**3.4 Cytolysin (Cyt)**

*Microorganisms*

**Figure 4.**

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

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

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

dead-end form of the enzyme called compound II [59].

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

(*oppBC*) and/or stress response (*etaR*) [62].

**3.3 Hyaluronidase (EC 4.2.2.1)**

**114**

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 protect from Cyt mediated bacterial cell death [83].

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 access [68, 84, 85].
