**Abstract**

Several authors reported that the absence of normal physical activity promotes a faster functional loss of several organs and systems, such as the cardiorespiratory system. It is known that scheduling physical activities and regular exercise for DMD patients, when performed based on a thorough functional evaluation, is fundamental for maintaining the quality of life of these children, as well as other associated resources that should be used, whenever possible. Exercise can help DMD patients to maintain and improve muscular strength for performing activities of daily living (ADL) such as stair climbing, slow the rate of increased weakness or contracture development that can prolong ambulation, maintain enough respiratory capacity and strengthened postural muscles, which can slow the onset of scoliosis. There is a need to pass throughout the message to professionals, staff and families who are in this context or who have children with developmental disabilities that exercise and physical activity are an essential factor for maintaining health and well-being throughout the lifespan. That's what we wish and hope with this chapter.

**Keywords:** exercise, Duchenne muscular dystrophy, physical activity, movement, life quality

## **1. Introduction**

It is known that there are a growing number of parents looking for ways to improve their children's quality of life because of Duchenne muscular dystrophy's consequences.

As a tool, exercise in aquatic environment allows children to achieve skills that can be difficult on the ground. With this in mind, a literature review was carried out to systematize the pediatric sequelae that can be treated with the benefits of physical activity in Duchenne muscular dystrophy [1–4].

Aquatic activity has been a way of stimulating the development of children and expanding the experiences of healthy, disabled or at-risk children [5]. The premature child and those who are at high risk of neurological injury or developmental delay may already have experienced hydrotherapy as an intensive care intervention. Very young babies participate together with their parents in aquatic programs. Within the exercise techniques, it is hydrotherapy that enables the accomplishment

#### *Muscular Dystrophies*

of activities of greater degree of difficulty, providing patients with psychological benefits. If compared to techniques performed on the ground, hydrotherapy due to the physical principles of water facilitates and improves balance, coordination and posture and gives the patient the feeling of safety. For the child the feeling of safety is found in the arms of the parents and in the contact with the body. This was experienced in the sensation of water environment, before birth, and is now again found in the heat of the water.


**Author details**

Emarti Kumari MBM University, Jodhpur, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Perspective Chapter: Dynamic Analysis of High-Rise Buildings Using Simplified Numerical… DOI: http://dx.doi.org/10.5772/intechopen.108556*


**5. Family businesses and self-employment**

A small business is not necessarily a family business. In the family business, ownership and management are concentrated in the family members, predominantly a family-based intraorganizational relationship. Family nature issues are prioritized against other objectives. For example, the company's property control by the family will not be sacrificed to fund the development and growth of the business. Indeed, family property is not diluted to include non-family investors. Therefore, the available financial resources are those generated by the company and those of the family itself. However, credit may be used with banking institutions to finance the growth of the business.

The uniqueness of the family business lies in the integration of family and business in the same context. The collaboration of the family members in the company may be an indication that the admission criteria are not governed by the higher academic and professional qualifications, which are more suitable for the performance of duties [19]. However, instead of prevailing the replacement of generations in the management of the company, the owners can choose to hire professional managers to create wealth but preserving the intra-family relationship in the company. On the other hand, family firms often find it difficult to attract and retain highly skilled managers, partly because of the limitations of career advancement and the absence of personal reward policies, as well as the lack of goal-oriented professional management and continuous improvement.

#### **5.1 Self-employment: advantages and disadvantages**

There are immense advantages and disadvantages of self-employment (being your own boss), and the entrepreneur must be aware of both before starting a business. We start by highlighting the three main advantages:

1.*Autonomy*: the need for independence and freedom to make decisions are the main advantages. The feeling of being your own boss is very satisfying to most entrepreneurs.

If self-employment were easy to achieve, there could be a much larger number of self-employed people. In fact, it is one of the most difficult professional careers anyone can choose. Some of the disadvantages of this life option are described below:

In short, small businesses' ability to innovate stems from their learning of the market, recognition of the need for rapid change, and organizational flexibility [20]. These valences are a support to the decision-making in the self-employment before the obtained feedback of the market.


*Aquifer Characterization: The Case of Hawassa City Aquifer DOI: http://dx.doi.org/10.5772/intechopen.91211*

#### **Figure 3.**

*(A) SWL contour map and vector map showing flow direction (2006) with color scale; (B) SWL contour map and vector map showing flow direction (2009) with color scale; (C) SWL contour map and vector map showing flow direction (2012) with color scale; (D) SWL contour map and vector map showing flow direction (2013) with color scale; (E) SWL contour map and vector map showing flow direction (2014) with color scale; (F) DWL contour map and vector map showing flow direction (2006) with color scale; (G) DWL contour map and vector map showing flow direction (2009) with color scale; (H) DWL contour map and vector map showing flow direction (2012) with color scale; (I) DWL contour map and vector map showing flow direction (2013) with color scale; and (J) DWL contour map and vector map showing flow direction (2014) with color scale.*

S-W areas. For the year 2009, the map presented shows turbulence, so the flow direction has no clear trend except for the lake shore area that receives water from the nearby aquifer. For the year 2012, relatively medium magnitude water flows into the Gara Riqata area, which produces huge water discharge for the next year 2013. The two years 2013 and 2014 results clearly reveal that significant amount of

**48**

*Resources of Water*

**3. Conclusion**

**Author details**

Hawassa, Ethiopia

**51**

Shemsu Gulta1 and Brook Abate2

Technology University, Addis Ababa, Ethiopia

provided the original work is properly cited.

\*Address all correspondence to<EMAIL_ADDRESS>
\*

1 School of Biosystems and Environmental Engineering, Hawassa University,

2 College of Architecture and Civil Engineering, Addis Ababa Science and

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

The main aquifers tapped by the Hawassa City ground water system is unconfined and semi-confined type since no confining beds like clay are clearly identified up to 200 m depth. Weathered and fractured pumice, basalt scoriaceous rocks, fine-to-coarse-grained sand, and weathered ignimbrites are major waterbearing formations. The first water striking point is the shallowest around the lake shore (west and S-W) and as the well site goes apart to the west direction, the water striking depth is increasing and ash, fractured basalt and ash with scoriaceous basalt are dominating. The Hawassa City ground water system is of high performance and potential due to the very small drawdowns, fast recovery percentage (up to 100%), high transmissivities, and saturated thicknesses of the aquifer. The aquifer materials are highly porous and the high aquifer porosities imply aquifers of high storativity and better yield. The protective capacity of the overburden rock materials in the area is very low. Transmissivity and hydraulic conductivity values are generally high in the lake shore and central parts. Since the aquifer materials in the study area are highly permeable and relatively shallow, the groundwater has a high susceptibility of being contaminated over large area. The ground water flows from the E and S-E parts toward the central and western side of the city with a very

similar profile with the surface water flow direction.

*Aquifer Characterization: The Case of Hawassa City Aquifer*

*DOI: http://dx.doi.org/10.5772/intechopen.91211*

ground water flows from the large area of the city toward the lake shore. Thus, the ground water system feeds the lake which will in turn accelerate the lake level rise. For those months in which the pumping test of the wells conducted (i.e., March, April, and May) large amount of infiltrated water from the discharge spots join the ground water reservoir as this is the time immediately after the rainy season stops in the area. Combining these graphical results and the one for the surface water flow direction (**Figure 3A–J**), groundwater flow direction follows similar tendency of the surface water movement, that is, it follows the surface profile. This also implies the fact that the highlands are recharge areas and the lowland areas as nice spots for discharging.

### **2.3 Guide to drilling depth in Hawassa City**

Using the wire frame and the grid map of the well depth on Surfer 8 Software, one can interpolate the required depth to be drilled at specific GPS location in the study area. This is an important guide for borehole drillers and clients to estimate the depth of the water striking formation at that specific site. To do so, the Surfer 8 Software can be used to display the map as shown in **Figure 4**.

The gridding method in Surfer 8 Software uses weighted average interpolation algorithms. This means that with all other factors being equal, the closer a point is to a grid node, the more weight it carries in determining the Z value at that grid node. The difference between gridding methods is how the weighting factors are computed and applied to data points during grid node interpolation. The coefficient of determination for this analysis is found to be R2 = 0.87, which indicates the strong acceptability of the guide.

To increase the likelihood that these data are honored, one can increase the number of grid lines in the X and Y direction. This increases the chance that grid nodes coincide with data points, thereby increasing the chance that the data values are applied directly to the grid file.

The geological formation at the depth to be drilled and the other parameters determined by this and other studies shall be combined to get more detailed information. Certainly, the more the depth drilled, the better will be the safe yield.


**1. Introduction**

Pyruvylation is a widespread non-carbohydrate modification of monosaccharides found in various classes of glycoconjugates. In most cases, the modification is present as a pyruvate (Pyr) ketal (cyclic acetal/ketal) bridging two hydroxyl groups of a monosaccharide residue and forming a ring structure [1], where pyruvate is most frequently placed across the 2,3-, 4,6-, or 3,4-positions (Figure 1I–III). The best-known example of pyruvylation, however, occurs as enol pyruvate (Figure 1IV), which is elaborated during the biosynthesis of the bacterial cell wall component peptidoglycan [2]. In both modes of pyruvylation, a dedicated pyruvyltransferase catalyses the transfer of the pyruvate moiety to the monosaccharide target.

**Figure 1.** Overview on the most common modes of monosaccharide pyruvylation. Shown is pyruvate ketal at bridging positions 2,3 (**I**), 4,6 (**II**), and 3,4 (**III**), and enol pyruvate (**IV**). Arabic numbers indicate ring positions.

Pyruvate-ketal-modified (henceforth abbreviated as "pyruvylated") glycoconjugates are found in various phylogenetic orders of life, including bacteria, yeast, and algae, but not in humans. Pyruvylated glycoconjugates are typically present in the cell envelope to which they impart a net negative charge; this is necessary for vital biological functions, such as regulation of the cell influx/efflux processes and cell–cell interactions including cell aggregation and pathogenic adhesion. Notably, besides pyruvylation, nature offers various alternate strategies to create anionic cell surfaces, including a wide range of acidic saccharides (e.g., muramic acid, hexuronic acids, sialic acids) and saccharide modifications (e.g., succinate, lactate, phosphate) [3]. These compounds further lead to an increased capability of cells to electrostatically bind cations at the surface, which, in turn, may foster the packing density of the saccharide portion of glycoconjugates [4].

The repertoire of monosaccharide targets of pyruvylation is quite diverse. The most abundant pyruvylated monosaccharide with 59 hits in the Carbohydrate Structure Database (CSDB, http: //csdb.glycoscience.ru/) [5,6] is galactose (Gal). Examples of pyruvylated galactose include, among others, the capsular polysaccharide of *Bacteroides fragilis* [7] and *Streptococcus pneumoniae* [8], *N*-glycans of the fission yeast *Schizosaccharomyces pombe* [9], as well as carragenans [10], and galactans from algae [11–13]. Recently, pyruvylated *N*-acetylmannosamine (ManNAc) has emerged as an important epitope on bacterial "non-classical" secondary cell wall glycopolymers, serving as a cell wall ligand for cell surface (S-) layer proteins, such as those of the pathogen *Bacillus anthracis* and the honeybee saprophyte *Paenibacillus alvei* [14,15]. There are also examples of pyruvylated monosaccharides on capsular polysaccharides serving as an immunostimulatory effector [7,16], or contributor to virulence as in the case of the secondary cell wall polymer of *Bacillus cereus* [17] or the exopolysaccharide xanthan of *Xanthomonas* spp., where pyruvylation is essential for successful colonization and pathogenesis in planta [18]. Pyruvyl groups on terminal glucose (Glc) and *N*-acetylgalactosamine (GalNAc) residues in the lipooligosaccharide of *Pseudomonas stutzeri* OX1, in contrast, are assumed to have biosynthetic implications [4]. All these examples are, among others, discussed in detail below.

Several studies dealing with pyruvylated glycans and their structural elucidation are available in the literature. However, knowledge of the enzymatic machinery governing pyruvylation is scarce. This is mainly due to missing sequencing data of the organisms, which produce pyruvylated glycoconjugates of known structure. Thus, despite their predictably widespread occurrence, pyruvyltransferases are an under-investigated class of enzymes.

This review summarizes the current state of knowledge about pyruvylated glycoconjugates in nature—focusing on bacterial sources—with an emphasis on the pyruvyltransferases involved in their biosynthesis.

#### **2. Enol-Pyruvylation in Peptidoglycan Biosynthesis**

The UDP-*N-*acetylglucosamine-3-*O*-enol-pyruvyltransferase MurA (EC <IP_ADDRESS>) targets UDP-*N*-acetylglucosamine (UDP-GlcNAc) as an acceptor substrate for enol-pyruvyl transfer from a phosphoenolpyruvate (PEP) substrate, thereby releasing free phosphate and yielding the UDP-activated form of the essential bacterial cell wall compound *N*-acetylmuramic acid (enolpyruvyl-UDP-*N*-acetylglucosamine; MurNAc). This first committed step of peptidoglycan biosynthesis is inhibited by the epoxide antibiotic fosfomycin [19]. As a PEP analogue, fosfomycin binds covalently to the key cysteine residue at position 115/116 (position depending on the source of enzyme) in the active site of MurA, preventing the formation of UDP-MurNAc [19–21]. The co-crystal structure of a Cys (cysteine)-to-Ser (serine) mutant of *Enterobacter cloacae*, MurA, together with its substrates, revealed that the Cys residue is essential for product release and not directly involved in the chemical reaction of enol-pyruvyl transfer. The comparison of the product state with the intermediate state and an unliganded state of MurA indicated that the dissociation of the products is an ordered event, with inorganic phosphate leaving first, followed by conformational changes that lead to the opening of the two-domain structure of MurA and the final release of UDP-MurNAc [20]. A recent study on MurA of the opportunistic pathogen *Acinetobacter baumannii* revealed that the enzyme exists as a monomer in solution and has a pH optimum of 7.5 at 37 ◦C. The Km for UDP-GlcNAc is 1.062 ± 0.09 mM and 1.806 ± 0.23 mM for PEP [21]. The relative enzymatic activity is inhibited approximately threefold in the presence of 50 mM fosfomycin. Superimposition of a model for the *A. baumannii* enzyme with MurA of *Escherichia coli (E. coli)* confirmed the structural similarity in the fosfomycin binding site. Because of the worldwide spread of antimicrobial resistance and the paucity of novel drugs in the development pipeline, there has been a renewed interest in fosfomycin as an alternative option for the treatment of infections caused by multidrug-resistant Gram-negative bacteria [22]. However, it has to be considered that natural MurA mutants exist that render the respective organisms fosfomycin resistant. This includes *Mycobacteria* and *Chlamydia* species, where Cys-to-Asp mutants occur [20].

Interestingly, NikO, another enol-pyruvyltransferase that is structurally closely related to the common MurA enzymes and, consequently, inhibited by fosfomycin, plays an essential role in the biosynthesis of nikkomycins. Nikkomycins are peptide-nucleoside antibiotics, which strongly inhibit chitin synthesis and, therefore, are effective against fungi and insects. NikO was shown to transfer the enol-pyruvyl moiety from PEP to the 3 -hydroxyl group of UMP and to be inactivated by fosfomycin because of alkylation of Cys130. However, the degree of inactivation is not as pronounced as in the case of common MurA enzymes [23].

#### **3. Ketal-Pyruvylated Glycoconjugates**

In this section, an overview of the different classes of pyruvylated glycoconjugates is given, including glycan composition and structure, as well as functional and biosynthetic aspects, when known.

There is a recent interest in understanding the biosynthetic pathways of pyruvylated glycoconjugates from bacterial sources, as these pathways might unravel novel targets for therapeutic intervention. However, the current biosynthesis models for the different classes of glycoconjugates are in most cases only fragmentarily available and, frequently, they consider in silico predictions of involved components without experimental evidence.

#### *3.1. Exopolysaccharides*

Many organisms produce extracellular polysaccharides (exopolysaccharide, EPS) that are actively secreted during growth, including bacteria, yeasts, and microalgae [24]. EPSs are a diverse class of carbohydrate polymers that are composed of either linear or branched repeating units that are connected with varying stereochemistry. Monosaccharide constituents include pentoses (ribose and arabinose—especially in *Mycobacterium* spp.), hexoses (mannose (Man), glucose, fructose, galactose), deoxysugars (rhamnose (Rha), fucose (Fuc), uronic acids (glucuronic and galacturonic acids), and amino

sugars (glucosamine, galactosamine, in several cases modified by *N*-acetylation) [24]. Depending on the monosaccharide composition, homo- or hetero-polymers are differentiated.

EPSs have a "jelly-like" appearance and are part of the glycocalyx—with which the "cellular sugar coat" is referred to [24]; as a common feature, they create a protective matrix around cells. The shielding effect against macromolecules that is conferred by EPS makes some bacteria 1000 times more resistant to antibiotics than their EPS-free counterparts [25].

Given the high application potential of microbial EPSs in medical fields, biomaterials, food applications, and in the replacement of petro-based chemicals [26], these glycoconjugates are currently of high interest.

#### 3.1.1. Xanthan

Xanthan is the main EPS produced by *Xanthomonas campestris* and other phytopathogenic *Xanthomonas* spp. that cause various economically important diseases in mono- and di-cotyledonous crops. Xanthan enhances the attachment to plant surfaces through its effect on biofilm formation, promotes pathogenesis by Ca2+ chelation and, thereby, suppression of the plant defence responses in which Ca2+ acts as a signal [27]. In practical applications, xanthan is frequently used as a viscosifying agent [28,29].

The pentasaccharide-repeating unit of xanthan consists of two β-(1→4) linked d-Glc residues as backbone and a trisaccharide side chain, α-(1→3)-linked to every other glucose. The side chain is composed of α-d-Man, β-d-glucuronic acid (GlcA), and β-d-Man, which are β-(1→2)- and β-(1→4)-linked to another, respectively [30]. In its natural state, the α-d-Man residue is acetylated and the β-d-Man is either acetylated or pyruvylated. It was found that the 4,6-ketal-pyruvate (4,6Pyr) specifically and, to a lesser extent, the acetyl groups that decorate the mannose residues are involved in Ca2+ chelation [27] and affect bacterial adhesion and biofilm architecture and, hence, contribute to the bacterium's virulence [18]. Furthermore, the rheological properties of xanthan are influenced by its pyruvylation and acetylation pattern [28,31].

Xanthan biosynthesis is encoded in a so-called *gum*-cluster. Of the 13 encoded genes, *gumDMHK* are involved in the synthesis of the pentasaccharide repeat, and *gumBCEJ* in polymerization and xanthan export across the outer membrane in a flippase/polymerase (Wzx/Wzy)-dependent pathway. Regarding the modifications of xanthan, the predicted pyruvyltransferase GumL is hypothesised to catalyse pyruvylation of β-d-Man residues, while GumF and GumG are involved in β-d-Man acetylation [28,32]. It remains to be determined at which stage of xanthan biosynthesis the modifications are elaborated; it might be either at the cytoplasmic membrane or in the periplasmic space [32]. To this end, it was shown that GumK, a glucuronic acid transferase, is active on the lipid-linked trisaccharide precursor α-Man-(1→3)-β-Glc-(1→4)-β-Glc-P-P-polyisoprenyl, and shows reduced activity on the acetylated precursor substrate 6-*O*-acetyl-α-Man-(1→3)-β-Glc-(1→4)-β-Glc-PP-polyisoprenyl [33]. This suggests that mannose acetylation occurs after the completion of the trisaccharide side chain [32,33]; this might also hold true for pyruvylation. The xanthan biosynthetic enzymes seem to be highly conserved among different organisms, except for the mannose-transferase GumI and the pyruvyltransferase GumL, for which no homologues are found in other organisms [34].

Interestingly, *Bacillus* sp. strain GL1, which utilizes xanthan for its growth, produces an extracellular xanthan lyase, which catalyses the cleavage of the glycosidic bond between 4,6Pyr-β-d-Man and β-d-GlcA residues in xanthan side chains and, thus, contributes to depolymerisation of xanthan [35,36].

#### 3.1.2. Succinoglycan

Succinoglycan is a pyruvylated EPS that is produced by *Agrobacterium* [37,38], *Alcaligenes*, *Pseudomonas* [39], and *Rhizobium* strains [40], and is of great importance in plant symbiosis.

It is a heteropolymer that is multiply decorated with pyruvate, succinate, and acetate substituents. While the extent of acetylation and succinylation depends on the strain and the cultivation conditions, pyruvate is always found in a stoichiometric manner at the terminal β-Glc residue [32]. The repeat

unit structure of succinoglycan is composed of β-Glc and β-Gal in a molar ratio of 7:1. Nineteen genes are involved in the polymer's biosynthesis, which are referred to as *exo* genes and encoded in a 16 kb gene cluster. The biosynthesis starts with the production of the nucleotide-activated sugars UDP-Glc and UDP-Gal, where ExoC (phosphoglucomutase), ExoB (UDP-glucose-4-epimerase), and ExoN (UDP-pyrophosphorylase) are involved [32,41,42] (Figure 2). The initial step in the biosynthesis is executed by ExoY, a priming galactosyltransferase transferring a single, reducing-end Gal residue onto an undecaprenylphosphate (undp-P) carrier. *ExoA*,*exoL*,*exoM*,*exoO*,*exoU*, and *exoW* encode subsequent glycosyltransferases, which complete the octa-saccharide repeat in a step-wise manner, with each enzyme transferring a single monosaccharide, each, except for ExoW which transfers the subterminal and terminal glucoses. Prior to the export of the octasaccharide via a Wzx-dependent pathway, pyruvylation (at the terminal, non- reducing-end glucose), acetylation, and succinylation reactions catalysed by ExoV, ExoZ, and ExoH, handed over to ExoQ, which is responsible for polymerization of the fully modified repeats [43]. Studies on ExoV, the pyruvyltransferase of *Shinorhizobium* (previously *Rhizobium*) *meliloti*, suggest that pyruvylation is important for polymerization of repeating units and efficient succinoglycan export [44].

**Figure 2.** Scheme of succinoglycan biosynthesis in *Shinorhizobium meliloti* [43]. The pyruvylation step occurs in the cytoplasm at the stage of the undp-PP-linked RU prior to export and polymerization in the periplasmic space. Pyruvylation (ExoV) is indicated by a star. The order of pyruvylation, acetylation (ExoZ), and succinylation (Exo) is unknown. RU: repeating unit. Monosaccharide symbols are shown according to the Symbol Nomenclature for Glycans (SNFG) [45].

For *Rhizobium leguminosarum*, it was shown that missing pyruvylation on the terminal glucose residue of the succinoglycan impairs the formation of the nitrogen-fixing symbiosis with *Pisum sativum*, supportive of a signalling role of pyruvylation in this process [46]. PssK was identified as the pyruvyltransferase involved in succinoglycan modification of *R. leguminosarum* [46].

#### 3.1.3. Salecan

The salt-tolerant soil bacterium *Agrobacterium* sp. ZX09 is the producer of salecan, a soluble, succinylated, and pyruvylated EPS with a β-(1→3) glucan structure that is of interest because of its multiple bioactivities and unusual rheological properties. Its basic repeating unit structure was initially elucidated as →3)-β-d-Glc*p*-(1→3)-[β-d-Glc*p*-(1→3)-β-d-Glc*p*-(1→3)]-α-d-Glc*p*- (1→3)-α-d-Glc*p*-(1→ [47]. On the basis of amino acid homology with the respective *exo* genes, it can

be concluded that succinyl- and pyruvyl-groups are conferred to salecan upon catalysis of SleA (succinyl-transferase) and SleV (pyruvyltransferase), respectively, both of which are located in a 19.6-kb gene cluster [48]. The exact positions of the salecan modifications remain to be determined.

#### 3.1.4. Colonic Acid

Colonic acid (CA) or M-antigen is another class of pyruvylated EPS mostly found in *Enterobacteriaceae*, including the majority of *Escherichia coli* strains. CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. CA is composed of hexasaccharide repeat units consisting of glucose, two fucoses, two galactoses, and glucuronic acid [49]. Additionally, acetylation is found on fucose or/and galactose, while pyruvylation is found on the terminal galactose only, with both modifications occurring non-stoichiometrically [32,50]. The overall structure of CA is 4,6Pyr-α-Gal*p*-(1→4)-β-Glc*p*A-(1→3)-*O*Ac-α-Gal*p*-(1→3)-α-Fuc*p*-(1→4)-*O*Ac-*O*Ac-α-Fuc*p*-(1→3)-β-Glc-(1→7)-α-Hep*p*-(1→6)-α-Glc*p*-(1→2)-α-Glc*p*-(1→3)-[α-Gal*p*-(1→6]-α-Glc*p*-(1→3)-[α-Hep*pP*-(1→7)] -α-Hep*pP*-(1→3)-[PEtN]α-Hep*p*-(1→5)-αKdo*p*-(1→, where Kdo is 3-deoxy-d-*manno*-oct-2-ulosonic acid.

The genetic determinants for CA biosynthesis reside in a 19-gene *wca* (*cps*) cluster and are tightly regulated by a complex signal transduction cascade [51]. The gene cluster encodes six glycosyl-transferases, named WcaJ, WcaI, WcaE, WcaC, WcaL, and WcaA. Furthermore, a putative pyruvyl-transferase (WcaK) is encoded next to two predicted acetyltransferases (WcaF and WcaB), although there are up to three acetylation positions described in CA [50]. Interestingly, WcaF seems to contribute to biofilm formation of the bacterium, since knocking out of this enzyme led to biofilm disruption under *in vitro* conditions [52].

#### 3.1.5. Unclassified Pyruvylated EPS

Up to now, there are several unclassified types of pyruvylated EPSs. The repeating unit structure of the acidic EPS produced by a mucoid strain of *Burkholderia cepacia* isolated from a cystic fibrosis patient was established as →3)-β-d-Gal*p*-(1→3)-4,6Pyr-α-Gal*p*-(1→ [53].

The freshwater biofilm isolate *Pseudomonas* strain 1.15 produces considerable amounts of an acidic EPS that is composed of repeating units with the structure →4)-[4,6Pyr-α-d-Gal*p*-(1→4)-β-d-GlcA*p*- (1→3)-α-d-Gal*p*-(*O*→3]-α-l-Fuc*p*-(1→4)-α-l-Fuc*p*-(1→3)-β-d-Glc*p*-(1→. Furthermore, of the four different *O*-acetyl groups present in non-stoichiometric amounts, two were established to be on O-2 of the 3-linked galactose and on O-2 of the 4-linked fucose [54].

*Enterobacter amnigenus*, a bacterium isolated from sugar beets, produces an EPS that is rich in l-Fuc and has a terminal, pyruvylated α-d-Man residue [55].

The cystic fibrosis lung pathogen *Inquilinus limosus* produces two EPSs with unique structures—an α-(1→2)-linked mannan and a β-(1→3)-linked glucan—both fully substituted with 4,6-linked pyruvate ketals [56,57]. Cystic fibrosis is an autosomal recessive disorder and its mortality is due to chronic microbial colonisation of the major airways that leads to exacerbation of pulmonary infection [58]. While *Pseudomonas aeruginosa* is one of the most threatening microbes colonizing cystic fibrosis lungs, the EPS of *I*. *limosus* is suspected to play a role in the pathogenesis of the disease.

The bacterium *Azorhizobium caulinodans* produces a linear homopolysaccharide-type EPS composed of α-(1→3)-linked 4,6Pyr-d-Gal residues. The bacterium undergoes a symbiotic interaction with *Sesbania rostrata* as a legume host plant, which results in the development of root nodules, accompanied by a massive production of H2O2. In situ H2O2 localization demonstrated that increased EPS production during early stages of invasion prevents the incorporation of H2O2 inside the bacteria, suggesting a role for EPS in protecting the microsymbiont against H2O2 [59].

A special K-antigen-like EPS is found in the marine bacterium *Cobetia marina* DSMZ 4741, with its repeating unit composed of ribose and pyruvylated Kdo [60].

Within the EPS structure of the lactic acid bacterium *Pediococcus pentosaceus* LP28, a pyruvate modification was described to occur on one of the four constituting monosaccharides (Glc, Gal, Man, and GlcNAc) [61]. The EPS biosynthetic gene cluster consists of 12 ORFs containing a priming enzyme, five glycosyltransferases, and a putative polysaccharide: pyruvyltransferase [61].

EPSs produced by an *Erwinia* spp. in association with the bacterium *Coniothyrium zuluense* are linked to a fungal canker disease of *Eucalyptus* [62]. One of these EPSs is that of *Erwinia stewartii*; another is that of *Erwinia futululu*, whose structures are identical except for the replacement of one terminal Glc residue by 4,6Pyr-Gal*p* in the latter, yielding →3)-β-d-Gal*p*-(1→3)[4,6Pyr-α-d-Gal*p*-(1→4) β-d-Glc*p*A-(1→4)][β-d-Glc*p*-(1→6)]-α-d-Gal*p*-(1→6)-β-d-Glcp-(1→ [63].

*Agrobacterium radiobacter* (ATCC 53271) produces an anionic EPS that gives aqueous dispersions, exhibiting high viscosity at low concentrations. The *A. radiobacter* EPS is composed of a complex heptadekasaccharide repeating unit, which exposes a subterminal 4,6Pyr-α-d-Glc residue on each of the two identical tetraglycosyl branches [64].

*Methylobacterium* sp. is a slime-forming bacterium isolated from a Finnish paper machine, which is a high EPS producer. Its EPS repeating unit has the structure →3)-4,6Pyr-α-d-Gal*p*-(1→3)-4,6Pyr-α-d-Gal*p*-(1→3)-α-d-Gal*p*-(1→ [65].

The marine bacterium *Alteromonas macleodii* subsp. *fijiensis* isolated from deep-sea hydrothermal vents displays a pyruvylated mannose in its EPS hexasaccharide repeating unit structure →4)-β-d-Glc*p*-(1→4)[4,6Pyr-β-d-Manl*p*-(1→4)-β-d-Glc*p*A-(1→3)-α-d-Glc*p*A-(1→3)]α-d-Gal*p*A-(1→4) α-d-Gal*p*-(1→ [66]. Aside from its use in the food industry, this marine polymer has been suggested to be used for the treatment of cardiovascular diseases and bone healing [67].

#### *3.2. Capsular Polysaccharides*

Capsular polysaccharides (CPSs) are also part of the glycocalyx but, in contrast to EPSs, are covalently connected to the bacterial cell surface via membrane phospholipids [24]. Because of their prominent cellular localization, CPSs are the first interaction zone of bacteria with the host immune system, and thus are important virulence factors of many bacteria. Very often, encapsulated bacteria are pathogenic, whereas capsule-deficient isolates are not [68]. Hence, CPSs are frequently used for the production of polysaccharide conjugate vaccines [69].

Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of CPSs with different structures, and these aid in distinguishing isolates by serotyping [68]. The widespread occurrence and the high structural differences of CPSs are reflected by 84 capsular serotypes (K-antigens) found alone in *E. coli* strains. Essentially, there are four groups of capsules [70]. Group I- and IV-CPS, which are often found in organisms leading to gastrointestinal diseases, use the Wzx/Wzy-dependent export pathway and their biosynthesis proceeds on a polyprenol linker. Capsules from groups II and III use the ABC-transporter export pathway and are frequently present in mucosal pathogens such as *Neisseria meningitidis*. Interestingly, a CPS attached via a novel β-linked poly-3-deoxy-d-*manno*-oct-2-ulosonic acid linker to the phospholipid *lyso*-phosphatidylglycerol is present, which in earlier studies was described as a diacylglycerol because of hydrolysis experiments [68].

#### 3.2.1. *Streptococcus pneumoniae* CPS

*Streptococcus pneumoniae* (pneumococcus) is a leading cause of bacterial-induced pneumonia, meningitis, and bacteraemia globally [71]. Prevnar 13, the most broadly protective pneumococcal conjugate vaccine, is composed of 13 protein-polysaccharide conjugates consisting of pneumococcal CPS serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F—each individually linked to the genetically inactivated diphtheria toxoid CRM197. Currently, approaches on the basis of biocon-jugation and glycosylation engineering are being pursued as manufacturing alternatives to enable the production of vaccines with higher protection rates, especially in children [69,72,73].

*S. pneumonia* CPS serotype 4 (ST4) is a prevalent serotype in vaccine formulations, containing 2,3-pyruvate ketal on the Gal residue of its repeating units—with the structure

→3)-β-d-Man*p*NAc-β-(1→3)-α-l-Fuc*p*NAc-(1→3)-Gal*p*NAc-α-(1→4)-2,3Pyr-α-d-Gal-(1→—which has been shown to be the key component of its specific immunogenic motif [74].

Thus, the pyruvate modification is essential for designing minimal synthetic carbohydrate vaccines for ST4, as vaccine formulations without pyruvylation would not recognize the natural CPS [8]. It is, therefore, highly recommended to include the pyruvate ketal epitope in glycoconjugate vaccines [16].

#### 3.2.2. *Acinetobacter baumannii* CPS

A clinically relevant producer of CPS is the opportunistic pathogen *Acinetobacter baumannii*, which triggers infections in immunocompromised patients causing severe nosocomial, bloodstream, pneumonia, urinary tract infections, and septicaemia [75]. Its clinical importance is related to its low susceptibility towards most of the antibiotics commonly used [76].

There are seven different capsule loci—KL1, KL2, KL4 KL6, KL7, KL8, KL9—in *A. baumannii* genomes. Five of these were found in clonal group 2, whereas two were found in clonal group 1, indicating that isolates with developing antibiotic resistance have a lot of variations of these loci [77]. The K4 CPS of isolate D78, which is a multiple antibiotic resistant strain, contains the KL4 cluster. The KL4 CPS backbone repeating structure is composed of a trisaccharide of α-*N*-acetyl-d-quinovosamine (d-Qui*p*NAc), α-*N*-acetyl-d-galactosamine uronic acid (α-d-Gal*p*NAcA), and α-d-Gal*p*NAc, which contains a branching 4,6-pyruvylated GalNAc residue. The trisaccharide structure was elucidated as →4)[4,6Pyr-α-d-Gal*p*NAc-(1→6)]-α-d-Gal*p*NAc-(1→4)-α-d-Gal*p*NAcA-(1→3)-α-d-Qui*p*NAc-(1→. The pyruvate ketal is predicted to be transferred by the putative pyruvyltransferase PtrA, however, without biochemical evidence [78].

#### 3.2.3. *Klebsiella* CPSs

Pyrogenic liver abscess-causing *Klebsiella pneumoniae* produces a CPS, which is composed of trisaccharide repeating units with the structure →4)-β-d-Glc-(1→4)-2,3(*S*)Pyr-β-d-GlcA-(1→4)-β-l-Fuc-(1→, in which each glucuronic acid residue is pyruvylated and additional acetylation of the fucose residue occurs at the C2-OH or C3-OH [79]. The CPS induces secretion of tumour necrosis factor and interleukin-6 by macrophages through the Toll-like receptor 4 dependent pathway, which is abandoned when pyruvylation is missing in the trisaccharide. This finding indicates that pyruvylation on glycoconjugates may be relevant for immune system stimulation [80]. Previously, the recognition of pyruvylated CPS from *K. pneumoniae* by IgM antibodies has been described [81].

The structures of several other pyruvylated *Klebsiella* CPS structures have been elucidated, however, without any functional information.

The structure of the CPS from *Klebsiella* serotype K14 was the first report on the rare case of a *Klebsiella* polysaccharide to contain a Gal*f* residue. The repeating hexasaccharide structure was shown to terminate with a glucose residue carrying a 4,6Pyr modification—→4)-β-d-Glc*p*A-(1→3) β-d-Gal*f*-(1→3)-β-d-Glc*p*-(1→4)[4,6Pyr-β-d-Glc*p*-(1→2)][α-l-Rha(1→3)]β-d-Man*p*-(1→ [82].

Also in the doubly pyruvylated CPS of *Klebsiella* K12, Gal*f* residues are found; its repeating unit has the structure 5,6Pyr-β-d-Gal*f*-(1→4)-β-d-Glc*p*A-(1→3)-β-d-Gal*f*-(1→6)-β-d-Glc*p*-(1→3)-α-l-Rha- (1→3)-α-d-Gal*p*-(1→2)[5,6Pyr-β-d-Gal*f*-(1→4)-β-d-Glc*p*A-(1→3)]-β-d-Gal*f*-(1→6)-β-d-Glc*p*-(1→3)-α-l -Rha-(1→3)-α-d-Gal*p* [83].

The structure of the CPS from *Klebsiella* serotype K70 is composed of linear hexasaccharide repeating units that contain a Pyr group attached to a (1→2)-linked α-l-Rha residue in every second repeating unit. The full structure of the*Klebsiella* K70 CPS is→4)-β-d-Glc*p*A-(1→4)-α-l-Rha*p*-(1→2)-α-l-Rha*p*-(1→2)-α-d-Glc*p*-(1→3)-β-d-Gal*p*-(1→2)-3,4Pyr-α-l-Rhap(1→ [84].

The *Klebsiella* serotype K64 CPS consists of hexasaccharide repeating units, composed of a →4)-α-d-Glc*p*A-(1→3)-α-d-Man*p*-(1→3)-β-d-Gal*p*-(1→4)-α-d-Man*p*-(1→ backbone with a 4,6Pyr-β-d-Gal*p* and a l-Rha residue attached to the (1→4)-linked α-d-Man*p* residue at O-2 and O-3, respectively; the repeating unit further contains one *O*-acetyl substituent [85].

The structure of the CPS from *Klebsiella* type K46 consists of a hexasaccharide repeating unit, which is unique in having a 4,6Pyr residue on a lateral, but non-terminal sugar residue—[β-d-Glc*p*- (1→3)-4,6Pyr-β-d-Man*p*-(1→4)]→3)-α-d-Glc*p*A-(1→3)-α-d-Man*p*-(1→3)-α-d-Gal*p*-(1→ [86].

The *Klebsiella* K33 CPS revealed to be a tetrasaccharide alditol with the structure β-d-Glc*p*-(1→4)[3,4Pyr-β-d-Gal*p*-(1→6)]-β-d-Man*p*-(1→2)-Ery-ol, where Ery-ol is erythritol [87].

Interestingly, there are two human monoclonal macroglobulins, IgMWEA and IgMMAY [88], which show specificity for *Klebsiella* polysaccharides containing 3,4-(K30, K33) and 4,6-(K21, K11) pyruvylated d-Gal in a pH-dependent manner and with differences in co-precipitation in dependence of the number of the CPS repeating units (i.e., CPS length) [81]. Of note, agar, which has an internal 4,6Pyr-Gal residue in its repeating unit, cross-reacts with IgMWEA [81].

*Klebsiella rhinoscleromatis* is a heavily capsulated bacterium that possesses a K3-type capsule. The repeating unit of K3 is a pentasaccharide with the structure →2)-4,6-*S-*Pyr-α-d-Man-(1→4)-α-d-GalA-(1→3)-α-d-Man-(1→2)-α-d-Man-(1→3)-β-d-Gal-(1→ [89]. The *Klebsiella* K3 capsule has been shown to be one of the few *Klebsiella* K types that are able to bind to the eukaryotic mannose receptor [90].

### 3.2.4. *Bacteroides fragilis* CPS A

*Bacteroides fragilis* is an opportunistic anaerobe, most frequently isolated from intra-abdominal abscesses [7,91–93]. Its most prominent CPS—CPS A—is composed of tetrasaccharide repeating units with the structure →4)-α-d-2-*N*-acetylamido-4-amino-galactopyranose (AAdGal*p*)-(1→3)-4,6PyrGal*p*-(1→3)-[β-d-Gal*f*-(1→3)]α-d-Gal*p*NAc-(1→) [94]. CPS A has been shown to have a tremendous effect on the immune system of a mammalian host and to be internalized by antigen-presenting cells [7]. Upon genetic deletion of CPS A, the abscess-inducing capability of the bacterium was drastically reduced [93]. CPS A from *B. fragilis* caught recent interest as a carbohydrate antigen to be used in vaccine formulations instead of conventional cationic proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KHL) [92].

CPS A biosynthesis is encoded by a single ~10.7-kb gene locus on the *B. fragilis* genome [93], and predictably employs a Wzx/Wzy-dependent pathway, on the basis of genomic evidence (Figure 3).

The gene locus encodes four transferases (WcfN, WcfP, WcfQ, and WcfS), where WcfS is responsible for the transfer of the AAdGal*p* residue from its nucleotide activator to an undp-P-lipid carrier as the first step in the synthesis of the CPS A repeating unit, and WcfR is responsible for the prior transfer of the amino group on the AdGal*p* residue to yield AAdGal*p*, which is crucial for virulence. A recent *in vitro* study of the individual enzymatic steps involved in the repeating unit biosynthesis of CPS A yielded first insight into the sugar pyruvylation reaction, with phosphoenolpyruvate (PEP) serving as a donor substrate. There is evidence that pyruvylation occurs on the undp-PP-linked disaccharide repeat unit precursor prior to tetrasaccharide repeat completion, export, and polymerization by a Wzx/Wzy-dependent system [7]. The pyruvyltransferase WcfO from the CPS A biosynthesis of *B. fragilis* [7] is one of the few biochemically characterized enzyme ketal-pyruvyltransferases (for details, see Section 5.1.2).

**Figure 3.** Scheme of capsular polysaccharide (CPS) A biosynthesis in *Bacteroides fragilis*. The pyruvylation step occurs in the cytoplasm at the stage of the lipid-PP-linked RU precursor. Pyruvylation (WcfO) is indicated by a star. Notably, in contrast to succinoglycan biosynthesis (Figure 2), pyruvylation of the internal Gal*p* of the RU needs to proceed prior to completion of the lipid-PP-linked tetrasaccharide repeat. RU: repeating unit. Monosaccharide symbols are shown according to the Symbol Nomenclature for Glycans (SNFG) [45].

#### 3.2.5. *Rhodococcus equi* CPS

The bacterial horse pathogen *Rhodococcus equi* elaborates a serotype-specific CPS that functions as a potential virulence factor [94]. This CPS is a high-molecular-weight acidic polymer composed of d-Glc, d-Man, pyruvate, and 5-amino-3,5-dideoxynonulosonic acid (rhodaminic acid, Rho) in the molar ratio of 2:1:1:1. Structural analysis revealed that the CPS consists of linear pyruvylated tetra-saccharide repeats with the structure →3)-β-d-Man*p*-(1→4)-β-d-Glc*p*-(1→4)-α-d-Glc*p*-(1→4)-7,9Pyr-α-RhoAmAc-(2→ [95].


Some cow groups contained both heifers and cows; \* Seroprevalence category defined as: Low, <20%; Moderate, 20–80%; and High, >80% seropositive.

The frequency of management groups likely to have experienced an outbreak of BVDV infection (i.e., >30% of AGID test results were ≥3; Table 2) varied between years. In 2009, approximately 3 in 10 and 2 in 10 heifer and cow groups, respectively (Table 2), were likely to have experienced an outbreak of BVDV infection. However, in 2011 only 1 in 10 and 1 in 20 heifer and cow groups, respectively (Table 2), were likely to have experienced an outbreak of BVDV infection. Outbreaks of BVDV infection in groups of heifers are relatively common due to the recognised generally lower age-specific seroprevalence [6]. A serological study of groups of mating age heifers and first-lactation cows in 12 herds in the Northern Territory found that in nearly half of the herds most of the cattle were likely to be susceptible to infection [8]. Further, these estimates of the prevalence of outbreaks of BVDV infection are similar to those derived from collation of laboratory investigations conducted in Queensland and the Northern Territory [9]. Differences between years in the prevalence of outbreaks of infection are likely to reflect the cyclical changes in proportion of naïve and PI cattle in management groups, strongly influenced by herd culling and heifer replacement practices.

Overall, there is no evidence that the prevalence of infection in beef cattle in northern Australia, either at the herd or animal level, has changed significantly between 1967 and 2009 to 2011.

**Table 2.** Distribution of management groups of unvaccinated heifers and cows by prevalence of recent BVDV infection [1].


**Performance-Based Design in Structural Fire Engineering**

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**2. Understanding the process of NETs formation**

The neutrophil extracellular traps (NETs) structures were described as another type of neutrophil cell death, different from apoptosis and necrosis. The research field on NETs has steadily been growing since 2004, when Brinkmann et al. reported for the first time this new function of activated neutrophils, demonstrating, by electron microscopy, that, when neutrophils are in the presence of bacteria, fungi, protozoa, or viruses, they acquire the capacity to form fibrillary structures, resembling nets or webs. These structures are composed mainly of nuclear material, chromatin fibers with diameters of 15–17 nm containing DNA decorated with neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G, proteinase 3 (PR3), high-mobility group protein B1 (HMGB-1), tryptase or antimicrobial peptide LL37, histones, and cytoplasmic proteins such as histones H1, H2A, H2B, H3, H4, G, lactoferrin, and gelatinase, among others [7].

Two mechanisms for the formation of NETs have been described: the suicide or lytic and vital NETosis [8]. In the first case, NETs release results from the activation of PMN by IL-8 or chemical compounds, such as phorbol myristate acetate (PMA). PMA activates neutrophils through the protein kinase C (PKC) and follows the Raf-MEK-ERK mitogen-activated protein kinase signaling pathway; the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase induces the translocation of elastase from the cytosolic granules to the inner nucleus, helping the rupture of the chromatin through histones. Induction of NETs with PMA by this mechanism can be observed from 30 min post-activation and, by 6–8 h postactivation, a high number of extracellular traps (ETs) are well formed (**Figure 1**).

In contrast, vital NETosis has been demonstrated following pathogen recognition by host pattern recognition receptors (PRRs). Gram-negative bacteria products, such as lipopolysaccharide Beneficial and Deleterious Effects of Neutrophil Extracellular Traps on Infection http://dx.doi.org/10.5772/intechopen.68634 29

phagocytic capacity and, more recently, on their role as neutrophil extracellular traps (NETs)

When neutrophils fail to kill invading pathogens by the classical phagocytosis mechanism, PMNs can accomplish this function by neutrophil extracellular traps (NETs), a process reported as a novel form of cell death called NETosis, which is dependent of the generation of reactive oxygen species [2–5]. Neutrophils forming NETs have been demonstrated by activating neutrophils with phorbol myristate acetate (PMA), interleukin 8 (IL-8), lipopolysaccharide (LPS), or under contact of neutrophils with Gram-negative and Gram-positive bacteria. NETosis induction has also been described for viral infections, and some of the signaling pathways involved have been analyzed, finding the involvement of pathogen-associated molecular patterns (PAMPs), TLR-4, TLR-7, and TLR-8. Rodríguez-Espinosa et al. have shown that NETs formation takes place in two separate metabolic steps: the first one involves chromatin decondensation, which is independent of external glucose and glycolysis, whereas the second, which involves the chromatin release, is a process that is dependent on external glu-

The neutrophil extracellular traps (NETs) structures were described as another type of neutrophil cell death, different from apoptosis and necrosis. The research field on NETs has steadily been growing since 2004, when Brinkmann et al. reported for the first time this new function of activated neutrophils, demonstrating, by electron microscopy, that, when neutrophils are in the presence of bacteria, fungi, protozoa, or viruses, they acquire the capacity to form fibrillary structures, resembling nets or webs. These structures are composed mainly of nuclear material, chromatin fibers with diameters of 15–17 nm containing DNA decorated with neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G, proteinase 3 (PR3), high-mobility group protein B1 (HMGB-1), tryptase or antimicrobial peptide LL37, histones, and cytoplasmic proteins such as histones H1, H2A, H2B, H3, H4, G, lactoferrin, and gelatinase, among

Two mechanisms for the formation of NETs have been described: the suicide or lytic and vital NETosis [8]. In the first case, NETs release results from the activation of PMN by IL-8 or chemical compounds, such as phorbol myristate acetate (PMA). PMA activates neutrophils through the protein kinase C (PKC) and follows the Raf-MEK-ERK mitogen-activated protein kinase signaling pathway; the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase induces the translocation of elastase from the cytosolic granules to the inner nucleus, helping the rupture of the chromatin through histones. Induction of NETs with PMA by this mechanism can be observed from 30 min post-activation and, by 6–8 h post-

In contrast, vital NETosis has been demonstrated following pathogen recognition by host pattern recognition receptors (PRRs). Gram-negative bacteria products, such as lipopolysaccharide

activation, a high number of extracellular traps (ETs) are well formed (**Figure 1**).

forming cells, in innate and adaptive immunity.

28 Role of Neutrophils in Disease Pathogenesis

**2. Understanding the process of NETs formation**

cose and glycolysis [6].

others [7].

**Figure 1.** Human peripheral blood neutrophils non-activated and activated with PMA (100 ng/ml) for different lengths of time. Neutrophil extracellular traps formation starts by 30 min post-activation; extracellular traps are more extended by 6 h post-activation (photographs taken by Moreno-Altamirano).

(LPS), activate neutrophils, by the ligation of TLRs (TLR-4 in the case of LPS), inducing the liberation of NETs. In the case of Gram-positive bacteria, the complement receptor 3 (CR3) and TLR-2 are required to induce vital NETosis; platelets are also inducers of vital NETosis, through CD11a. This mechanism maintains the external membrane integrity and thus the function of neutrophils, until cells are devoid of nucleus [7, 8].

A third mechanism for the induction of NETs, recently reported, is through autophagy [9, 10]. It is worth mentioning that neutrophils are not the only cells that form extracellular traps (ETs), and other immune cells, such as mast cells, eosinophils, and macrophages, can also release ETs. Although the molecular principles underlying the formation of ETs by mast cells [11], eosinophils [12], and monocytes/macrophages [13] are similar to those observed in neutrophils, there are some notable disparities. The most remarkable mechanism of ET formation has been described in eosinophils. In these cells, ETs are formed by both nuclear and mitochondrial DNAs, in a reactive oxygen species (ROS)-dependent manner.

Neutrophil extracellular traps are able to capture microorganisms trap microorganisms, killing them or not, this much depends on the type of pathogen involved. NETs are produced by the neutrophils of mice, humans, and some other animals, and can be induced by chemical compounds, bacteria, fungi, protozoa, and viruses. The role of NETs in viral infections is not yet clear. However, some viruses induce the release of NETs [14, 15].

While some viruses are immobilized and inactivated by NETs, others such as HIV induce the production of an IL-10-like protein that inhibits the formation of NETs [15], and dengue virus inhibits PMA-induced formation of NETs. Interestingly, neutrophils seem to be arrested at the chromatin decondensation step, failing to liberate NETs, thus suggesting a metabolicrelated mechanism of NETs inhibition [16].

Controversy surrounding neutrophil extracellular traps as a host defense mechanism makes it necessary to analyze how NETs limit the growth of various infectious agents, whereas, apparently, they have no effect on others. On the other hand, how NETs may cause damage and autoimmune diseases also needs to be investigated.