Fsr-Independent Production of Protease(s) May Explain the Lack of Attenuation of an Enterococcus faecalis fsr Mutant Versus a gelE - sprE Mutant in Induction of Endocarditis

Abstract

An Enterococcus faecalis gelE insertion disruption mutant (TX5128), which produces neither gelatinase (GelE) nor the cotranscribed (in the wild type) serine protease (SprE), was found to be attenuated in a rat endocarditis model with a significant decrease in the endocarditis induction rate versus wild-type E. faecalis OG1RF (GelE ؉ , SprE ؉ ). TX5266, which has a nonpolar deletion in fsrB and, like TX5128, is phenotypically GelE ؊ under usual conditions, was also studied; fsrB is a homologue of agrB of staphylococci and participates in regulation of gelE-sprE expression. Unexpectedly, TX5266 approximated wild-type OG1RF in the endocarditis model and was significantly less attenuated than TX5128. This is in contrast to other models which have found fsr mutants to be as or more attenuated than TX5128. Further study found that the fsrB mutant produced very low levels of gelatinase activity after prolonged incubation in vitro versus no gelatinase activity with TX5128 and did not show the extensive chaining characteristic of TX5128. Reverse transcription-PCR confirmed that gelE was expressed in TX5266 at a very low level versus wild-type OG1RF and was not expressed at all in TX5128. Possible explanations for the increased induction of endocarditis by TX5266 versus TX5128 include the production of low levels of protease(s) or some other effect(s) of the inactivation of the E. faecalis fsr regulator. The equivalent ability of OG1RF and its fsr mutant to initiate endocarditis may explain why we did not find naturally occurring fsr mutants, which account for ca. 35% of E. faecalis isolates, unrepresented in endocarditis versus fecal isolates (J. C. Roberts, K. V. Singh, P. C. Okhuysen, and B. E. Murray, J. Clin. Microbiol. 42:2317-2320, 2004).We have previously described the fsr locus, a regulatory system of Enterococcus faecalis and a homologue of staphylococcal agr loci, and shown that it encodes a quorum-sensing system that positively regulates the expression of gelatinase and serine protease (encoded by the cotranscribed gelE and sprE genes) via the Fsr-dependent gelE promoter (20, 21). We have also reported absence of a 23.9-kb region, as originally described by Nakayama and colleagues (16), in ca. 35% of 215 E. faecalis isolates tested; this deletion results in loss of fsrA, fsrB, and part of fsrC, resulting in an fsr-gelE locus functionally equivalent to constructed fsr mutants (24). OG1RF mutants with an insertion disruption in the gelE or fsr genes have shown significant delays in mortality in a mouse peritonitis model (21, 27), and a nonpolar fsrB deletion mutant (TX5266) (20) showed even more attenuation than a gelE disruption mutant in a Caenorhabditis elegans infection model (26) and a rabbit endophthalmitis model (6,7,14,26 Kristich et al. (11). We have also shown that Fsr mutants show decreased biofilm production (12, 31), albeit to a slightly but significantly lesser extent than the gelatinase and serine protease mutant (Gel Ϫ , Spr Ϫ ) TX5128, and both TX5128 and Fsr mutants showed markedly decreased t...

Vancomycin-Resistant Enterococcus faecalis Endocarditis: Linezolid Failure and Strain Characterization of Virulence Factors

“…In contrast, two subsequent studies did not show an increased prevalence of fsr in E. faecalis endocarditis and bloodstream isolates (11,23). In a rat endocarditis model, an E. faecalis mutant that did not produce gelatinase or serine protease had an endocarditis induction rate that was significantly reduced compared to that of wild-type E. faecalis (28). Further investigation is needed to elucidate the role of the fsr locus in the pathogenesis of E. faecalis infective endocarditis.…”

“…A quorum-sensing fsr locus has recently been described that regulates the transcription of a gelatinase gene (gelE) and a serine protease gene (sprE) and could contribute to E. faecalis virulence (22,26,28). The fsr locus regulates biofilm formation (14,20).…”

“…These included the gelatinase gene (22,26,28,30); recently described pilus-encoding genes (16); genes encoding putative MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) with predicted immunoglobulin (Ig)-like folds (17,18,27; J. Sillanpää, S. R. Nallapareddy, and B. E. Murray, unpublished data); genes, including esp (33), in a predicted pathogenicity island (PAI) (15); and an acquired gene that contributes to biofilm formation (32) ( Table 1). The strain was examined for phenotypic production of gelatinase (22), hemolytic activity on Bacto Tryptic Soy Agar (Becton Dickinson and Company, Sparks, MD) plus 5% human blood agar plates, and biofilm formation (14).…”

Influence of the fsr locus on biofilm formation by Enterococcus faecalis lacking gelE

“…Biofilm formation in E. faecalis has been reported to be influenced by various genes such as esp, initially reported by Toledo-Arana et al (2001). Gelatinase, which strongly influences virulence in models of peritonitis, endocarditis (Singh et al, 1998(Singh et al, , 2005, endophthalmitis (Engelbert et al, 2004) and in vitro translocation (Zeng et al, 2005), has been reported to influence biofilm formation (Mohamed et al, 2003(Mohamed et al, , 2004Hancock & Perego, 2004;Kristich et al, 2004). Other E. faecalis genes such as epa, atn (Mohamed et al, 2004), bop (Hufnagel et al, 2004), salA and salB (Mohamed et al, 2006) have also been shown to influence biofilms.…”

“…Most strains of E. faecalis are gelE + , although only about 60 % of them have the fsr locus and are gelatinase producers by standard assay (Roberts et al, 2004). We have shown recently that all three fsr mutants (phenotypically GelE 2 SprE 2 by standard assay; Qin et al, 2000;Singh et al, 2005), as well as gelE mutants (GelE 2 SprE 2 ), showed decreased biofilm production (Mohamed et al, 2003(Mohamed et al, , 2004, a finding confirmed by Hancock & Perego (2004); the latter also showed that the introduction of fsrABC and fsrABC/gelE/ sprE into strain FA2-2 (GelE 2 , with point mutations in the fsrB and fsrC loci) resulted in gelatinase production and increased biofilm production (Hancock & Perego, 2004). However, our initial results had also suggested that fsr may have a role, in addition to activating gelatinase-mediated biofilm formation, as the fsr mutants formed slightly more biofilm than the gelatinase/serine protease double mutant TX5128 (Mohamed et al, 2004).…”