystems). The resulting sequences were searched against NCBI nucleotide or non-redundant protein database by using BLAST tool.

# RESULTS

# Planktonic Growth and Biofilm Formation Capacity on Abiotic Surfaces by *B. pertussis* Tohama I Strain and Clinical Isolates

Using a collection of eight clinical isolates recovered in Argentina from whooping cough patients and the reference strain *B. pertussis* Tohama I, a comparative growth analysis under planktonic conditions in SS broth was performed. **Figure 1** shows the growth kinetics for Tohama I strain and the clinical isolates *B. pertussi*s 2723 (Bp 2723), *B. pertussis* 892, *B. pertussis* 1918, and *B. pertussis* 492. The four isolates are depicted as representative for the clinical isolates, which showed similar growth behavior (data not shown). At stationary phase the planktonic biomass of the isolates -measured by optical density- was approximately 70% higher than the biomass reached by *B. pertussis* Tohama I strain. From batch cultures, specific growth rate (μ) for each strain was calculated. *B. pertussis* 2723, as well as the other clinical isolates, exhibited similar specific growth rates of 0.091 ± 0.003 h−1 while the μ of Tohama I strain under the same experimental conditions was significantly lower (0.052 ± 0.002 h−1). Next, we compared the adhesion and the mature biofilm biomass of the clinical isolates on abiotic surface. All clinical isolates showed higher

adhesion to polypropylene beads after 4 h of static incubation (data not shown) and higher biofilm biomass production after 72 h of culture (mature biofilm) compared to the reference strain (**Figure 2**). However, the final sessile biomass was different for each isolate. The *B. pertussis* 2723 strain was selected for further analysis since it exhibited fivefold more biofilm biomass as well as 70% more biomass under planktonic conditions compared to the reference strain Tohama I. The growth kinetics of this clinical isolate and *B. pertussis* Tohama I strain growing as biofilm were also studied. The clinical isolate Bp2723 showed a final biomass of 2.6 × 1010 CFU/cm2 while the biomass for reference strain was 2.5 × 109 CFU/cm2. In addition, the specific growth rate for Bp2723 was 0.033 ± 0.002 h−1 and for Tohama I strain 0.028 ± 0.001 h−1. Next, and to differentiate the structure of mature biofilms produced by both Bp2723 clinical isolate and Tohama I strain, micrograph images of biofilms were obtained from CLSM stacks (Supplementary Figure S1). The images were then analyzed by using COMSTAT software. The images obtained showed characteristic biofilm architecture with channels for both, Bp Tohama I and Bp2723. A more profound analysis revealed that the clinical strain produced a bigger biofilm, characterized by a maximum thickness of around the double of that achieved by the reference strain. Five architectural parameters calculated for the two biofilms are provided in **Table 3**. These parameters show not only significant differences in the thickness of the biofilms but also in the covered surface and the roughness coefficient that were higher for the clinical strain biofilm. This analysis revealed an apparent higher complexity of the biofilm produced by Bp2723 compared to the biofilm produced by the reference strain and confirm its enhanced biomass production under this culture condition. To gain insight into the nature of the biofilm developed by the clinical isolate we employed FT-IR spectroscopy for the comparison of the chemical composition of the biofilms developed by the reference strain and the clinical isolate growing in similar environmental

conditions. FT-IR spectroscopy, one of the most frequently used spectroscopic techniques to compare biochemical composition among biological samples, in association with PCA showed clear chemical differences between the FT-IR spectra obtained from both biofilms communities (**Figure 3**). Differences in biomass composition were detected between spectral areas assigned to protein and carbohydrates. The protein:carbohydrate ratio was 1.684 for Bp2723 and 1.142 for *B. pertussis* Tohama I strain, respectively. Based on this information we decided to explore the molecular basis for these significant phenotypic differences between the reference strain and the clinical isolate selected.

# 2D-DIGE Analysis and Protein Identification

*Bordetella pertussis* Tohama I strain and the clinical isolate *B. pertussis* 2723 were grown in parallel as planktonic cells for 24 h in SS medium (exponential phase) and as biofilm on polypropylene beads for 72 h (mature stage). To investigate the differentially expressed proteins in the reference strain and the clinical isolate under both culture conditions, a comparative

TABLE 3 | Biometric parameters obtained from 72 h biofilms formed by *Bordetella pertussis* wild type (Bp Tohama I) and the clinical isolate *B. pertussis* 2723.

*The features of the biofilms were quantified using the program COMSTAT. Parameters correspond to mean values. Standard errors are indicated.*

proteomic analysis was performed. The soluble cellular protein fraction was isolated from three replicates per strain and growth condition, and subjected to differential 2D DIGE analysis in two pH ranges (4–7 and 6–9) and a PCA was carried out. As shown in Supplementary Figure S2, the statistical analysis of each biological replicate, clearly indicates a distinct clustering of the four groups demonstrating a high reproducibility between the replicate samples. In addition, the analysis demonstrates that the highest variation was found to be strain-dependent (PC1), whereas PC2 discriminates the different growth conditions. Representative 2D electrophoresis patterns of bacterial proteins based on the internal standard sample for both pH ranges are depicted in **Figure 4**. The global proteome analysis showed that out of a total of 1275 spots analyzed, 65 proteins (5.1%) were differentially expressed in *B. pertussis* 2723 compared to the reference strain growing attached to surface and under planktonic culture conditions. Forty eight differentially expressed protein spots were selected for protein identification based on a combination of selection criteria as published elsewhere (Radwan et al., 2008). MALDI–ToF–MS–MS analysis resulted in the identification of 35 different proteins and/or protein species (Supplementary Table S1). The clinical isolate showed, in comparison to Tohama I strain 10 up-regulated proteins (*p* < 0.05) and five down-regulated proteins (*p* < 0.05) growing under biofilm conditions, and 27 proteins up-regulated and eight proteins down-regulated growing under planktonic conditions. These differentially expressed proteins can be assigned to five functional categories, namely metabolismenergy production, amino acid and protein synthesis, transport, virulence, and cellular process (Supplementary Table S1). More specifically, within the metabolic group, four proteins (aconitate hydratase –*acnB*-, dihydrolipoamide succinyl transferase component of 2-oxoglutarate dehydrogenase complex –*odhB*-, citrate synthase –*gltA*- and enoyl-CoA

hydratase/isomerase –*acnA*-), related to the energy production were found in higher abundance in the clinical isolate under both culture conditions. Three out of four proteins belonging to "amino acids and protein biosynthesis" pathways were found down regulated in the clinical isolate 2723. These proteins correspond to enzymes involved in phenylalanine, tyrosine, tryptophan, and lysine biosynthesis. The fourth protein, cystathionine beta-lyase -*metC*-, which is related to methionine synthesis and sulfur metabolism, was up-regulated in *B. pertussis* 2723. In addition, three proteins involved in amino acids transport [leu/ile/val (branched chain amino acid-) –*Q7VYN1*- binding protein –*livJ*-, amino acid- binding periplasmic protein –*Q7VS83*-, ABC transporter ATP binding protein –*Q7VTG4-*] and four proteins related to stress response and adaptation (putative Zinc protease –*Q7VVY4*-, antioxidant protein –*Q7VZE7*-, chaperone protein DnaJ –*Q7VVY3*-, and protein tex –*Q45388*-) were found to be up-regulated in the clinical isolate under both culture conditions (Supplementary Table S1). Noteworthy, four BvgAS-activated virulence factors, BcrH2, OmpQ, Vag8, and BrkA, were found under both growth conditions in higher abundance in the clinical isolate 2723 than in the reference strain. One of these proteins, namely BcrH2, is a chaperone protein member of the Type III secretion system (T3SS). T3SS correspond to an injection system that delivers virulence factors into the host cells changing its physiological functions (Mattoo et al., 2004; Medhekar et al., 2009). It is known to protrude from bacterial outer membrane of many Gram-negative bacteria. Another BvgAS regulated protein found to be differentially expressed in the clinical isolate is OmpQ, an outer membrane porin protein that so far has no clear assigned function (Finn et al., 1995). In addition, Vag8 and BrkA were also found in higher abundance in the clinical isolate. Vag8 is an autotransporter recently described to participate in serum resistance and described to bind C1; and BrkA, another serum resistance protein of *B. pertussis*, was reported as necessary for efficient colonization of mice (Finn and Amsbaugh, 1998; Barnes and Weiss, 2001; Marr et al., 2011). These latter observations were particularly interesting since we detected four BvgAS-activated proteins highly expressed in the clinical isolate compared to the Tohama I strain in soluble fraction. As BvgAS-activated genes play an essential role in biofilm development, we next studied the expression of BvgAS regulated adhesins in both strains under biofilm and planktonic culture conditions.

# qRT-PCR for *bvgAS* and its Positively Regulated Genes

qRT-PCR was performed to measure mRNA expression levels of BvgAS-regulated genes: *fhaB*, *fim* (*fim2* for Tohama I and *fim3* for Bp2723), *prn*, and *bipA* in cells grown under biofilm and planktonic conditions. In addition *vag8*, *brkA*, *bcrH2,* and *ompQ* genes, encoding the four proteins which showed increased levels of expression by *B. pertussis* 2723 compared to Tohama I strain in the cytosolic proteome (Supplementary Table S1), as well as *bvgS, bvgA,* and *ptx* genes were included in the analysis. This approach revealed a higher mRNA expression levels of *vag8*, *bcrH2, prn,* and *brkA* in planktonic cells of the clinical isolate compared to Tohama I strain (**Figure 5A**). Interestingly, significantly higher mRNA expression levels of *fhaB*, *fim3, ptxS1*, *bipA, vag8*, *prn, bvgA, bsp22,* and *brkA*, genes were found in sessile cells of the clinical isolate compared to the reference strain (*p* < 0.05; **Figure 5B**). The higher expression of *fhaB, prn*, *fim3*, and *bipA* genes, might probably be associated with increased biofilm formation capacity depicted by the clinical isolate, since these genes encode adhesins reported to participate in the development of *B. pertussis* mature biofilm (Serra et al., 2011; Sugisaki et al., 2013; de Gouw et al., 2014).

# Sequence Analysis of *bvgA*, *bvgS*, Virulence Genes, and Promoters

To determine whether specific nucleotide changes within virulence genes, or their promoter regions had occurred in Bp2723, the respective DNA sequences and their flanking regions were analyzed. No changes were detected when compared with

the published sequences of Tohama I strain (GenBank accession number BX470248.1). However, one nucleotide polymorphism, which corresponds to a single exchange of an A for a G at position 2113 of the *bvgS* gene (**Figure 6**), resulting in the replacement of K by E in the amino acid sequence of BvgS protein was observed for Bp 2723. Based on this result, we sequenced the *bvgS* gene of all clinical isolates used in this study and surprisingly found the same mutation in each clinical isolate (**Figure 6**). Interestingly, the same nucleotide exchange has been previously described by Herrou et al. (2009) for strains circulating in the Netherlands.

# Effect of E at 705 Position of BvgS Sensor on the Biofilm Formation Ability of *B. pertussis*

To evaluate whether the mutation detected in the *bvgS* gene could affect biofilm formation ability, *B. pertussis* 2723 and *B. pertussis* Tohama I strain were tested for their capacity for biofilm development. Growth was evaluated in tube-bioreactors containing polypropylene beads. The mutant strain BpK705E with an E at position 705 of BvgS and the wild type strain (BPSM) with a K at position 705 were also included in this analysis. Our results did not reveal any significant differences in the mature biofilm biomass of BpK705E strain, the wild type

isolates analyzed are shown. The single nucleotide mutation at position 2113 is highlighted in yellow.

BPSM and Tohama I strain after 72 h of culture (data not shown), suggesting that this point mutation in the *bvgS* gene is not associated to the increased biofilm biomass shown by the clinical isolate. Since this mutation has been previously described to confer a sensitive response to modulatory agents, such as MgSO4 and nicotinic acid, we investigated whether this mutation could trigger a faster attachment of bacteria when they are transferred from a modulatory environment to a non-modulatory one. The four strains described above were therefore modulated using SS culture medium with the addition of 40 mM MgSO4 and then incubated in a non-modulating SS medium under static conditions with polypropylene beads for 4 h. Under this condition, the surface adhesion of each strain was monitored every 30 min. Noteworthy, the clinical isolate Bp2723 and the mutant BpK705E strain adhered to polypropylene beads significantly faster than Tohama I and BPSM wild type strains (**Figure 7**). The kinetic adhesion of the Tohama I and BPSM wild type strains showed a lag period of 120 min while an increase of biomass attached was already observed for the clinical isolate and BpK705E strain after 30 min. These findings suggest that the *bvgS* allele coding for the 705 E protein variant might be associated with an accelerated expression of adhesins involved in the initial adhesion steps.

# DISCUSSION

In the current work, we investigated the biofilm formation capacity of eight argentinean *B. pertussis* clinical isolates recovered over a seven years time period in a local children hospital compared to a laboratory adapted strain grown under biofilm conditions. Clinical strains showed an increased ability to grow attached to polypropylene surfaces compared to the laboratory strain. The architecture of biofilm developed by the reference strain was compared with the one developed by the isolate Bp2723 growing in similar culture conditions by using CLSM. We found a marked variability in three dimensional biofilm structures between the two strains studied. The measurement of parameters extracted from confocal stack images analyzed by COMSTAT software such as thickness, roughness coefficient, surface to biovolume ratio, revealed that the biofilm developed by clinical isolate is significantly thicker than the formed by the reference strain. In addition to the differences between the architectures of both biofilms a FT-IR spectroscopic analysis also showed clearly phenotypic variations between these two biofilms. Thus, to better understand the different growth performances between clinical isolates and Tohama I strain we carried out a proteome investigation. The major functional groups of differentially expressed proteins in the clinical isolate included energy metabolism, transport, stress and regulation, and virulence factors. Seven proteins involved in metabolism and energy production were found upregulated in sessile cells of the clinical isolate *B. pertussis* 2723. Among them the citrate synthase that catalyzes the first reaction in the TCA cycle represents an important control point for determining the metabolic rate of the cell (Park et al., 1994). The higher expression of proteins associated to energy production under respiratory conditions was attributed primarily to the cell biosynthetic needs to produce higher biomass quantities. Our results indicate that the clinical isolate 2723 could have different metabolic and energetic requirements than the reference strain, which is supported by the higher final biomass reached for this isolate both in liquid medium and biofilm growth conditions. However, this higher capacity of biofilm formation is not associated with a higher growth rate. The TCA cycle is not only a central point in the metabolism of living organisms but also important for the survival of infectious biofilms. Therefore, its inhibition could be a promising strategy for the control of biofilms (Yahya et al., 2014). Similar results were previously reported in comparative proteomic studies of two *Burkholderia cenocepacia* isolates retrieved from a chronically infected cystic fibrosis patient. *B. cenocepacia* isolate obtained after 3 years of persistent infection and antibiotic therapy, showed an upregulation of citrate synthase (Madeira et al., 2011), which was reported to be important for biofilm formation and virulence (Subramoni et al., 2011). The results from the *B. cenocepacia* study as well as the results from our current study on *B. pertussis*, point toward a tight link between primary metabolism and biofilm formation capability.

Our proteome analysis revealed an increased expression of the Bvg-activated factors BcrH2, OmpQ, BrkA, and Vag8 in the clinical isolate *B. pertussis* 2723. These proteins are positively regulated by the *B. pertussis* BvgAS two-component signal transduction system. This system is known for its key role in the regulation of *Bordetella* virulence gene expression including adhesins and toxins, and it has also been shown to be determinant for the ability of *Bordetella* species to produce biofilms (Irie et al., 2004; Mishra et al., 2005; Serra et al., 2007). If BvgAS is not active *B. pertussis* is unable to adhere to respiratory tract and colonize the host (Bassinet et al., 2000; Scheller et al., 2015). Using quantitative real-time PCR assays we analyzed the relative expression level of adhesin genes, known to be positively regulated by the BvgAS system. After 72 h of biofilm growth these genes were transcribed at higher levels in the clinical isolated compared to Tohama I strain. In addition the *bvgA* regulatory gene showed three times higher transcription in *B. pertussis* 2723 sessile cells compared to reference strain, although no significant increase was found on the protein level. The latter results underpin the importance and value of combinatory analysis on transcriptional and translation levels. Although beyond the scope of our current work, these studies should be expanded in the future to post-translation and metabolic level to gain a holistic picture of pathogen host adaptation mechanisms.

The adhesins tested, namely FHA, Fim3, Prn, and BipA, showed higher transcriptional levels by qRT-PCR in the clinical isolate grown as biofilm compared to the reference strain Tohama I. FHA, one of the main adhesins described for *B. pertussis,* is involved in different steps of biofilm formation *in vitro* and *in vivo* in mouse nasopharynx, contributing not only to the first adhesion to the surface but also enhancing cell–cell interactions (Serra et al., 2011). Recently, we also reported that BipA is a common signature of *B.* pertussis biofilms (de Gouw et al., 2014). Interestingly, although Prn negative strains are now increasingly being isolated from patients with whooping cough (Barkoff et al., 2012; Pawloski et al., 2014), our results showed that this protein is up-regulated in biofilm culture for *B. pertussis* 2723. Proteomics and targeted transcriptomics approaches provide a picture of the changes between reference strain and a clinical *B. pertussis* isolate growing in similar culture conditions. These results are in agreement with the ones from FT-IR analysis, which show a chemical abundance of proteins in the mature structure of the biofilm produced by the clinical isolate. Therefore, it is tempting to speculate that: (i) the high expression of adhesins, mediating a faster and enhanced attachment, as well as (ii) the higher expression of enzymes involved in energy metabolism, leading to the augmented biomass, are responsible for the robust biofilm structure of the clinical isolate. When bacteria are under stress conditions, they often get together to form biofilms, which suggests that this bacterial lifestyle increases the fitness of the cells in harsh environments. Differential gene expression patterns between Tohama I strain and clinical isolates planktonic cells were previously attributed to either sequence divergence in *cis*regulatory regions or variation in the levels, activity, or encoding of transcriptional regulatory proteins (Cummings et al., 2006). However, in our current study, the higher transcription of adhesin genes could not be assigned to specific polymorphisms in the sequences of structural genes or promoters, suggesting that trans-acting factors could be involved. A single nucleotide mutation was found in *bvgS* gene of all clinical isolates tested, resulting in an exchange of lysine by glutamic acid at position 705 in the linker domain of the sensor protein. Interestingly, Herrou et al. (2009) reported the same mutation previously. Using an experimental infection model, they demonstrated that this mutation in the BvgS sensor of *B. pertussis* BPSM strain does not lead to a better pulmonary survival of the pathogen, though a faster response to modulatory agents like MgSO4 and nicotinic acid was observed. The latter results are in agreement with our current findings. After being modulated by MgSO4, the clinical isolate Bp2723 were transferred to SS medium and incubated under non-modulating conditions with polypropylene beads. Under the latter culture condition cells showed faster adherence to abiotic surfaces than the reference strain. In our experimental conditions, both, the clinical isolate Bp2723 and the Bp*K*075*E* strain showed an accelerated adhesion kinetic to polypropylene beads compared to Tohama I and the wild type BPSM strain. This high phase variation capability might represent an important adaptive advantage during pathogen colonization of its host. Fast adhesion suggests that the phases are tuned to different environmental niches favoring spatially defined regulation. This timing for attachment could promote persistence by protecting bacteria from the clearance occasioned by hydrodynamic forces in upper respiratory tract and the killing activity of host defense mechanisms. It is important to note that the increase of adhered cells (Bp2723 and Bp*K*075*E* strain) detected after 30 min of incubation under our experimental conditions is not due to growth on the surface since these sessile bacteria have a reduced specific growth rate (μ = 0.033 h−1, and duplication time of 21.0 h). *B. pertussis* transmission from human to human can probably occur by aerosolized respiratory droplets containing modulated bacteria. Thus, the results shown in our work could be important for the understanding of the rapid adaptation of clinical isolates to new environment. Our combinatory approach of proteomics, targeted transcriptional and genetic analysis revealed that multiple realms of regulation are governing the adaptation of *B. pertussis* to biofilm lifestyle.

# CONCLUSION

The divergent biofilm responses between *B. pertussis* clinical isolates and the laboratory adapted reference strain suggest that clinical isolates probably evolved in order to increase their potential and capacity to form biofilm and eventually to adapt rapidly to the fluctuations that they encounter at the site of infection. To date the emergence of pertussis remains a critical issue that should gear researchers to develop novel control measures by considering particularly the biofilm as a *B. pertussis* way of life.

# AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: OY and ME-S. The experiments were performed by LA, TG, NC, DdG, MV, DS. Analysis of data: all the authors. Contributed reagents/materials/analysis tools: OY, ME-S, and FM. Contributed to the writing of manuscript: all the authors.

# FUNDING

OY is supported by grants from the Ministry of Science and Technology of Argentina (ANPCyT-PICT 2012-2514 and MINCYT-Dirección de Relaciones Internacionales). LA and NC are fellows of CONICET-Argentina. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

# ACKNOWLEDGMENTS

We would like to acknowledge the support of the Science and Technology Cooperation Program between Argentina and Germany as well as Argentina and Austria. We are grateful to Francoise Jacob-Dubuisson from Institut Pasteur, Lille, France, for providing the bacterial strain BpK705E. We thank Nicole Guiso from Institut Pasteur, Paris, France for the analysis of PtxS1, Prn and Fim of clinical isolates.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.01352

# REFERENCES

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factors P.*69*/pertactin and pertussis toxin in The Netherlands: temporal trends and evidence for vaccine-driven evolution. *Infect. Immun.* 66, 670–675.

**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.

*Copyright © 2015 Arnal, Grunert, Cattelan, de Gouw, Villalba, Serra, Mooi, Ehling-Schulz and Yantorno. 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.*

# *Candida albicans* Shaving to Profile Human Serum Proteins on Hyphal Surface

Elvira Marín1 , Claudia M. Parra-Giraldo1 † , Carolina Hernández-Haro1 , María L. Hernáez 2 , César Nombela1, 3 †, Lucía Monteoliva1, 3 \* and Concha Gil 1, 2, 3

#### *Edited by:*

Nelson Cruz Soares, University of Cape Town, South Africa

#### *Reviewed by:*

Gustavo Antonio De Souza, University of Oslo, Norway Maria Dolores Moragues, Universidad del País Vasco, Spain

> *\*Correspondence:* Lucía Monteoliva<EMAIL_ADDRESS>
#### *†Present Address:*

Claudia M. Parra-Giraldo, Grupo de Enfermedades Infecciosas, Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Colombia; César Nombela, Universidad Internacional Menéndez Pelayo, Madrid, Spain

#### *Specialty section:*

This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology

> *Received:* 04 September 2015 *Accepted:* 16 November 2015 *Published:* 08 December 2015

#### *Citation:*

Marín E, Parra-Giraldo CM, Hernández-Haro C, Hernáez ML, Nombela C, Monteoliva L and Gil C (2015) Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface. Front. Microbiol. 6:1343. doi: 10.3389/fmicb.2015.01343 1 Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain, 2 Unidad de Proteómica, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain, 3 Instituto Ramón y Cajal de Investigación Sanitaria, Madrid, Spain

Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream.  This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in NS than in HIS, as validated by immunofluorescence.

Keywords: *Candida albicans*, shaving, surface proteins, human serum, complement pathways, coagulation pathways, GPI-anchored proteins, host-pathogen interaction

# INTRODUCTION

The yeast Candida albicans is the most important opportunistic fungi, causing a wide variety of infections ranging from superficial to invasive candidiasis, and it is often found in the mucosal microbiota. C. albicans infections produce high morbidity and mortality in intensive care, post-surgery and cancer patients or other types of immunocompromised patients (Leleu et al., 2002; Almirante et al., 2005; Hube, 2006; Brown et al., 2012). The high mortality of these infections is determined by the diagnostic limitations, the scarcity of antifungal agents and the emergence of resistance to them (Viudes et al., 2002; Wilson et al., 2002). For these reasons, the incidence of invasive candidiasis is still high.

The cell wall of C. albicans is a dynamic and complex multilayered structure located external to the plasma membrane. It is responsible for maintenance of the shape that characterizes each growth form (mainly yeast and hyphae) of the fungus (Klis et al., 2009). The cell wall mediates the first contact with the environment and integrates multiple cues into complex signaling networks that are coordinated by various transcription factors. Consequently, C. albicans differentially expresses cell surface proteins and virulence factors. The dimorphic transition that is the ability of C. albicans to reversibly switch from yeast to hyphal growth is essential for virulence; strains that are locked in either form are avirulent (Yan et al., 2013; Lu et al., 2014). The hyphal form allows the pathogen to penetrate into tissues to acquire nutrients or escape from the host defense, and yeast cells disseminate across the human body. There are many signals capable of inducing the dimorphic phenotypic switch: N-acetyl-D-glucosamine, physiological temperature and pH (37◦C and neutral), high CO2 concentration, hypoxia and nutrient starvation; nonetheless, growing in human serum is the most physiological condition to study this process (Kumamoto and Vinces, 2005; Karkowska-Kuleta et al., 2009; Sudbery, 2011; Mayer et al., 2013; Ene et al., 2014).

C. albicans is able to adhere to host cells and tissues; for this reason, it exposes surface proteins such as adhesins and many other pathogenic factors (Chaffin, 2008). Adhesion to host tissue is a prerequisite for tissue invasion and infection. Among adhesins, there are the Als family (agglutinin-like sequence), the Hwp family and the Iff/Hyr family (de Groot et al., 2013). Pathogenic factors include tissue-digesting hydrolytic enzymes such as the Sap family (Secreted Aspartyl Proteinases) (Kretschmar et al., 2002; Buu and Chen, 2014), lipases and phosholipases. The Sap family is composed of 10 proteins with different expression patterns; they act optimally at acidic pH. Their main role is related to digesting molecules for nutrient acquisition and for digesting or distorting host cell membranes to avoid or resist antimicrobial attack by the host immune system (Naglik et al., 2003). Some adhesins are attached to the wall through a C-terminus glycosylphosphatidylinositol (GPI) anchorage sequence (De Groot et al., 2003; Pardo et al., 2004; Klis et al., 2009).

Human serum is a very complex body fluid; its qualitative and quantitative composition was a hot topic of study during years (Anderson and Anderson, 2002; Mitchell, 2010). In the Plasma Proteome Database, proteomic data of 10,546 proteins detected in human serum and plasma are collected (Nanjappa et al., 2014). The dynamic range of abundance of these proteins has more than 10 orders of magnitude of variation. The complement system is an efficient component of the innate immune response and establishes a link with the adaptive system. The three conventional pathways by which complement is activated (classical, alternative and lectin) are composed of around 30 proteins (Engleberg et al., 2012). It mediates several physiological processes such as tissue regeneration and clearance of immune complexes. C2 and factor B (FB) are the main heatsensitive complement components (Soltis et al., 1979). C2 is the central component in the activation of classical and lectin pathways and FB is for alternative pathway; both are necessary for the formation of C3- and C5-convertases in the respective complement cascades aforementioned.

C. albicans develops several strategies to evade the complement system, delay the detection by the host immune systems and even capable of manipulate the host defenses for its own purpose (Collette and Lorenz, 2011). In this way, C. albicans acquires host-derived inhibitory proteins such as factor H (FH), factor H-like protein 1 (FHL-1) and C4b-binding protein (C4BP), to prevent activation of complement pathways on its surface. These three protein regulators are closely related molecules composed of variable numbers of short consensus repeat (SCR) domains.  With the exception of Hgt1, the other three are able also to bind plasminogen. Additionally, Pra1 and Hgt1 bind C4BP (Lesiak-Markowicz et al., 2011; Luo et al., 2011). Factor H mediates binding to polyanions (sialic acids and human heparin) present on C. albicans cell wall (Soares et al., 2000; Rodríguez de Córdoba et al., 2004; Green et al., 2013). Pra1 also binds fibrinogen and C3 directly (Zipfel et al., 2011).

The aim of this study is to know the human serum protein coat on C. albicans surface as well as which C. albicans proteins could be candidates for diagnostic markers, potential vaccines or therapeutic targets, as they are detected when human serum is present. The interaction between human serum proteins and other microorganism surface was tested by different proteomic approaches in Staphylococcus aureus and Streptococcus pyogenes (Dreisbach et al., 2011; Sjöholm et al., 2014). In this work, we carried out a gel-free proteomic approach by shaving live C. albicans cells with trypsin after serum interaction to obtain exposed peptides of C. albicans and human proteins. With this shaving strategy that was applied in our laboratory to C. albicans and S. cerevisiae cultured in synthetic media (Hernáez et al., 2010; Insenser et al., 2010; Gil-Bona et al., 2015), it is not necessary to perform sub-cellular fractionation. In the present work, we have optimized this protocol for the detection of C. albicans and human proteins together in conditions that mimic in vivo interaction during systemic infections; overall, 372 C. albicans and 214 human proteins were identified. Among C. albicans proteins, 147 are described as located at the cell surface. Interestingly Pra1 and Gpd2 are able to attach inhibitors of complement cascades and plasminogen; while a further 7 proteins are also capable of attaching plasminogen. Regarding human proteins, all main proteins of complement pathways were identified, and many proteins involved in coagulation and metabolism, as well as immunoglobulins or cytoskeletal proteins. Furthermore, in this work proteins of complement and coagulation pathways were observed surrounded the surface of C. albicans hyphal cells.

# MATERIALS AND METHODS

# *Candida albicans* Strain and Growth Conditions

The C. albicans strain used in this work was SC5314, from a clinical isolate (Gillum et al., 1984). This strain was grown on YPD plates (2% D-glucose, 2% peptone, 1% yeast extract, and 2% agar) and incubated at 30◦C. A colony was picked and grown in YPD medium at 30◦C and 200 rpm during 6–8 h. The OD600 nm was measured and diluted on minimum medium (MM) at 0.00003 OD600 nm (2% D-glucose, 0.17% yeast nitrogen base, 0.5% (NH4)2SO4, 0.192% synthetic complete mixture (Kaiser) drop-out -ura and 0.01% uracil). Culture was grown at 30◦C and 200 rpm overnight to obtain and collected at a concentration ∼106 cells/ml. 1.5 × 107 cells were resuspended in 9 ml of Lee medium at pH 6.7 (Lee et al., 1975). One milliliter of human serum was added and the culture was grown during 5 h at 37◦C and 200 rpm. Human sera were from healthy donor volunteers who did not have any clinical or microbiological evidence of infection. They were used in the experiments as normal serum (NS) or heat inactivated serum (HIS). Serum inactivation was done by heating at 56◦C during 30 min in a water bath.

# Surface Shaving

Surface shaving procedure was adapted from previous experiment of our group (Hernáez et al., 2010; Gil-Bona et al., 2015). Briefly, after incubation of C. albicans with 10% human serum (normal serum-NS or heat inactivated serum-HIS), the cultures were centrifuged at 3500 rpm 5 min. Cell pellet was resuspended in 1 ml of phosphate buffer saline (PBS) with 0.1% Tween 20, centrifuged again and washed 6 times more with PBS. The last pellet was resuspended in 400µl of ammonium bicarbonate (AMBI buffer; NH4HCO3) 25 mM pH 8.0 and 7.5µl dithiothreitol (DTT) 1 M and 9µg of recombinant sequencing grade trypsin (Roche) were added. DTT was added during the digestion as reducing agent to consent deep digestion of non-covalently or disulphide bridges associated protein. The samples were incubated at 37◦C, 30 min and 600 rpm. After incubation, samples were centrifuged at 5000 rpm 5 min and the supernatant was filtered with a filter unit of 0.22µm. The cell pellet was resuspended in 400µl of fresh AMBI buffer and 100µl of trifluoroacetic acid (TFA) 0.1% (v/v) were added to stop the proteolytic reaction. It was centrifuged and the supernatant was filtered again. Both peptide-supernatants were put together and processed for further proteomic analysis. Subsequently, originated peptides were cleaned up with a Poros R2 resin (ABSciex, Framingham, MA). Peptides were eluted with 80% acetonitrile in 0.1% TFA, dried in a Speed-Vac and resuspended in 0.1% formic acid. The samples were stored at −20◦C before nano-LC-MS/MS analysis.

# LTQ-Orbitrap Velos Analysis, Protein Identification, and Bioinformatics Analysis

Peptides were analyzed by nano-LC-MS/MS analysis. Peptide samples were concentrated and desalted using C18-A1 ASYcolumn 2 cm pre-column (Thermo Scientific) and then eluted onto a Biosphere C18 column (Nano-Separations). Peptides were separated with a 140 min gradient (110 min from 0 to 40% Buffer B; Buffer A: 0.1% formic acid/2% acetonitrile; Buffer B: 0.1% formic acid in acetonitrile) at a flow-rate of 250 nl/min on a nano-Easy HPLC (Proxeon) coupled to a nano-electrospray ion source (Proxeon). Mass spectrometry experiments were performed using a LTQ-Orbitrap Velos (Thermo Scientific) in the positive ion mode. Full-scan MS spectra (m/z 400/1400) were acquired in the Orbitrap apparatus with a target value of 1,000,000 at a resolution of 60,000 at m/z 400 and the 15 most intense ions were selected for collision induced dissociation (CID) fragmentation in the LTQ with a target value of 10,000 and normalized collision energy of 38%. Precursor ion charge state screening and monoisotopic precursor selection were enabled. Singly charged ions and unassigned charge states were rejected. Dynamic exclusion was enabled with a repeat count of 1 and exclusion duration of 30 s.

Protein identification was carried out with mass spectra raw files using a licensed version of search engine MASCOT 2.3.0 with Proteome Discoverer software version <IP_ADDRESS> (Thermo Scientific). Searchers were made against C. albicans SC5314 (6221 sequences) and human database (20233 sequences) available on Candida Genome Database (Assembly 21 of CGD, http://www.candidagenome.org/) and Uniprot-SwissProt (http://www.uniprot.org) respectively, to identify peptides and proteins. Search parameters were oxidized methionine as variable modification, peptide mass tolerance 10 ppm, 2 missed trypsin cleavage sites and MS/MS fragment mass tolerance of 0.8 Da. In all protein identification, the FDR was <1%, using a Mascot Percolator, with a q-value of 0.01. Gene Ontology (GO) terms at CGD (http://www.candidagenome.org/cgi-bin/ GO/goTermFinder) were used to classify C. albicans proteins.

As an estimation of the relative protein abundances the normalized spectral abundance factor (NSAF) was used (Zybailov et al., 2007), and the average of the normalized values was calculated. Mass spectrometry proteomics data have been deposited in C. albicans PeptideAtlas (Vialas et al., 2014) with the data set identifier PASS00446.

# Cell Permeability

Before and after trypsin treatment, C. albicans cell wall permeability was evaluated by staining with propidium iodide (PI). 106 cells/ml were incubated with 10µl of PI 5 mM and the positive fluorescent cells (red staining) were checked, at least 200 cells of each sample were counted in a fluorescence microscope. Cells treated with 70% ethanol-PBS (v/v) were used as positive control.

# Epifluorescence and Confocal Fluorescence Microscopy

For detecting complement proteins on C. albicans surface after interaction with human serum, a solution of 106 cells/ml on Lee medium at pH 6.7 with 10% of human serum (NS or HIS) was prepared. 105 cells of this suspension were laid during 30 min, 1.5 or 5 h at 37◦C on glass coverslips pre-coated with poly-L-lysine (1 mg/ml). Cells were fixed with formaldehyde 4% (w/v) (in PBS) for 20 min at room temperature (RT). Glass slides were washed twice with PBS. To stain cell membrane, PKH26 dye was added following technical instructions (Sigma Aldrich). Then, slides were blocked during 45 min at RT with buffer B (PBS plus bovine serum albumin (BSA) at 1 mg/ml). The slides were washed twice again with PBS and then incubated for 1.5 h at RT in the same buffer with an anti-C3, anti-factor B polyclonal serum (dilution 1:75 and 1:50, respectively) or only buffer. The slides were washed three times with PBS and further incubation for 1 h with an antirabbit IgG conjugated with Alexa-488 diluted at 1:500 in buffer B was done. Nuclei were stained with DAPI dye (5µg/ml; 5 min at RT). Mounting medium Fluoromount-G (SouthernBiotech) was added to the preparations. Cells were then examined with epifluorescence and confocal microscopy images were collected using an Olympus FV1200 microscope.

# Flow Cytometry Analysis

C. albicans cells were grown as previously described on Immunofluorescence assay section. In this case the incubation with human serum (NS or HIS) was done during 30 min to avoid larger hyphae formation. After that, cells were washed with PBS and fixed with formaldehyde 4% in PBS during 20 min at RT. Then, three washes with PBS were performed and samples were blocked with 0.1% BSA in PBS at 4◦C overnight with gentle shaking. Samples were incubated with primary antibody or with buffer alone as negative control, for 2 h at RT. The primary antibodies were prepared in PBS with 0.1% BSA, anti-C3 and anti-FB at 1:75 and 1:50 dilutions, respectively. Four PBS washes were performed and incubated with secondary antibody below, an anti-rabbit conjugated with Alexa-488 diluted at 1:500, 2 h at RT with gently shaking. Then, samples were washed with PBS 4 times more. These cells were analyzed with a Guava easyCyte cytometer of Millipore.

# RESULTS

# Gel-free Proteomic Approach to Study Protein Interactions between *C. albicans* and Human Serum

In order to break through the broad spectrum of human serum proteins attached to C. albicans cells and the fungus surface proteins, we analyzed C. albicans cell surface after 5 h of incubation with 10% human serum by a gel-free proteomic strategy. Normal serum (NS) was used to mimic physiological conditions of C. albicans during invasive candidiasis and heat inactivated serum (HIS) to determine human proteins deposited on the C. albicans surface without conventional complement cascade activation. As human serum is the most important inductor of C. albicans dimorphic transition, after 5 h of incubation all C. albicans cells were in the hyphal form. To obtain the protein peptides, a shaving methodology was used, which consists of direct cell digestion with trypsin and MS/MS identification. Before digestion, cells were washed with PBS plus Tween-20 and several additional washes with PBS were done to remove human serum proteins weakly attached to C. albicans cells. We optimized parameters such as time and trypsin concentration during shaving of C. albicans cells, in order to control cell integrity and identify the highest number of proteins (from both, C. albicans and human). With short times of trypsin treatment, between 5 and 10 min, only human proteins were identified (data not shown). Otherwise, after 30 min, human and C. albicans proteins were detected; thus, this duration of trypsin incubation was selected. Before and after trypsin treatment, cells were treated with propidium iodide (PI) and the cell permeability state was analyzed under the microscope. Less than 1% positive staining was obtained in all conditions. Cell integrity was assessed with the purpose of avoiding cytoplasmic protein contamination after trypsin treatment.

Peptides obtained after C. albicans cells interaction with 10% human serum (NS or HIS) by shaving were recovered and identified by LC-MS/MS on a LTQ Orbitrap. Four biological replicates were made using NS and three using HIS. Mass spectra raw files were searched against C. albicans SC5314 and human databases (CGD and Uniprot-SwissProt, respectively) and proteins identified in at least two biological replicates with two or more peptides in one of them were selected. C. albicansidentified proteins are listed in **Table 1** and Table S1, and human proteins in **Table 2** and Table S2.

This analysis rendered the identification of 372 unique C. albicans proteins, 371 in NS and 134 in HIS (Table S1). There were 133 proteins identified in both conditions, accounting for 36% of the total C. albicans-identified proteins. The Orf19.8 (an ortholog of C. dubliniensis CD36) was the only protein identified with HIS and not with NS. Noteworthy, around ∼33% of the C. albicans proteins were identified in at least six of the seven analyzed samples. Furthermore, of the 372 C. albicans-identified proteins, 60 proteins presented a signal peptide (SP) at CGD. On the other hand, 214 human proteins were identified; of these, 128 were common to both conditions (60%), 43 were present only after incubation with NS and the other 43 only with HIS (**Table 2** and Table S2).

# Analysis of *C. albicans* Cell Surface Protein Pattern after Interaction with 10% human Serum

Only those C. albicans identified proteins with a Gene Ontology (GO) annotation at CGD pointing either to surface localization, either to surface exposure or with the ability to induce human serum antibodies, were included in **Table 1**. We focused our study on these 155 C. albicans proteins classified into 5 categories: glycosylphosphatidylinositol (GPI)-anchored proteins; proteins

TABLE 1 | Selected *C. albicans* proteins identified after incubation with 10% NS or HIS related with cell surface and immunogenicity.

The classification is hierarchical and exclusive. Proteins were included if they were detected in at least two replicates of one condition with at least 2 peptides in one replicate. There are four replicates of Normal Serum (NS) and three replicates of Heat Inactivated Serum (HIS). Protein names from Candida Genome Database (CGD) (Inglis et al., 2012).

aGPI-cell wall proteins reviewed in De Groot et al. (2003), Pardo et al. (2004), Chaffin (2008), Klis et al. (2009).

bGene Ontology (GO) terms (in parentheses the GO identifier) at CGD.

Proteins able to induce human serum antibodies in patients with invasive candidiasis are indicated in bold (Pitarch et al., 2004, 2006, 2008, 2011; Mochon et al., 2010).

involved in cell wall organization or biogenesis; cell surface proteins; plasma membrane proteins and other immunogenic proteins not present in the previous group. The groups are hierarchical and exclusive.

The first category includes 23 proteins attached to the plasma membrane or cell wall by a GPI-anchor; most are involved in biogenesis and maintenance of the C. albicans cell wall. All GPIanchored proteins identified have a detected SP for entry into the secretory pathway at CGD. In the second category, there are 26 other proteins involved in cell wall organization or biogenesis. Among the 62 proteins included in the cell surface category, there are proteins located at the cell surface, although most of them do not have SP and could not reach this cell localization via the secretory pathway. Several of them have been described as "moonlighting" or multifunctional proteins. It is interesting that antibodies against a total of 52 C. albicans-identified proteins have been detected in human blood or serum from patients with invasive candidiasis (these immunogenic proteins are highlighted in **Table 1**). Many of these are abundant at the cell surface (as they were detected with more than 10 unique peptides) and were not secreted by the classical secretory pathway (not have SP), such as Eno1, Fba1, Hsp70, and 17 additional proteins.

It is also remarkable that two out of the four proteins described in C. albicans with the ability to fix the complement inhibitor factor H (FH) were identified. Pra1 was identified in both samples (NS and HIS), but Gpd2 was only in NS samples. Furthermore, we detected many C. albicans proteins described with the ability to bind plasminogen: Adh1, Eno1, Fba1, Gpd2, Pgk1, Pra1, Tdh3, Tef1, and Tsa1.

# Analysis of Human Serum Proteins that Interact with *C. albicans* Cell Surface Relevant to *C. albicans*-host Interaction

The 214 identified human proteins were classified into the following classes according to their function: complement pathway, coagulation pathway, metabolism, immunoglobulins, cytoskeleton, and others. Proteins belonging to complement and coagulation cascades are listed in **Table 2** and the rest of the proteins are in Table S2. The percentages of proteins identified in each category in the two conditions tested are shown in **Figure 1**. The complement and coagulation pathways represent higher percentages of the total identified proteins when HIS was used and, on the other hand, the immunoglobulin

#### TABLE 2 | Human proteins of complement and coagulation pathways identified on the surface of *C. albicans* after incubation with human serum.

(Continued)

#### TABLE 2 | Continued

aAccession number, gene symbol and function from UniProtKB/Swiss-Prot database (Uniprot, 2014).

bProteins were included if they were identified in at least two replicates of one condition with at least 2 peptides in one replicate.

group represents a higher percentage when NS was used.

Close to all components of the three complement pathways (33 proteins) were identified, most of them in the two conditions tested. However, there are some complement proteins that have been detected only in the NS sample (Collectin-11) or in the HIS sample [complement C1q subcomponent subunit A (C1qA), complement C1r-like protein, complement factor D (FD), ficolin-2, phosphatidylinositol-glycan-specific phospholipase D (GPLD1) and plasma protease C1 inhibitor (SERPING1 or C1INH)] (**Table 3**). The identification of many complement inhibitors such as alpha-2-macroglobulin (A2M), C1INH, FH, and factor I (FI) is also interesting. In relation to the coagulation pathway, 31 proteins were identified, with most of them also being detected in the two conditions. Although heparanase was only identified in NS sample and coagulation factor XIII B chain, hyaluronan-binding protein 2 and plasma serine protease inhibitor (SERPINA5) were only identified in the HIS one. Interestingly, antithrombin-III (SERPINC1) and plasminogen (PLG), two coagulation proteins that are relevant complement inhibitors, were identified. Focusing on human immunoglobulins IgG and IgM, they were identified in NS and HIS samples and with a similar number of peptides in both of them (Table S2). The category of metabolic proteins includes many apolipoproteins detected on the surface of C. albicans (Table S2).

Interestingly, 13 proteins belonging to the serpin family were identified. Serpins are a relevant group of proteins with similar structures able to inhibit proteases. Some of them had only been observed in the HIS sample, such as SERPINA5, C1INH and cortisol-binding globulin (SERPINA6) (**Table 4**).

To estimate the relative abundance of human proteins identified on C. albicans surface, the normalized spectral abundance factor (NSAF) (Zybailov et al., 2007) of each protein was calculated and the average NSAF values among samples were assessed. We compared these NSAF values with the ranking of the original protein abundance in human plasma using data of the dynamic range of most abundant proteins adapted from Mitchell (2010). As shown in Figure S1, the relative abundance TABLE 3 | Comparison of complement proteins identified on the cell surface of *C. albicans* by shaving after incubation with NS vs. HIS.

Proteins indicated in bold are only identified in one condition.

Proteins with \*are identified only in one replicate of the indicated condition.

#### TABLE 4 | Identified proteins belonging to the SERPIN family.

of proteins in the plasma is different to the relative abundance at the hypha surface after interaction with human serum. For example, C3 protein of complement pathways was identified as one of the most abundant proteins on the surface of C. albicans in both conditions and it was not among the most abundant proteins in plasma. Also, in the apolipoprotein family, the relative abundance of some proteins is different in the plasma vs. the fungal surface. ApoB100 and ApoE are less abundant in human plasma than ApoCIII and both proteins showed higher NSAF than ApoCIII.

The abundance ranking of the proteins involved in the complement and coagulation pathways identified on C. albicans surface is shown in **Figure 2**. C3, C4-B, and C4-A had the highest NSAF results among complement proteins in both serum conditions. Complement components of Membrane Attack Complex (MAC: C6, C7, C8-A, C8-B, C8-G, and C9) had higher NSAF in NS than in HIS samples and only C5 had higher NSAF in HIS than in NS samples. In the coagulation pathway, it is notable that SERPINA1 (alpha-1-antitrypsin) is much more abundant in the HIS sample than other proteins such as HRG (histidine-rich glycoprotein), HCF2 (SERPIND1) or F2 (prothrombin), which are more abundant in the NS sample than SERPINA1.

During complement activation, C3, C4, and C5 are proteolyzed by cleavage at a specific site and small fragments are produced; these fragments are called anaphylotoxins or complement peptides, and are indicated with the letter "a" (Walport, 2001a,b). The analysis of the identified peptides belonging to: (i) anaphylotoxin regions (C3a, C4a, and C5a), (ii) released domains during proteolysis activation of complement

with 10% human serum at 37◦C (normal serum-NS or heat inactivated serum-HIS) and NSAF was calculated for proteins belonging to complement and coagulation proteins. The proteins were ordered according to their NSAF obtained in NS samples, from more to less abundant. Gradient color ranges from red (more abundant) to green (less abundant).

pathways (C2b and FBa), and (iii) of the MAC-interaction regions (MACPF) is shown in **Table 5**. The tendency was the identification of more peptides belonging to these regions in HIS samples than in NS samples. It is remarkable that peptides from C2b and C5a were not identified in NS samples.

# Validation of Proteomic Results

To validate the proteomic results, firstly the deposition level of C3 and FB on the C. albicans surface was analyzed by immunofluorescence microscopy and flow cytometry assays. C3 is a common component of the three complement cascades and together with A2M, are the two highest abundant complement proteins in human serum. Factor B (FB) is an abundant complement protein specific of the alternative pathway. C. albicans cells were incubated with NS and HIS and treated with anti-C3 and anti-FB antibodies (**Figure 3**). We observed the deposition of C3 and FB along the surface of all C. albicans cells when incubated with human serum (NS or HIS) and some areas with higher intensity in the case of C3 after 5 h of incubation with NS (**Figure 3A**, left panels). These differences TABLE 5 | Analysis of peptides corresponding to cleavage fragments produced during complement activation and to contact regions in MAC formation.

Identified peptides belong to the indicated released region or remain part of the full-length protein.

Gray background highlights identification in HIS condition.

between the fluorescence intensity of FB and C3 can be correlated with the number of peptides of these proteins in the samples at 5 h. When cells were incubated with HIS, the intensity of FB was slightly higher than in the case of NS incubation (it is well observed in short incubation times) (**Figure 3A**, right panels). To achieve flow cytometry assays, the incubation time of C. albicans cells with human serum was decreased to avoid the formation of large filaments. Within 30 min of incubation, the deposition of C3 was higher among the population in NS samples than in HIS (Figure S3). The intensity of FB was lower than that of C3, as expected, and less homogenous in both samples (NS and HIS) (upper panels). Mean fluorescence intensity (MFI) of two independent experiments were collected and showed that, in all cases, the MFI for C3 and FB was higher when cells were incubated with NS than with HIS at short times, although the differences are not significant in the case of FB (bottom panel).

All of these data together showed that C3 and FB deposition was clearly observed at short incubation times, and C3 enhanced its accumulation on the surface with longer incubation time and more dramatically when the incubation was with NS. However, FB accumulation on C. albicanssurface did not appear to increase with time, maybe because it can be masked by the rest of the complement proteins (**Figure 3A**).

Furthermore, to address whether C3 and FB are close to the cytoplasmic membrane, fluorescence co-localization was evaluated with confocal microscopy images by the analysis of transversal sections across cells (**Figure 3B**). As observed, both C3 and FB proteins are localized around the plasmatic membrane, staining when C. albicans cells were incubated with NS and also with HIS.

Afterwards, in order to evaluate the deposition of human IgGs on C. albicans surface after 5 h of incubation with 10% human serum, IgGs were detected in NS and HIS samples, with higher intensity on the yeast surface than on the hyphal surface in both samples (Figure S2).

# DISCUSSION

Our work shows, for the first time to our knowledge, the identification by a shaving approach of 147 C. albicans surface proteins and 214 human serum proteins attached to the surface of the fungus in the same report. The more relevant C. albicans proteins identified are GPI-anchored proteins and proteins involved in cell wall organization or biogenesis. The most relevant human proteins identified belong to complement and coagulation pathways. Indeed, all main human complement proteins were identified on the surface of C. albicans. The identified proteins were reproducible among the experiments and would represent the physiological mode of action of the immune system against C. albicans or components of the immune system used for C. albicans to evade, disturb or use on its own purpose the host defenses.

# Analysis of *C. albicans* Surface Proteins

With the objective of unraveling the protein surface pattern of C. albicans hyphal cells, we analyzed our data in comparison to the data obtained by our group using the same methodology of cell shaving over yeast and hyphal cells (growth also in Lee's medium, but without human serum) (Gil-Bona et al., 2015). This analysis rendered 304 proteins identified in all C. albicans assays (yeasts and hyphae with and without human serum) that we consider the C. albicans common core of cell surface proteins and not morphotype specific (Table S3). Also, a hyphal protein pattern of 55 proteins identified on C. albicans hypha forms, but not identified in C. albicans yeasts, was obtained. Among them, Als3, Hyr1, and Sod5 were associated with hyphae in different works (Heilmann et al., 2011; Sosinska et al., 2011; Sudbery, 2011). They are proteins that are expected to be detected, indicating that the other 52 proteins are at least more abundant or accessible in hypha than in yeast cells. Another example is Ece1, which is a hyphal-specific protein with unknown function (Fan et al., 2013) (Table S3).

Other proteins were associated with hyphae in some works and described as morphotype-independent in others, for example, Phr1 and Rbt1 (Heilmann et al., 2011; Ragni et al., 2011; Monniot et al., 2013). We identified both proteins in all shaving C. albicans experiments. Furthermore, Phr2 (a homolog of Phr1) was also identified in all conditions. The differences observed at the level of detection of Phr1 and Phr2 among the samples were possibly related to the optimum pH for expression of each one (Sosinska et al., 2011; Dühring et al., 2015) (Table S1). On the other hand, Rhd3 and Ywp1 were reported as yeast-specific and were found to be part of the common core pattern in our study (Heilmann et al., 2011). A relevant protein found in C. albicans common core pattern is Msb2; this stabilizes the fungal cell wall and inactivates human antimicrobial peptides (Dühring et al., 2015).

It is interesting to know that GPI-anchored proteins identified in this work are part of the common core of C. albicans surface proteins or of the hyphal-form specific ones. There are 17 proteins belonging to the common core in our analysis, and also Ece1 and Sod5, which are part of the hyphal-specific group, whose genes were up-regulated under blood growth (Fradin et al., 2003).

Remarkably, 12 proteins were only identified on the C. albicans hyphal surface when it was grown with 10% human serum and we highlighted Pra1, Sap5, Tef1, and 5 more Orfs with unknown function (Table S3). In this work, two members of the Sap family, Sap9 and Sap5, were identified. Sap9 is part of C. albicans common core while Sap5 shows up in hyphae induced with the human serum group. Both genes were up-regulated in C. albicans biofilm associated with bloodstream infections (Joo et al., 2013). Ten out of these twelve proteins are identified for the first time on the surface of C. albicans; Pra1 and Tef1 have been previously identified in the cell wall of C. albicans.

Many proteins described as "moonlighting" or multifunctional proteins in C. albicans were identified in this work (Nombela et al., 2006; Chaffin, 2008) (indicated with ∗ in Table S1). Among them, Gpd2 has been described as a virulence factor on microorganism surface because it mediates complement evasion; meanwhile, there is a glycerol 3-phosphate-dehydrogenase in the cytoplasm that is involved in the intracellular accumulation of glycerol to control osmotic pressure (Luo et al., 2013). Tsa1 is involved in oxidative stress response in the cytoplasm and in hyphal cell wall biogenesis (Urban et al., 2005). Tdh3 is a glycolytic enzyme (glyceraldehyde-3-phosphate dehydrogenase) in the cytoplasm and able to attach fibronectin and laminin at the cell surface (Gozalbo et al., 1998).

Regarding C. albicans proteins that interact with complement inhibitor proteins (FH and PLG), the expression of Gpd2 and Pra1 is more prominent at the hyphal tip (Zipfel et al., 2011; Luo et al., 2013). Pra1 is also able to bind C4BP and it is also known that the hyphal tip is a prominent binding site for C4BP (Meri et al., 2004; Luo et al., 2011). For these reasons and because it facilitates host tissue penetration, the hyphal tip is considered an important factor of pathogenesis. We identified these two proteins on the C. albicans surface grown with 10% human serum that activate hyphal growth. Focusing on the mechanism to evade complement activation mediated by plasminogen coated surfaces, as plasminogen is an important complement cascade inhibitor in the fluid phase and on the human surface (Barthel et al., 2012), the identification of nine out of the eleven C. albicans proteins with the ability to bind PLG (Adh1, Eno1, Fba1, Gpd2, Pgk1, Pra1, Tdh3, Tef1, and Tsa1) is another highlight of this study (Crowe et al., 2003; Jong et al., 2003; Barthel et al., 2012).

# Human Serum Proteins with the Ability to Interact with *C. albicans* Surface

We detected human plasma proteins from different abundance strata on C. albicans surface; most of them are from the most abundant proteins or from intermediate stratum (Anderson and Anderson, 2002; Mitchell, 2010). Due to the heat treatment of HIS serum, FB and C2 become non-functional; in this situation, other human serum proteins could interact further with the surface of C. albicans. Remarkably, C. albicans surface could be less accessible to trypsin digestion when it was grown with 10% HIS as the number of coated of human proteins is greater. This could explain the observation of higher sum of peptide-spectrum match (PSMs) of human serum proteins in the HIS condition in comparison with the NS condition. In any case, it is not possible to discard that some proteins differentially observed in HIS are a result of changes in their binding properties due to protein denaturation.

Albumin is the most abundant protein in human plasma with close to two orders of magnitude more than the rest of proteins (Mitchell, 2010), but it was not detected as the highest abundant protein on C. albicans surface incubated with human serum (either NS or HIS) (Figure S1). Also, there are some proteins only detected on C. albicans surface in HIS samples that have intermediate abundance in human plasma such as C1INH, C1qA, and RBP4. Serotransferrin and fibrinogen (FGG, FGA and FGB) were more abundant in NS condition and in the other side apolipoprotein A-I and transthyretin in HIS condition. All of these data indicate that the interaction between human proteins and C. albicans surface did not respond solely to their protein abundance in human serum. The study of C3 and FB deposition on C. albicans surface after incubation with 10% human serum was carried out by immunofluorescence with specific antibodies. The clearly deposition of C3 and FB was observed at short times of interaction in **Figure 3A**.

A graphical summary of the identified proteins at C. albicans surface from the different complement pathways in NS and HIS conditions is presented in **Figure 4**. The enrichment on the surface of C. albicans in proteins belonging to the lectin, classical and alternative complement pathways is visibly observed in normal conditions (NS); almost all of the main proteins of complement pathways were identified (**Figures 4A,B**). Peptides belonging to anaphylotoxin region are less frequently identified in NS than in HIS condition; this could be related to that C2b, C3a, FBa, and C5a fragments were released to the medium under this condition (**Table 5**). Furthermore, two peptides (CCEDGMR and CCEDGMRENPMR), which are part of the LRK26 peptide derived from C3a and with antimicrobial effects on C. albicans surface (Sonesson et al., 2007), were only identified in NS.

Proteins mainly identified on heat inactivated serum (HIS) in (C). Identified proteins on samples are indicated with gray background and not-identified in white. Complement fragments released during complement activation are indicated with triangles. Peptides belonging to complement fragments that can be released or be part of a full-length protein are indicated with \*. Numbers in (A,B) correspond to sequential steps during complement pathways activation. (A) Collectin-11 was only identified in NS samples. MASP2 was identified in NS and MASP1 was not identified in any sample. Fc (constant region of Ig) of IgGs or IgMs start classical complement pathway. (B) Properdin was identified in NS and Factor D (FD) was not. (C) Ficolin-2, Ficolin-3, C1qA, C1INH, and FD were identified only in HIS samples. C1INH dissociates the C1qrs complex, blocks alternative C3 convertase and inhibits C3 activation by kallikrein. C2 was identified in HIS, including 4 peptides of C2b. Five peptides belonging to FBa were identified in HIS condition. Signally, peptides belonging to C5a fragment were only identified in HIS samples.

Peptides belonging to the C5a region were only identified in HIS samples, just as more peptides belonging to the MACPF region of C6, C7, and C8-A. Furthermore, lower NSAF for all components of MAC, except C5, were obtained with HIS (**Figure 2**), which could be correlated with the enhanced identification of C5a peptides in this condition. In contrast, no identification of peptides belonging of C5a region and higher NSAF for the rest of components of MAC in NS samples may indicate MAC formation. MACPF-domains would not be accessible to trypsin digestion because they are points of interaction for MAC formation and would be protected in this condition. Furthermore, we identified clusterin and vitronectin (VTN) with higher NSAF in NS condition and both are able to interact with MAC in plasma, and thus prevent the cytolysis of host cells (Chauhan and Moore, 2006). Further experiments are required to determine the formation and the functionality of MAC on C. albicans surface.

In the same line of thought, in NS samples, properdin (FP) and not factor D (FD) were identified, while the opposite happened with HIS samples (**Figure 4B** vs. **Figure 4C**). The results are in agreement with a functional alternative complement pathway in NS condition on the surface of C. albicans. On the other hand, in HIS samples, FD could still be attached to the C3bB complex and we suppose that C3 convertase is not formed. In this situation, FP could not stabilize the complex and would be not identified on HIS samples. C1INH is able to bind to immobilized C3b and block progression of the complement cascade (Jiang et al., 2001). Serpin role of C1INH produces dissociation of C1qrs complex and the identification of these proteins in the HIS condition was enhanced, possibly for this reason. C3 and C4 (C4-A and C4-B) were identified with less NSAF and C2 and FB with higher NSAF in HIS samples (**Figure 4C**). In a current study about Streptococcus pyogenes interaction with human plasma, C2, C1qA, and FD were not detected on the surface of the microorganism (Sjöholm et al., 2014). In our experiments, when C. albicans cells were incubated with HIS, we detected not only these proteins, but also C1INH, and the enrichment of FB, C1qB, and C1qC. This is an interesting observation that may be due to the absence of functional convertases; FB and C2 might be more accessible to trypsin digestion. We assume that the unsuitable formation of complement convertases, or at least in a less extended manner, is responsible for the enhanced peptide identification of C2, FB, FD, C5, and the C1qrs complex in HIS samples.

Coagulation and complement pathways are highly connected, with many cross-talks (Kazatchkine and Jouvin, 1984; Amara et al., 2008, 2010; Peerschke et al., 2008; Diamond, 2013; Verschoor and Langer, 2013). Many complement inhibitors were identified on the surface of C. albicans and in general more abundant in HIS samples. The innate immune system strives to respond and activates complement cascades by non-conventional mechanisms (thrombin (F2), PLG, Factor XII (F12), kallikrein (KLKB1), ficolin-2 and -3) and bypass locking points in absence of functional FB and C2 (Nielsen et al., 1996; Rittirsch et al., 2008) (**Figures 2**, **4C**). In HIS samples, KLKB1 inhibitors were enriched, such as SERPINA5, SERPINC1, A2M, and C1INH (Gadjeva et al., 1998; Paréj et al., 2013).

A relevant group of proteins is serpin family (serine protease inhibitors), which are broadly distributed among eukaryote organisms. Serpins are able to inhibit proteases by a specific mechanism where covalent complexes are formed with target proteases (Law et al., 2006). Since serpin has to be cleaved to inhibit the target substrates, inhibition also consumes the serpin; serpins are therefore irreversible enzyme inhibitors. For these reasons, the identification of many serpins on C. albicans surface and more interestingly in HIS samples is an interesting observation because they may be inactivating their substrates attached to the surface of C. albicans or themselves coating C. albicanssurface in the sense to avoid interactions between their substrates and C. albicans (**Table 4**).

Interestingly, the apolipoproteins classified in the metabolism group are involved in lipid transport and also play a relevant role in host defense as part of the innate immune system (Grunfeld and Feingold, 2008). Furthermore, C. albicans grows better in lipoprotein-depleted plasma due to two factors: greater availability of lipids and reduced neutralizing candidacidal factor (Vonk et al., 2004). ApoE has a relevant role in systemic candidiasis infection and showed higher NSAF in NS samples (Figure S1). ApoE-/- mice showed an increased mortality in comparison with wild type (85 vs. 52%) (Vonk et al., 2004), and also an impaired immune response in Klebsiella pneumonia infection (de Bont et al., 2000).

In summary, the mechanisms developed by C. albicans to evade, interfere or use for its benefit host defenses are very wide and part of its pathogenic evolution. The analysis of C. albicans surface after incubation with human serum to obtain a partial breakdown of the immunological crosstalk between C. albicans and human serum proteins is a relevant target of study. C. albicans mimic human immune evasion model using many surface proteins to fix plasminogen and complement inhibitors (FH and C4BP) to protect against complement activation. Furthermore, the identification of Pra1, Sap5, Tef1, and 9 more proteins only identified in C. albicans hyphal surface induced with 10% human serum and not in other published conditions with an equivalent approach is a relevant highlight of this analysis.

# AUTHOR CONTRIBUTIONS

EM designed and performed the experiments, analysis of results, and writing of the manuscript. CP designed and performed the experiments. CH performed the experiments. MH performed the experiments and analysis of results. CN research support. LM designed the experiments, analysis of results and critically revised the manuscript. CG designed the experiments, analysis of results, and critically revised the manuscript.

# ACKNOWLEDGMENTS

This work was supported by BIO2012-31767 from the Ministerio de Economía y Competitividad, PROMPT (S2010/BMD-2414) from the Comunidad Autónoma de Madrid, REIPI-Spanish Network for the Research in Infectious Diseases (RD12/0015/0004) and PRB2 (PT13/0001/0038) funded by the ISCIII and FEDER. CP was the recipient of a fellowship from the program Continuous training for teachers of the Pontificia Javeriana University (Colombia) in conjunction with COLCIENCIAS-LASPAU University of Harvard. The proteomic analyses were carried out in the Proteomic Units of Complutense University of Madrid and of Centro de Investigaciones Biológicas (CSIC), members of ProteoRed network. The Immunofluorescence Microscopy images were carried out in Centro Nacional de Microscopía Electrónica (ICTS)-UCM. The authors would like to thank Dr. Fernando Vivanco [Fundación Jimenez Diaz, Universidad Complutense,

# REFERENCES

Madrid (Spain)] and Dra. Pilar Sánchez-Corral Gómez [Unidad de Investigación de Inmunopatología, Hospital Universitario La Paz, Madrid (Spain)] for the gift of C3 and FB antibodies, respectively. These results are lined up with the Spanish Initiative on the Infectious Diseases-Human Proteome Project (B/D-HPP).

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.01343

information for Candida albicans and Candida glabrata. Nucleic Acids Res. 40, D667–D674. doi: 10.1093/nar/gkr945

clinical outcome and risk factors for death. Eur. J. Clin. Microbiol. Infect. Dis. 21, 767–774. doi: 10.1007/s10096-002-0822-1

**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.

Copyright © 2015 Marín, Parra-Giraldo, Hernández-Haro, Hernáez, Nombela, Monteoliva and Gil. 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.

# Structure and Interactions of a Dimeric Variant of sHIP, a Novel Virulence Determinant of Streptococcus pyogenes

Carl Diehl 1, 2, Magdalena Wisniewska1, 3 , Inga-Maria Frick 4 , Werner Streicher 1, 5 , Lars Björck 4 , Johan Malmström4 and Mats Wikström1 \* †

1 Protein Function and Interactions Group, Faculty of Health and Medical Sciences, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark, 2 SARomics Biostructures, Lund, Sweden, 3 Malopolska Centre of Biotechnology, Krakow, Poland, 4 Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden, 5 Novozymes A/S, Bagsvaerd, Denmark

#### Edited by:

Jonathan M. Blackburn, University of Cape Town, South Africa

#### Reviewed by:

Kai Papenfort, Ludwig-Maximilians-Universität München, Germany Didier Soulat, Universitätsklinikum Erlangen, Germany

#### \*Correspondence:

Mats Wikström<EMAIL_ADDRESS>
†Present Address: Mats Wikström, Amgen Inc., Thousand Oaks, CA, USA

#### Specialty section:

This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology

> Received: 22 October 2015 Accepted: 18 January 2016 Published: 05 February 2016

#### Citation:

Diehl C, Wisniewska M, Frick I-M, Streicher W, Björck L, Malmström J and Wikström M (2016) Structure and Interactions of a Dimeric Variant of sHIP, a Novel Virulence Determinant of Streptococcus pyogenes. Front. Microbiol. 7:95. doi: 10.3389/fmicb.2016.00095

Streptococcus pyogenes is one of the most significant bacterial pathogens in the human population mostly causing superficial and uncomplicated infections (pharyngitis and impetigo) but also invasive and life-threatening disease. We have previously identified a virulence determinant, protein sHIP, which is secreted at higher levels by an invasive compared to a non-invasive strain of S. pyogenes. The present work presents a further characterization of the structural and functional properties of this bacterial protein. Biophysical and structural studies have shown that protein sHIP forms stable tetramers both in the crystal and in solution. The tetramers are composed of four helix-loop-helix motifs with the loop regions connecting the helices displaying a high degree of flexibility. Owing to interactions at the tetramer interface, the observed tetramer can be described as a dimer of dimers. We identified three residues at the tetramer interface (Leu84, Leu88, Tyr95), which due to largely non-polar side-chains, could be important determinants for protein oligomerization. Based on these observations, we produced a sHIP variant in which these residues were mutated to alanines. Biophysical experiments clearly indicated that the sHIP mutant appear only as dimers in solution confirming the importance of the interfacial residues for protein oligomerisation. Furthermore, we could show that the sHIP mutant interacts with intact histidine-rich glycoprotein (HRG) and the histidine-rich repeats in HRG, and inhibits their antibacterial activity to the same or even higher extent as compared to the wild type protein sHIP. We determined the crystal structure of the sHIP mutant, which, as a result of the high quality of the data, allowed us to improve the existing structural model of the protein. Finally, by employing NMR spectroscopy in solution, we generated a model for the complex between the sHIP mutant and an HRG-derived heparin-binding peptide, providing further molecular details into the interactions involving protein sHIP.

Keywords: proteomics, structural biology, virulence factors, protein-protein interactions, host-pathogen relationship

# INTRODUCTION

Streptococcus pyogenes, also known as group A streptococci (GAS) is a significant human pathogen that infects and colonizes the skin and the upper respiratory tract where it causes relatively mild clinical conditions such as impetigo and pharyngitis. Some infections caused by invasive strains, such as the AP1 strain of the M1 serotype, can lead to severe and potentially life-threatening diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome (STSS), whereas acute rheumatic fever and glomerulonephritis are sequelae to acute S. pyogenes infections. S. pyogenes causes an estimated 700 million cases of mild and non-invasive infections each year, of which ∼650,000 progress to severe invasive infections with a mortality of at least 25% (Carapetis et al., 2005; Ralph and Carapetis, 2013). S. pyogenes produces a number of proteins that enable the bacterium to attach to host tissues, evade the immune response, and spread by penetrating host tissue layers.

In a recent study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice (AP1), with a non-virulent strain (SF370). We found that one protein in particular was present at significantly higher levels in the stationary growth medium from the virulent strain. The amount of sHIP in the medium fraction is similar to the secreted mitogenic exotoxin SmeZ (Kamezawa et al., 1997), and shows an overall abundance profile resembling the profile observed for the surface associated proteins H and M1 (Lancefield, 1962; Åkesson et al., 1990; Wisniewska et al., 2014). Through the use of affinity pull-down mass spectrometry analysis of human plasma, we could demonstrate that the new bacterial protein interacts with the antimicrobial human protein histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal Histidine-rich glycoprotein Interacting Protein) was therefore introduced (Wisniewska et al., 2014). HRG is an abundant plasma glycoprotein of approximately 60 kDa that interacts with several other protein ligands such as tropomyosin, heparin, plasminogen, plasmin, fibrinogen, and IgG (Jones et al., 2005). HRG has been shown to exhibit broad antimicrobial potency (Rydengård et al., 2007), including activity against S. pyogenes (Shannon et al., 2010). It was shown that sHIP binds both intact HRG and HRG-derived peptides (peptides containing consensus heparin-binding sequences) with high affinity. Moreover, the antibacterial activity of HRG is blocked by protein sHIP, which represents a new mechanism that can contribute to the virulence of AP1 bacteria. In addition, we could show that patients with severe S. pyogenes infection, in contrast to patients with superficial and uncomplicated infections, are more prone to develop antibodies against sHIP, which suggests that sHIP represents a novel virulence determinant (Wisniewska et al., 2014). Furthermore, the determination of the threedimensional structure of sHIP, showed that it has a tetrameric organization composed of four helix-loop-helix motifs. A similar structural unit can be found in the adhesion factor FadA from Fusobacterium nucleatum. However, the two proteins differ significantly in their respective oligomeric organization. In FadA, the helix-loop-helix motifs form elongated fibers whereas the sHIP monomers are organized into a compact tetrameric structure. In order to understand the molecular prerequisites for the observed interaction between sHIP and HRG, we have performed extensive crystallization trials but not been able to obtain any diffracting crystals of complexes between sHIP and HRG or any HRG derived peptides. Due to the size of the tetrameric sHIP, NMR experiments in solution have proven to be very challenging and would benefit from access to a sHIP variant with lower molecular weight and retained activity. We have previously made the observation that the tetrameric structure of sHIP can also be described as a dimer of dimers, and postulated that three residues in the dimer-dimer interface could represent important determinants for the stabilization of the tetrameric form of sHIP. In this study, we have mutated these three interfacial residues and thereby been able to produce a stable dimeric variant of sHIP that was shown to be active both in biophysical binding experiments and an antimicrobial assay. The generation of stable dimers enabled further characterization of the interaction between sHIP and a peptide from HRG through NMR experiments, providing the first molecular details of an interaction involving this novel virulence determinant.

# MATERIALS AND METHODS

# Mutagenesis

The DNA sequence corresponding to residues Lys3-Met98 in the S. pyogenes protein sHIP (UniprotID: Q99XU0) was expressed as described previously and denoted sHIPwt (Wisniewska et al., 2014). A protein sHIP variant, a quadruple mutant named sHIPqp, in which Leu84, Leu88, Tyr95 were mutated to alanine, and Cys65 was mutated to serine, was constructed using the Quikchange XL site-directed mutagenesis kit (Agilent) following the manufacturer's instructions. We have previously observed that the wildtype protein can form larger aggregates through the exposed Cys65 residue in the absence of a reducing agent. The mutation Cys65Ser was introduced in order to avoid the need to use a reducing agent in subsequent experiments. As previously noted, this mutation has no effect on the structure and function of sHIP (Wisniewska et al., 2014). We are therefore using the Cys65Ser mutant in all studies involving both the wildtype sHIP and in the sHIP variant. All mutations were verified by DNA sequence analysis. The resulting expression construct contained a His-tag and a TEV protease cleavage site preceding the protein sequence of interest.

# Protein Expression and Purification

The protein sHIP variants sHIPwt and sHIPqp were expressed in E. coli strain Rosetta BL21 (DE3) and purified in two steps using standard immobilized metal ion affinity chromatography (IMAC) followed by proteolytic removal of the His-tag, and reverse phase chromatography (RPC), as described previously (Wisniewska et al., 2014). For the NMR studies, protein was produced using minimal medium for cell growth in order to introduce the stable isotopes 15N and 13C enabling tripleresonance NMR experiments (Neidhardt et al., 1974). The Histag was removed by treatment with tobacco etch virus (TEV) protease giving the following amino acid sequences for the wildtype protein sHIP, sHIPwt:

### SMKQDQLIVEKMEQTYEAFSPKLANLIEALDAFKEHYEE YATLRNFYSSDEWFRLANQPWDDIPCGVLSEDLLFDMIG DHNQLLADILDLAPIMYKHM,

and the quadruple mutant protein sHIP, sHIPqp:

SMKQDQLIVEKMEQTYEAFSPKLANLIEALDAFKEHYEE YATLRNFYSSDEWFRLANQPWDDIPSGVLSEDLLFDMIG DHNQLAADIADLAPIMAKHM, respectively.

The purity and mono-dispersity of the recombinant proteins were verified by SDS-PAGE electrophoresis and mass spectrometry.

# Crystallization, X-Ray Data Collection, and Structure Determination

Crystallization of the protein sHIP variant was carried out using the sitting drop vapor diffusion method at 18◦C. The best crystals were obtained from 20% PEG 6000, 0.2 M calcium chloride and 0.1 M Tris, pH 8.0. Prior to plunge freezing, the crystals were soaked for approximately 30 s in a drop of a reservoir solution containing 20% v/v ethylene glycol as cryo-protectant. The crystals belonged to the space group P22121 and contained one monomer per asymmetric unit. The highest resolution data set of 180 frames (1◦ oscillation range) was collected from an orthorhombic crystal at Max II lab beamline I911-2, Lund, Sweden, using a MAR CCD detector. Data were indexed and integrated using iMOSFLM (Battye et al., 2011) and scaled using SCALA (Evans, 2006) from the CCP4 suite of programs. The structure was solved by molecular replacement using wild type sHIP as the search model (PDB ID: 4MER), and refined using REFMAC (Murshudov et al., 1997). Refinement rounds were complemented with manual rebuilding using COOT (Emsley et al., 2010). Water molecules were automatically inserted using Arp/wArp and visually inspected. For further details on data processing and refinement statistics, see **Table 1**. Geometry of the model was analyzed with Molprobity (Chen et al., 2010). Molecular graphics images were generated using the program Pymol. The atomic coordinates and structure factors for the sHIP mutant (sHIPqp) crystal structure have been deposited with the protein data bank (PDB ID: 4PZ1).

# Analytical Ultracentrifugation

Sedimentation velocity experiments were performed at 20◦C and 50000 rpm using a Beckman XL-I instrument. Samples containing either sHIPwt or sHIPqp were in 20 mM MES buffer TABLE 1 | Crystallographic data collection and refinement statistics.

aValues for the highest resolution shell values are indicated in parentheses.

bRMSD, root mean square deviation.

alpha, beta, gamma refers to the unit cell axes dimensions, while alpha, beta, and gamma in symbol refers to the inclination angles of the axes in the unit cell. The space group number P22121 refers to the description of the symmetry of the crystal.

(2-(N-morpholino) ethanesulfonic acid), pH 5.5. The data were analyzed with SEDFIT using a continuous c (S) distribution (Schuck, 2000). HYDROPRO (Ortega et al., 2011) was used to calculate the theoretical sedimentation coefficient using the PDB IDs 4MER (sHIPwt) and 4PZ1 (sHIPqp), respectively.

# Synthetic Peptide

The sequence for the HRG peptide used in the study referred to as HRGsingle, corresponding to a single heparin-binding motif, have the amino acid sequence GHHPHG. The peptide was synthesized by Biosyntan GmbH (Berlin, Germany) and purity and molecular mass were confirmed by MALDI-TOF MS.

# Antimicrobial Assay

The S. pyogenes strain AP1 (40/58), was from the World Health Organization Collaborating Centre for Reference and Research on Streptococci, Prague, Czech Republic. The bacteria were cultivated in THY (Todd-Hewitt broth (Difco) supplemented with 0.2% yeast extract (Oxoid) at 37◦C and 5% CO2 until reaching mid-log phase (OD 620 nm approximately 0.4). The bacterial cells were washed and resuspended in 10 mM Tris-HCl, pH 7.5, containing 5 mM glucose, to a concentration of 2 × 109 cfu (colony forming unit)/ml. Subsequently, the bacteria were diluted to a concentration of 2 × 106 cfu/ml in 10 mM MES buffer, pH 5.5, containing 5 mM glucose. Fifty microliters of the bacterial solution was incubated with recombinant His-tagged HRG (Creative Biomart) at a concentration of 0.45 µM together with various concentrations of protein sHIPwt or protein sHIPqp for 40 min at 37◦C. Serial dilutions of the incubation mixtures were plated on TH agar, plates were incubated over night at 37◦C and the number of cfu's were determined.

# Isothermal Titration Calorimetry (ITC)

ITC experiments were performed using a VP-ITC200 instrument (GE Healthcare). The samples were extensively dialyzed against 20 mM MES buffer, pH 5.5. 0.5 mM HRGsingle peptide was titrated into 0.05 mM of sHIPwt, or sHIPqp respectively. All experiments were performed at 25◦C and run until saturation was achieved. The data were fitted using a model describing one independent binding site using the software provided by the manufacturer (Wiseman et al., 1989).

# NMR Experiments

Protein samples were prepared in 20 mM MES pH 5.5 with 7% (v/v) D2O for the spectrometer lock and 0.02% (w/v) NaN3 to prevent bacterial growth in the samples. All NMR experiments were performed at 25◦C. For the studies of the sHIPqp/HRGsingle peptide complex, the sample contained 1.0 mM of double-labeled (15N, 13C) protein sHIPqp and 1.1 mM of the HRGsingle peptide. The backbone and side-chain resonance assignment experiments were performed at 800 MHz proton resonance frequency, whereas the experiments in order to acquire Nuclear Overhauser Enhancement (NOE) distance restraints were acquired at 900 MHz proton resonance frequency. The assignment of the signals from sHIPqp and the HRGsingle peptide, and collection of NOE restraints were performed by using a combination of standard 2D and 3D triple-resonance experiments including HNCA (Clore et al., 1990), HNCOCA (Bax and Ikura, 1991), HNCACB (Wittekind and Mueller, 1993), CBCACONH (Grzesiek and Bax, 1992), HCCONH (Montelione et al., 1992), CCONH (Montelione et al., 1992), 15N-NOESY-HSQC (Marion et al., 1989), 13C-NOESY-HSQC (separate for aliphatic and aromatic signals; Marion et al., 1989), 2D double-filtered NOESY (Ikura et al., 1992), and 2D singlefiltered NOESY (Ikura et al., 1992). In addition, 2D NOESY and TOCSY experiments were acquired for the HRGsingle peptide. Spectra were processed with NMRpipe (Delaglio et al., 1995), applying zero-filling and linear prediction in the indirect dimensions and solvent filter and polynomial baseline correction in the direct dimension. CCPNMR (Vranken et al., 2005) was used for visualization of spectra, resonance connectivity analysis, and distance restraints (NOE) assignments. Structure calculations were performed using an iterative procedure within the ARIA/CNS suite of programs (Brunger et al., 1998). The assessment of the quality of the NMR generated structural models, were performed using the Protein Structure Validation

Suite (PSVS, **Table 3**; Bhattacharya et al., 2007). Molecular graphics images were generated using the program Pymol (**Figure 5**).

# RESULTS

# The sHIPqp Mutant Forms Stable Dimers of Two Helix-Loop-Helix Motifs

The wildtype sHIP is composed of tetramers that can be described as the dimer of two dimers each harboring two helix-loop-helix motifs (**Figure 1A**). The hypothesis was that the three non-polar residues at the tetramer interface (Leu84, Leu88, Tyr95) represent important determinants for the protein oligomerization (**Figure 1B**), and these residues were therefore mutated to alanines in the sHIPqp mutant protein. The orthorhombic crystals of the sHIPqp mutant contain one monomer in the asymmetric unit, with a solvent content of 36%. The crystal packing shows the formation of a dimer with a total buried surface area of 6070 Å2 as calculated by

FIGURE 2 | Sedimentation velocity experiments for protein sHIP. The dotted lines indicate the theoretical sedimentation coefficient for a tetramer and a dimer respectively calculated using HYDROPRO and the structural coordinates for protein sHIPwt (PDB ID: 4MER) and protein sHIPqp (PDB ID: 4PZ1). The wild-type sHIPwt is shown in black, and the sHIPqp mutant in red respectively.

PISA server (Krissinel and Henrick, 2007). The final model comprises residues 3-98, corresponding to the entire sHIPqp molecule (**Figure 1C**). The structure is very well defined, with an average B factor of 12.2 Å2 (10.8 Å2 and 13.6 Å2 for main chain and side chains, respectively). Final electron density maps are of very high quality including the loop region, which was partially not visible in sHIP wild type (**Figure 1D**). All non-glycine residues exhibit main-chain angles in the favored regions of the Ramachandran plot as defined by the use of the program Molprobity (Chen et al., 2010; **Table 1**). To provide a quantitative analysis of the oligomeric states of the sHIP variants, analytical ultracentrifugation was employed on sHIPwt and the sHIPqp mutant. These experiments clearly show that the wildtype sHIP (sHIPwt) appear as tetramers in solution whereas the sHIP mutant (sHIPqp) exist as dimers (**Figure 2**). The largest differences in the monomeric helix-loop-helix motif between the two sHIP variants are observed in the loop region connecting the two helices. In the sHIPqp mutant, all residues, including side-chains, are very well defined, while in sHIPwt, some residues could not be defined due to the absence of interpretable electron density maps in this region (**Figure 1D**).

# The sHIPqp Mutant Inhibits the Antibacterial Activity of HRG

It is known that HRG has antibacterial activity including activity against S. pyogenes (Shannon et al., 2010), and when challenged by HRG, sHIP has been shown to rescue S. pyogenes bacteria (Wisniewska et al., 2014). To examine whether a sHIP mutant has the same ability, we performed an antimicrobial assay comparing sHIPwt and sHIPqp. As shown in **Figure 3**, sHIPqp inhibits the bactericidal activity of HRG to the same or even higher extent that wildtype sHIP indicating that the dimeric mutant sHIPqp retains its inhibitory capacity.

# sHIP Binds a Single Heparin-Binding Motif in HRG

In our previous studies we have shown that protein sHIP binds to both HRG and HRG-derived peptides with nanomolar to micromolar affinity, and that the histidine-rich region is important for the interaction (Wisniewska et al., 2014). To further dissect the molecular details in the interaction, we investigated whether the sHIP variants are also able to bind a single heparin-binding motif (GHHPHG) in HRG (denoted as HRGsingle). Isothermal titration calorimetry (ITC) experiments showed that both sHIPwt and sHIPqp binds HRGsingle with similar micro-molar affinities, clearly indicating that the heparinbinding motif represents an important recognition motif in HRG for the interaction with protein sHIP (**Figure 4**, **Table 2**).

# The HRG-Binding Site in sHIP Involves Both Helix-Loop-Helix Motifs of the Dimer

The wildtype protein sHIP is a tetrameric protein with a molecular weight of 46 kDa complicating NMR experiments in solution due to broad and overlapping resonances. The creation of the mutant dimeric form of sHIP, sHIPqp, and through the verification of its functional properties, we now have access to a smaller, yet biologically relevant system with a molecular weight of 23kDa, which enabled detailed NMR studies in solution.

A molecular model was created with a combination of the structure of sHIPqp (PDB ID: 4PZ1), and distance restraints (NOEs) extracted from the NMR experiments of the complex between sHIPqp and the HRGsingle peptide. A total of 1714 NOEs were used as distance restraints in the structure calculations of which 321 are long-range restraints and 56 represent NOEs detected between sHIPqp and the HRGsingle peptide (**Table 3**). It should be noted that this model represent

TABLE 2 | Summary of thermodynamic parameters from ITC experiments of the binding of the two sHIP variants sHIPwt and sHIPqp to the HRGsingle peptide respectively.

a preliminary description of the sHIPqp-HRGsingle complex that will need further refinement before considered a finalized complex structure. However, the assessment of the generated structure shows that it is of sufficient quality to represent a most relevant descriptive model for the complex (**Table 3**). The model shows that the HRGsingle peptide binds in a pocket created by residues from both monomeric helix-loop-helix motifs, suggesting that the dimeric form represents the smallest sHIP variant attainable with retained HRG-binding capacity. Each sHIP dimer binds two HRGsingle peptides in two identical sites (**Figure 5A**). The binding site includes the N-terminal part of helix 1 of one monomer (residues Asp5-Met12), and the end region of helix 1 of the second monomer (residues Ala41- Ala56). For the first monomer three residues are involved in NOE contacts with the HRGsingle peptide with primary contribution from Ile8. For the second monomer, thirteen residues give rise to detectable NOEs dominated by contacts involving Phe46, Glu51, and Arg54. Moreover, the binding site does not involve any residues in the proximity of the dimer-dimer interface in agreement with the observation that the mutant sHIPqp maintain its full inhibitory capacity of HRG in antimicrobial assays (**Figure 3**). The two external Glycine residues of the HRGsingle peptide, Gly-1 and Gly-6, are not involved in any interactions with sHIP. However, the core part of the peptide, (HHPH), forms multiple contacts with a binding pocket created by residues from both chains of the structural framework created by the sHIP dimer. In particular, residues His-2 and His-5 are positioned deeply in the binding pocket, a confirmation of the peptide backbone that is made possible due to the unique backbone properties of the connecting Pro-4 residue (**Figure 5B**).

# DISCUSSION

Bacterial infections represent a major challenge to human health worldwide, a challenge increasing at an elevated pace due to the alarming upsurge of antibiotic resistance. In order to treat bacterial infections in the future, new therapeutic strategies need to be developed that will require a better understanding of the complex molecular interplay between bacterial pathogens and their human host. S. pyogenes represents one of the most significant bacterial pathogens in the human population. Using mass spectrometry based proteomics, we previously identified a novel protein and virulence factor from this bacterium (Wisniewska et al., 2014). In this study we have extended the structural and functional characterization of this protein to

aAnalyzed for residues 2 to 98 (sHIPqp chain A), 102-198 (sHIPqp chain B), 301-306 (HRGsingle peptide 1), and 401-406 (HRGsingle peptide 2).

bPROCHECK (Laskowski et al., 1993, 1996).

cRamachandran statistics calculated by PROCHECK (Laskowski et al., 1993, 1996).

further our understanding of the interactions between sHIP and the human host.

The structure determination of the sHIPqp mutant showed that the fold of the sHIP mutant and the sHIP wild type monomers is essentially identical, displaying an elongated helixloop-helix motif (**Figure 1**). However, the most significant difference is related to the oligomeric states of the two protein variants. For wildtype sHIP, four monomers assemble into a compact homotetramer, which can be described as a dimer of dimers due to the interactions at the dimer-dimer interface. Our previous studies showed that this interface consists of amino acids with largely non-polar side-chains, such as Leu84, Leu88, Tyr95, and Met98, which can be responsible for protein oligomerization. The mutation of these residues to alanine's resulted in a shift in quaternary structure from tetramers to dimers further evidenced by sedimentation velocity experiments that clearly show that the sHIPqp mutant exists as dimers in solution (**Figure 2**).

To gain further understanding of the interaction between sHIP and HRG, a molecular model was created for the complex between sHIPqp and a single heparin-binding motif of HRG (HRGsingle), a peptide shown to be active in binding to both sHIPwt and sHIPqp (**Figure 4**). The model shows that the HRGsingle peptide interacts with both monomers of the helixloop-helix dimers, and that a proline situated in the middle of the peptide sequence allows two histidines, His-2 and His-5, to enter deeply into the binding pocket created by the

two sHIP monomers (**Figures 5A,B**). However, even if both monomers contribute to contacts with the HRGsingle peptide, one of the monomers clearly dominates the overall contribution to the binding epitope for the sHIP dimer. Future studies should involve mutagenesis experiments of both sHIP and HRG peptides to further define the critical determinants for the interaction.

# CONCLUDING REMARKS

Through structure-based protein engineering of a selected set of residues in the dimer-dimer interface of sHIP, we were able to generate a dimeric form of the protein with retained functional properties. The access to dimeric sHIP provides the opportunity to utilize NMR techniques in order to further elucidate interactions involving this protein. In conclusion, we have through a combination of protein engineering, biophysics, and structural biology methods, provided the first molecular details describing the interaction between the antimicrobial protein HRG and the novel virulence factor sHIP.

# AUTHOR CONTRIBUTIONS

CD and MagW performed the NMR and X-ray experiments, respectively. IF performed the antimicrobial assay, WS the AUC experiments, and JM the MS proteomics analysis. LB was providing the clinical data assessment. MW (corresponding author) supervised the project, performed the ITC experiments, and wrote the manuscript with input from all authors.

# FUNDING

This work was supported by the Swedish Research Council (projects 7480, 2008:3356, and 621-2012-3559), the Swedish Foundation for Strategic Research (grant FFL4), the Crafoord Foundation (grant 20100892), the Wallenberg Academy Fellow KAW (2012.0178), the European research council starting grant

# REFERENCES

(ERC-2012-StG-309831), the Novo Nordisk Foundation grant NNF14CC0001, the European Commission under the Seventh Framework Program (FP7) contract Bio-NMR 261863 providing access to the Swedish NMR Centre, Gothenburg, Sweden, and the MAX laboratory, Lund, Sweden enabling access to the MAX laboratory (ID 20120014 and 20130189).

# ACKNOWLEDGMENTS

We would like to thank SyBIT project of SystemsX.ch for support and maintenance of the lab-internal computing infrastructure, the ITS HPC team (Brutus).

**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.

Copyright © 2016 Diehl, Wisniewska, Frick, Streicher, Björck, Malmström and Wikström. 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.

# Proteogenomic Analysis of Mycobacterium smegmatis Using High Resolution Mass Spectrometry

Matthys G. Potgieter 1 , Kehilwe C. Nakedi 2 , Jon M. Ambler 1 , Andrew J. M. Nel 2 , Shaun Garnett 2 , Nelson C. Soares 2 , Nicola Mulder 1 \* and Jonathan M. Blackburn2 \*

1 Computational Biology Division, Department of Integrative Biomedical Sciences, IDM, University of Cape Town, Cape Town, South Africa, 2 Division of Chemical and Systems Biology, Department of Integrative Biomedical Sciences, IDM, University of Cape Town, Cape Town, South Africa

Biochemical evidence is vital for accurate genome annotation. The integration of experimental data collected at the proteome level using high resolution mass spectrometry allows for improvements in genome annotation by providing evidence for novel gene models, while validating or modifying others. Here, we report the results of a proteogenomic analysis of a reference strain of Mycobacterium smegmatis (mc2155), a fast growing model organism for the pathogenic Mycobacterium tuberculosis—the causative agent for Tuberculosis. By integrating high throughput LC/MS/MS proteomic data with genomic six frame translation and ab initio gene prediction databases, a total of 2887 ORFs were identified, including 2810 ORFs annotated to a Reference protein, and 63 ORFs not previously annotated to a Reference protein. Further, the translational start site (TSS) was validated for 558 Reference proteome gene models, while upstream translational evidence was identified for 81. In addition, N-terminus derived peptide identifications allowed for downstream TSS modification of a further 24 gene models. We validated the existence of six previously described interrupted coding sequences at the peptide level, and provide evidence for four novel frameshift positions. Analysis of peptide posterior error probability (PEP) scores indicates high-confidence novel peptide identifications and shows that the genome of M. smegmatis mc2155 is not yet fully annotated. Data are available via ProteomeXchange with identifier PXD003500.

Keywords: Mycobacterium smegmatis mc2155, mass spectrometry, proteogenomics, genome annotation, proteomics

# INTRODUCTION

Evidence for the existence of protein coding genes include ab initio gene predictions, transcriptomic analysis, and comparative genomics information (Krug et al., 2011). Although, gene annotation of model organisms often relies on transcript sequencing, it has become apparent that evidence of transcription may not equal evidence of translation (Castellana and Bafna, 2010). Proteomics data, on the other hand, gives direct evidence of which genes are translated. Using proteomics data, true genes can be separated from pseudogenes, and the translational frame can be determined. The location of TSSs (translational start sites) can be identified, while signal cleavage and other posttranslational modifications (PTMs) can be identified (Kucharova and Wiker, 2014). By mapping to uncharacterized genomic coordinates, novel genes can be identified (Borchert et al., 2010).

#### Edited by:

Biswarup Mukhopadhyay, Virginia Tech, USA

#### Reviewed by:

Leonard James Foster, University of British Columbia, Canada Julian Uszkoreit, Ruhr-Universität Bochum, Germany

#### \*Correspondence:

Nicola Mulder<EMAIL_ADDRESS>Jonathan M. Blackburn<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology

> Received: 25 October 2015 Accepted: 16 March 2016 Published: 05 April 2016

#### Citation:

Potgieter MG, Nakedi KC, Ambler JM, Nel AJM, Garnett S, Soares NC, Mulder N and Blackburn JM (2016) Proteogenomic Analysis of Mycobacterium smegmatis Using High Resolution Mass Spectrometry. Front. Microbiol. 7:427. doi: 10.3389/fmicb.2016.00427

Genomic six frame translation allows for the creation of a database containing, in the ideal case, all possible putative proteins, but at the cost of including many spurious entries. This leads to decreased sensitivity of identifications at the same FDR as standard proteomic databases with a higher proportion of non-spurious entries (Castellana and Bafna, 2010). Due to the large database sizes involved in proteogenomics, and large numbers of spectra that need to be assigned, many false positive identifications are obtained even at a low error rate. To limit the number of spurious entries, thus increasing the sensitivity of identifications, proteogenomic databases can be compacted, by excluding entries below a minimum length cutoff or only focusing on genomic regions identified by ab initio gene prediction tools (Castellana and Bafna, 2010).

Automated gene prediction at the early stage of genome annotation is prone to errors, with rates of incorrect TSS prediction of up to 44% reported (Gallien et al., 2009), while short protein-coding genes are difficult to predict (Renuse et al., 2011). Accurately identifying the TSS of a gene is complicated by the existence of different possible start codons, with many nonstandard start codons identified in prokaryotes (Castellana and Bafna, 2010). Mycobacteria are known to use GTG and TTG as initiator Met (fMet) start codons, in addition to translation of these codons with Val and Leu, respectively (Kelkar et al., 2011).

Wang et al. (2005) used databases of gene predictions to identify mass spectra, identifying 901 proteins in Mycobacterium smegmatis using the partially sequenced genome available at the time—validating many predicted genes. Gallien et al. (2009) identified 946 proteins in M. smegmatis, characterizing 443 Nterminal peptides, and revealed an error rate of 19% in predicted TSSs. Kelkar et al. (2011) reported 41 novel protein-coding genes in Mycobacterium tuberculosis strain H37Rv. By identifying Nterminal peptides, the authors were able to correct the TSS of 33 proteins, and validate the TSS of 727 annotated proteins. Strikingly, the authors identified eight proteins with evidence for translation initiation at two different sites.

The occurrence of false positive peptide identifications from tandem mass spectrometry data implies the need for statistical concepts such as the false discovery rate (FDR) and posterior error probability (PEP) score to gauge the reliability of identifications. The FDR is controlled by scoring and ranking peptide spectral matches (PSMs), and identifying the score above which a maximum allowable proportion of PSMs from a decoy database are identified—indicating the expected maximum proportion of incorrect PSMs—while posterior error probability (PEP) indicates the probability of error of a single PSM (Käll et al., 2008). The MaxQuant algorithms calculate PEP scores based on search engine PSM scores and peptide length, and determine a cutoff PEP score at a specific FDR (Cox and Mann, 2008).

Krug et al. (2013) compared the number and median PEP scores of novel peptide identifications with reverse sequence hit peptides in a proteogenomic analysis of Escherichia coli, and determined that the absolute number as well as the median PEP-values for both groups were very similar. The authors concluded that as a genome approaches complete annotation, the likelihood of any novel peptide identification being a false positive identification increases (Krug et al., 2013).