The fact that O-antigen of leptospires is twelve times less endotoxic than that of E. coli has been known for some time (Isogai et al., 1986). Leptospires O-antigen resembles that of gram negative bacteria, but it does not trigger TLR4 activation as the gram negative O-antigen. Instead, TLR2 is activated, which by itself is unable to mount an effective innate immune response following an infection (Chassin et al., 2009). It has been reported that lipoproteins and S. aureus peptidoglycan favor the induction of Th2 development by dendritic cells by triggering TLR2 (Moll, 2003; Re and Strominger, 2001). Here we found that some genes of gram positive bacteria are encoded in the rfb locus of leptospires i.e. Cap8EFG from S. aureus and NeuBC from Streptococcus agalactiae (S. agalactiae) respectively. It was reported that Cap8EFG provides antiphagocytosis properties in the O-antigen (Thakker et al., 1998; Cunnion et al., 2001; Cunnion et al., 2003) and NeuBC functions as binding inhibition of the activated complement factor C3b to the surface of bacteria to prevent activation of the alternative complement pathway and inhibit complement- mediated opsonophagocytosis (Marques et al., 1992; Takahashi et al., 1999; Von Hunolstein et al., 1999; Cieslewicz et al., 2001).
Besides this, leptospires have several mechanisms to survive attack by innate immune cells including macrophages or to promote survival after ingestion by phagocytic cells. KatE detoxifies H2O2 and protects against reactive oxygen species
(ROS) including hydrogen peroxide, superoxide and hydroxyl radicals (Johnson et al., 1993; Soler-Garcia & Jersie, 2004; Wu et al., 2009; Muench et al., 2009). ClpC encodes an ATPase promoting early escape form the phagosome of macrophages (Rouquette et al., 1996; Rouquette et al., 1998; Nair et al., 2000) RecN encodes a recombinational repair protein that protects against ROS and non-oxidative killing by
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neutrophils (Stohl et al., 2006; Criss et al., 2009). Transition from the environment to the host induces the up-regulation of several stress response genes (Matsunaga et al., 2007) such as catalase, KatE (Eshghi et al., 2012) and the molecular chaperone ClpB (Lourdault et al., 2011). Besides this, there are reports that leptospires survive and replicate in macrophages and induce macrophage apoptosis (Toma et al., 2011 and Li et al., 2010). Mutation of the leptospiral Hsp90 homologue, HtpG, also resulted in attenuation of virulence, but without a concomitant increase in susceptibility to oxidative stress (King et al., 2014); the basis of these differences remains unknown (Adler, 2014).
Four locus tags with sequence homology to the MtrD protein of Neisseria gonorrhea, which encodes resistance nodulation division (RND)-type of efflux pump were found (Shafer et al, 1998; Jerse et al., 2003). It is hypothesized to function as a supporter of growth of leptospires under hostile conditions encountered in vivo. Iron is essential for the viability of most, but not all, bacterial species. The ability to acquire iron in vivo is therefore a key virulence property. Pathogenic leptospires possess a heme oxygenase, HemO, which facilitates the acquisition of iron from heme and is required for virulence (Murray et al., 2008; Murray et al., 2009b). Here we found sequence homology to two proteins that function in iron uptake and assimilation in Legionella pneumophila i.e. FeoB (a Fe2+ transporter) and CcmF, which promotes iron assimilation and intracellular infection (Robey and Cianciotto, 2002; Viswanathan et al., 2002). Mechanisms and physiological role of Mgtb are not completely clear; however, Mgtb is a unique transport system for Mg (2+) with unusual mechanisms for mediating Mg (2+) movement through the membrane (Moncrief and Maguire, 1999).
Gram-negative bacteria are equipped with five types of double-membrane-spanning secretion systems (Costa et al., 2015), but only 2 secretion systems have been reported so far in leptospires: a Type 1 secretion system (T1SS) and a type 2 secretion system (T2SS). Although the role of T2SS in protein secretion has not been demonstrated, several components of the T2SS are encoded in the leptospiral genome. Besides T1SS and T2SS, other types of secretion systems are reported to be absent from the leptospiral genome (Abby et al., 2016; Picardeau, 2017). In this study, we found that there are sequence homologies of T2SS genes from other
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bacteria in the six strains of L. interrogans. The proteins are GspD and GspE from Shigella dysenteriae and XcpS from P. aeruginosa, the latter of which is required for the translocation of a variety of toxins and enzymes across the outer membrane into extracellular fluid (Chapon-Hervé, 1997; Filloux et al.,1998).
It is a mystery how leptospires invade the host cells without having the type 3 secretion system (T3SS) which is a significant virulent factor for bacterial invasion and survival in host cells. Here we found a significant sequence homology of the CdsN ptotein that act as an ATPase for T3SS in Chlamydia trachomatis (C. trachomatis) in all six L. interrogans strains with a conserve locus tag. In C.
trachomatis and C. pneumoniae, CdsN is a functional ATPase that catalyzes
unfolding of proteins and the secretion of effector proteins through the injectisome and interacts with a putative chaperone, Cpn0706, and with CopN, the putative plug and effector protein, which is suggestive of a functional T3SS system in C. pneumoniae (Stone et al., 2008). However, proteins with sequences similar to Cpn0706 and CopN have not been found in L. interrogans.
It is known that biofilms production by leptospires facilitates their persistence in the environment. It is also postulated that biofilm biosynthesis in vivo play a major role in promoting leptospiral colonization in proximal renal tubule of carrier animals and protect them from host defends mechanisms (Treuba et al, 2004 and Ristow et. al, 2008).
From the result of protein homology of alginate biosynthesis, the inner membrane and periplasmic proteins were found to be present in all six genomes but not the transmembrane proteins. The absence of proteins particularly associated with alginate export (AlgK and AlgE) suggested there might be a different gene pertaining to these functions or there are probably unspecific transport mechanisms used for alginate export through the cell wall. Figure A13 shows a summary of protein homology associated with alginate biosynthesis, regulatory and genotypic switching in reference to that of P. aeruginosa PA01.
Genes for alginate biosynthesis in L. interrogans were scattered across chromosome 1 and were not arranged in a gene cluster as in P. aeruginosa. Some genes only resulted in very low e-values on alignment blast analysis (i.e. AlgX), while some
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genes might have different sequence homology (e.g. Alg14, AlgZ). There is also a possibility that these genes might have undergone frameshift mutations due to high passage number. All product names in locus tags were the same for each gene, suggesting that they are conserved protein. The hypotheses of alginate biosynthesis mechanism in L. interrogans is displayed in Figure A13.
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Figure A13. Hypothesis of alginate biosynthesis, regulatory and genotypic switching of L.
interrogans in reference to alginates biosynthesis proteins of P. aeruginosa PA01. Alginate biosynthesis is a mechanism for survival of leptospires in environment. Biosynthesis of Alginates is initiated by conversion of Fructose-6-phosphate to GDP Mannuronic acid in the cytoplasm catalyzed by gene products of algA, algC and algD. Further conversion i.e. polimerisation (alg8 and alg44i), acetylation (algI, algJ, algF and algX), epimerization (algG) and exportation (algK and algE) takes places. Protein homology was shown to be high (red) for proteins involved in alginate biosynthesis process in the cytoplasm (algA and algD) and inner membranes (algI). There is lack of sequence homology between L. interrogans and P. aeruginosa for alginate enzymes action at polymerization (inner membrane), epimerization (periplasmic) and exportation (outer membrane) suggesting different enzymes or mechanisms being used.
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