Environmental Adaptation and Stress Response

Environmental adaptation traits and stress resistance mechanisms have been linked to virulence, as bacterial survival in the host is often reliant on these factors. Disruption of genes encoding such traits often leads to attenuation of the organism in an animal model [32-35]. As mentioned previously, E.faecalis is a very robust organism able to survive in many harsh environments. E.faecalis V583 contains a V-type and F^-type ATPase responsible for regulating the intracellular pH and proton motive force. Cation transport ATPases can also contribute to pH homeostasis and, interestingly, a homologue of a K+-ATPase is present on the pathogenicity island. A further mechanism for pH homeostasis is the arginine deiminase pathway, which can raise the pH of the environment due to the release of ammonia and its reaction with H+ [36]. The E.faecalis V583 genome shows the presence of all the putative members of this pathway, and has two putative ornithine cadamoyltransferases (EF0105, EF0732), and ornithine cyclodeaminases (EF0118, EF0616), and contains four putative carbamate kinases (EF0106, EF0386, EF0735, EF2575). The genome also contains an MscL-like protein (EF3152), thought to act as a electromechanical switch involved in sensing the state of lipid bilayers [37]. In addition, the genome contains five cold shock proteins and four universal stress proteins. The presence of 14 predicted metal ion P-type ATPases may account for the resistance of this organism to metals and ensure cation homeostasis [38].

Present in the E.faecalis V583 genome are a number of proteins involved in heat shock response, including homologues of heat shock proteins DnaK (EF1308) and GroEL (EF2633), gsp66 and gsp67, respectively [39]. The genome also contains a homologue of CtsR (EF3283) from B.subtilis which has been shown to control molecular chaperone gene expression [40]. Unlike Staphylococcus aureus, S. pneumoniae, S. pyogenes, and L. lactis, the groR operon does not show the presence of CtsR binding sites, and this suggests that in E.faecalis the groE and dnaK operons are primarily controlled by HrcA [39]. CtsR recognition sites have been noted upstream from the clpB (EF2355), clpP (EF0771), and clpE (EF0706) genes [40]. The Clp ATP-dependent proteases have been shown in B. subtilis to play essential roles in stress survival [41]. Recent data from L. monocytogenes suggests that in that organism, clpB is required for virulence and has a role in ther-motolerance, but is not involved in other stress responses [42]. The role of these proteins has not been investigated in E. faecalis.

Also present is an HtrA (DegP) homologue (EF3027), part of the widely distributed family of serine proteases. In Streptococcus mutans, htrA mutants have a reduced ability to withstand high temperature, and are also more sensitive to low pH and H2O2 [43]. In S. pyogenes the virulence of a degP knockout was reduced in a mouse model, and was sensitive to both temperature and oxidative stress [32]. It was hypothesized that this enzyme is responsible for degrading misfolded or aggregated proteins [32]. More recent work has suggested that DegP influences the expression of at least two virulence factors in S. pyogenes [44]. In S.pneumo-

niae, HtrA is involved in growth at high temperatures, resistance to oxidative stress, and the ability to undergo genetic transformation [45].

Gls24 is a general stress protein found in E.faecalis (EF0079), and has an adjacent homologue, GlsB (EF0080), which is 71% identical at the amino acid level [46]. A further two homologues of this protein are located on the E.faecalis pathogenicity island (EF0055 and EF0117), both having 55% identity to Gls24 and 54% identity to each other. Gls24 has been shown to be induced at the onset of starvation and also when exposed to bile salt and CdCl2 stress [46]. It has been observed that expression of the gls24 gene is increased when E. faecalis is grown in serum (15-fold increase) and urine (nine-fold increase) [47]. Recently disruption of gls24 has been shown to affect both virulence and stress response; however, disruption of glsB increased bile salt sensitivity, but had no effect on virulence [48]. The actual function of Gls24 and GlsB is unknown at present. Other genes present which may have a role in bile salt resistance include SagA [49], (EF0394), a putative ABC transporter (EF0675-EF0674) which has homology to the BilE bile exclusion system in L. monocytogenes [50], and a putative bile salt hydrolase which is present on the pathogenicity island.

E. faecalis has a respiratory pathway that in the absence of hematin or fumarate produces substantial amounts of superoxide [51]. Superoxide anion has a destructive effect on a wide variety of cells and tissues, in addition to biological compounds such as lipids, proteins, and nucleic acids, and because of this, E. faecalis has a number of strategies to combat oxidative stress. Firstly, absent from the E. faecalis genome sequence are superoxide-sensitive enzymes associated with the tricarboxylic acid (TCA) cycle. The E.faecalis V583 genome sequence also shows the presence of several enzymes and gene products that contribute to oxidative stress resistance, such as NADH peroxidase (npr) (EF1211), NADH:peroxiredoxin oxidoreductase (EF2738), alkyl hydroperoxidase resistance protein (aphCF), NADH oxidase (nox) (EF1586), and manganese superoxide dismutase (EF0463), which catalyzes the reduction ofsuperoxide to oxygen and hydrogen peroxide.

In addition to these genes, V583 contains a cydABCD operon (EF2061-EF2058), ohr (organic hydroperoxidase resistance protein) (EF0453) [52], and two Dps family proteins (EF3233, EF0606), which have been implicated in the protection of DNA from oxidative stress. A second ohr/OsmC protein (EF3201) may also aid in resistance to organic hydroperoxides. The genome contains an apoenzyme of cata-lase (katA) (EF1597), which can produce catalase upon the addition of heme [53]. Extracellular O2- generated by E. faecalis has been shown to damage colonic epithelial cell DNA both in vitro and in vivo [54], and electron spin resonance data suggests that nutrient conditions in the rat intestine favor the production of O2- [55]. Previously a link has been suggested between the invasiveness of E. faecalis isolates and their rates of superoxide production [56].

Despite its ability to adapt to many different environmental stresses, E. faecalis possesses a moderate number of regulatory genes, including only three alternative sigma factors, 17 two-component systems, and one orphan response regulator. Three response regulator systems are present on mobile elements, the previously mentioned VncRS homologue (EF1863-EF1864), VanRSB (involved in regulation of the van operon) (EF2298-EF2299), and KpdDE, which is found on the pathogenicity island. Two-component signal transduction systems allow bacteria to monitor their environment and respond to a wide array of stresses [57], and recent work has investigated the role of these two-component systems in stress response in E.faecalis V583 [58]. Generation of insertional mutations in 17 of the 18 response regulators demonstrated one response regulator that is essential for cell viability (vicR) (EF1193) [58]. Mutation in the CroRS two-component system has previously been linked to the intrinsic resistance of E. faecalis to cephalosporins [59], and insertional mutation of this locus increased susceptibility to bacitracin, cefotaxime, cefuroxime, and vancomycin, but not ampicillin [58], and has also been associated with defects in growth and cell morphology [60]. These data suggest a role for CroRS in the regulation of cell wall integrity in E. faecalis. The role of two-component systems in E.faecalis JH2-2 has also been examined, and in this genetic background four systems were induced by environmental stresses [60]. In E.faecalis OG1RF, an EtaRS (EF1050-EF1051) mutant was found to be more acid-sensitive, and was attenuated in a mouse peritonitis model [61].

No r or rB-like sigma factors have been found in the genome of E.faecalis, which raises the question of how this bacterium regulates gene expression during stress responses. A regulatory protein HypR (EF2958) has recently been characterized that is involved in oxidative stress response, and disruption reduced survival of E.faecalis in an in vivo-in vitro macrophage infection model [62].

Quorum sensing is an important mechanism used by many prokaryotes to adapt to different environments encountered during infection. E. faecalis has a homologue of LuxS, which is required for the biosynthesis of the type 2 autoinducer (AI-2). AI-2 has been shown to be detected by a sensory protein in a cell-density-dependent manner, and is thought to be involved in the regulation of a wide range of bacterial physiological functions [63]. In S. pyogenes, LuxS activity has been shown to play a role in the expression of virulence factors associated with epithelial cell internalization [64]. The role of LuxS in E.faecalis is at present unknown.

Adding to its ability to survive in diverse environments, E. faecalis contains 35 probable PTS-type sugar transporters and encodes pathways for the utilization of 15 different sugars. This is comparable to L. monocytogenes, of which 4% of the genome encodes PTS and ABC transporters [28]. As mentioned previously, the TCA cycle is absent in E. faecalis and energy is derived via glycolysis or the pentose phosphate pathway. E. faecalis has a catabolite repression pathway to actively regulate energy metabolism while growing on easily fermentable sugars such as glucose [51]. The ability of E.faecalis to respond to rapidly changing environments and stresses probably contributes to its survival through all stages of infection.

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