**3. Pathogenesis and virulence associated with enterococci**

Virulence factors are potential traits that define the pathogenesis of most infections which involves a series of events namely, colonization, adhesion to the host's cells, tissue invading and resistance to non-specific defensive mechanisms. Researchers are encouraged to characterize the factors involved in etiology of infections caused by pathogenic ENT in immunocompromised or impaired immunity patients. Two major classes of virulent factors have been well characterized: (1) surface factors that promote colonization in host cells, and (2) protein and peptides secreted by ENT that damage the tissues [46].

## **3.1 Gelatinase (***gelE***), serine protease (***sprE***) and** *fsr* **regulator**

Gelatinase is a zinc metalloprotease expressed extracellularly and hydrolyze gelatin, collagen and casein [47]. It is proved to be a full virulence factor expressed in mouse model of peritonitis, endocarditis [48, 49], endophthalmitis [50], in nematode [51] and in vitro translocation [52]. It is encoded by *gelE* and *sprE* operon and expressed in regulation by a quorum sensing system encoded by the *fsr* locus [53]. The *fsr* locus (*E. fl* regulator) is a well characterized locus containing *fsrA*, *fsrB*, *fsrC* and *fsrD* genes which is homologs to staphylococcal *agrBCA* loci [54]. A signaling peptide in *fsrB* liberates gelatinase biosynthesis activating pheromone (GBAP) peptide by auto-processing and a quorum sensing system. *gelE* and *sprE* genes are induced when GBAP accumulates from exponential to stationary phase. *Fsr* regulon is present above the *sprE* and *gelE* and encode a serine protease and gelatinase, respectively [55]. Possible molecular mechanism behind the expression of *gelE* and *sprE* is shown in **Figure 4** [56]. Epidemiological data suggests the involvement of *fsr* locus and gelatinase in virulence traits, like adhesion capacity (biofilm) established by processing of C-terminal gelatinase protein [57, 58].

#### **3.2 Catalase (EC 1.11.1.6)**

Catalase is a renowned enzyme present in all three domains of life. It catalyzes the decomposition of hydrogen peroxide (HP) to water and oxygen, protecting the cell from oxidative damage of HP. HP is a reactive oxygen species (ROS) in

**Figure 4.**

*Flow diagram showing the possible mechanism of* gelE *and* sprE *gene expression.*

biosphere. It is produced as a by-product in aerobic metabolism such as in oxygen activation, in photosynthetic and respiratory electron transport chain and as product of oxidases activity. First step of catalase reaction is the reduction of HP to water forming cationic heam radical and an oxoiron [compound 1 (FeIV═O ion)]. In the second step, dismutation is completed by the reaction of a second HP, resulting in the release of oxygen and water. The enzyme is regenerated in the resting FeIII state. NADPH binding catalases prevent the build-up of an inactive partially oxidized dead-end form of the enzyme called compound II [59].

Catalases are of three types: Prokaryotic Mn-catalases (minor bacterial protein family), bifunctional catalase peroxidases (not found in plants and animals and exhibit both catalytic and peroxidative activities) and haem catalases (most abundant group found in Archaebacteria, Eubacteria, Fungi, Protista, Animalia and Plantae). Despite catalyzing the same reaction (2H2O2 → 2H2O + O2), all three families differ in architecture of active site and mode of reaction [60]. Among G +ve lactic acid bacteria (LAB), *E. fl* are unable to make porphyrin compounds, including heam groups. It exhibits catalase activity but only when it is grown in heme containing medium [61]. *E. fl* catalase (*katA*) is a homo-tetrameric protein containing only one heme group (protoheme IX) and belongs to the group of heme containing mono functional catalases [62]. In the absence of heme, *E. fl* produces NADH peroxidase (*Npr*) that degrades HP to water. Factors involve in biogenesis of catalase was not known until Baureder and Hederstedt [62] carried out a research in which they used two different transposon systems to construct libraries of *E. fl* mutants and screened for clone defective in catalase activity by using colony zymogram staining procedure. They identified nine genes (in addition to *katA,* which codes for catalase enzyme protein) distributed over five chromosomal loci which are important for expression of catalase activity in *E. fl*. The proteins encoded by those genes have diverse functions such as NADH oxidation and HP detoxification (*npr*), global regulation of RNA turnover (*rnjA*, *srmB*), membrane transport (*oppBC*) and/or stress response (*etaR*) [62].

#### **3.3 Hyaluronidase (EC 4.2.2.1)**

These are the enzymes capable of degrading hyaluronate (Hyaluronic acid, hyaluronan) found in several body parts, like umbilical cord, synovial fluid, cartilage, brain, muscles and extracellular matrix (ECM) in connective tissues. Almost half of the total body hyaluronate is found in the skin. The viscous ground substance release by the connective tissues provides a barrier for the entry of bacteria or toxin into the body. However, ground substance contains hyaluronate as a major component which is degraded by hyaluronidases. Rooster's combs and certain bacteria like

**115**

access [68, 84, 85].

**3.5 Enterococcal surface protein (***esp***)**

*The Genus* Enterococcus *and Its Associated Virulent Factors*

protect from Cyt mediated bacterial cell death [83].

streptococci also produce hyaluronidases [63]. Many pathogenic bacteria release some extracellular products which helps them in damaging the tissue thus acting as a virulent factor and smoothen the progress of bacterial toxin into the tissues and are commonly named as "spreading factors." Bacterial hyaluronidases (BH) are among some of the spreading factors released by certain G +ve and G −ve bacteria. BH belongs to the third type of hyaluronidases commonly called as hyaluronate lyases. They eliminate β 1–4 linkage resulting in the production of unsaturated disaccharides by acting as endo-*N*-acetylhexosaminidases [63]. Different models of *E. fm* trans conjugant's virulence that harbors conjugative mega plasmid have been reported [64, 65] to carried *hyl* gene. According to some previous studies, the *hyl* gene was more prevalent in clinical isolates rather than community base isolates. According to a recent study, *hyl* gene is considered as a passive marker of virulence because deletion of this gene caused no effect on mouse peritonitis model [66, 67].

Enterococcal Cyt is a broad range prokaryotic and eukaryotic lysin usually plasmid encoded. It is reported to enhance virulence of *E. fl* in animal models. It was originally described as lanthionine-containing bacteriocins of G +ve bacteria [68]. The Cyt operon is a part of *E. fl* PAI consisting of 6 genes related to toxin biosynthesis and two promoters namely PL (involve in regulation of transcription of genes related to toxin structure and function) and PREG (involve in transcription of regulatory genes) and present near *esp* gene [69]. Like gelatinase, expression of Cyt is quorum sensing dependent and regulated by two component systems [70]. The regulatory system of Cyt consists of two open reading frames (ORFs) namely *cylR1* and *cylR2* which encodes a transmembrane protein of unknown function (cylR1) and a helix-turn-helix DNA binding protein (cylR2) [71]. The Cyt operon is either present on conjugative pheromone responsive plasmid such as pAD1 [72] or encoded by chromosome within 150 kb PAI [73, 74]. Todd et al. [75] conducted the first comprehensive study on hemolysin molecule after the observation of hemolysis zones on blood agar plates produced by *E. fl*. Increased virulence due to Cyt in *E. fl* was first described in the study of Ike and colleagues [76] through dose dependent intraperitoneal injections of *E. fl* strains harboring plasmid pAD1 which encodes Cyt. Later, various researchers showed the lyses of mouse erythrocytes, macrophages, and PMNs or death of experimental animals/organism like mouse, rabbits and *C. elegans* with Cyt [58, 73, 77–80]. Self-lysis of Cyt producing cells is prevented by an unknown mechanism. However, immunity proteins or ABC transporters protects other lantibiotic producing bacteria from self-lysis [81, 82]. In *E. fl*, a zinc metalloprotease and transmembrane protein, CylI (immunity factor) is shown to

Despite having a virulence face, Cyt can also act as beneficiary trait for both *E. fl* and its host. Possible beneficial activities might include, acting as colonization factor, providing self-defense against something which is more harmful (probably an intestinal parasite), facilitating nutrient acquisition from prokaryotic or eukaryotic sources, function as signaling molecule to monitor bacterial population size and probe the environment for target cells and last but not the least, bacteriocin activity of Cyt allows *E. fl* to occupy a novel host niche which non-cytolytic bacteria cannot

*Esp,* a putative virulent factor is found in both *E. fl* and *E. fm*. It is located on pathogenicity island (PAI) at the surface of the bacterium [56]. It was initially

*DOI: http://dx.doi.org/10.5772/intechopen.89083*

**3.4 Cytolysin (Cyt)**

streptococci also produce hyaluronidases [63]. Many pathogenic bacteria release some extracellular products which helps them in damaging the tissue thus acting as a virulent factor and smoothen the progress of bacterial toxin into the tissues and are commonly named as "spreading factors." Bacterial hyaluronidases (BH) are among some of the spreading factors released by certain G +ve and G −ve bacteria. BH belongs to the third type of hyaluronidases commonly called as hyaluronate lyases. They eliminate β 1–4 linkage resulting in the production of unsaturated disaccharides by acting as endo-*N*-acetylhexosaminidases [63]. Different models of *E. fm* trans conjugant's virulence that harbors conjugative mega plasmid have been reported [64, 65] to carried *hyl* gene. According to some previous studies, the *hyl* gene was more prevalent in clinical isolates rather than community base isolates. According to a recent study, *hyl* gene is considered as a passive marker of virulence because deletion of this gene caused no effect on mouse peritonitis model [66, 67].
