Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence

Abstract

Three agr-like genes (fsrA, fsrB, and fsrC, for Enterococcus faecalis regulator) were found upstream of the previously reported gelatinase gene (gelE) and a downstream putative serine protease gene (sprE; accession number Z12296) of Enterococcus faecalis OG1RF. The deduced amino acid sequence of fsrA shows 26% identity and 38% similarity to Staphylococcus aureus AgrA (the response regulator of the accessory gene regulator system in the agr locus), FsrB shows 23% identity and 41% similarity to S. aureus AgrB, and FsrC shows 23% identity and 36% similarity to S. aureus AgrC (the sensor transducer of Agr system). Northern blot analysis suggested that gelE and sprE are cotranscribed and that fsrB and fsrC are also cotranscribed in OG1RF. Northern blot analysis of fsrA, fsrB, fsrC, gelE, and sprE insertion mutants showed that fsrB, fsrC, gelE, and sprE are not expressed in fsrA, fsrB, and fsrC mutants, while insertion in an open reading frame further upstream of fsrA did not effect the expression of these genes, suggesting that agr-like genes may be autoregulated and that they regulate gelE and sprE expression, as further confirmed by complementation of fsr gene mutations with a 6-kb fragment which contains all three fsr genes in the shuttle vector, pAT18. Testing of 95 other isolates of E. faecalis showed that 62% produced gelatinase (Gel(+)), while 91% (including all Gel(+) strains) hybridized to a gelE probe; 71% (including all Gel(+) strains) hybridized to an fsr probe, corroborating the conclusion that both gelE and fsr are necessary for gelatinase production. Testing of fsrA, fsrB, and sprE mutants in a mouse peritonitis model showed that sprE and agr-like gene mutants resulted in highly significantly prolonged survival compared to the parent strain OG1RF, a finding similar to what we had previously shown for a gelE mutant. These results suggest that sprE and agr-like genes contribute to the virulence of E. faecalis OG1RF in this model.

FIG. 1

Open reading frames and their transcripts in gelE flanking regions. (A) Open reading frames. The solid line represents the chromosome, and the genes and open reading frames are indicated by boxes in different shades. The orientation of the genes and open reading frames are indicated by arrows. (B) Summary of the Northern blot results using RNA from different strains and different gene probes. −, No signal in Northern blot analysis.

FIG. 2

Sequence alignment of FsrA, FsrB, and FsrC with AgrA, AgrB, and AgrC from S. aureus. Identical amino acids are indicated by a symbol as “|”, and similar amino acids are indicated by a symbol as “:” or “.” between them. Conserved residues in H, N, and G2 blocks of histidine protein kinases and conserved Asp and Lys residues in response regulators of bacterial two-component systems (20) are boxed. The sequences from S. aureus were as follows: AgrA, accession number M21854 (14); AgrB, accession number AF001782 (5); and AgrC, accession number AF001783 (5).

FIG. 3

Gelatinase and serine protease activities in fsr and sprE mutants. These pictures are the negative images of gelatin and casein zymogram gels. (A) Gelatinase activity of mutants on gelatin zymogram gel. (B) Serine protease activity of mutants on casein zymogram gel. Lanes: o1, mutant with disruption of orf1; o, OG1RF; a, mutant with disruption of fsrA; b, mutant with disruption of fsrB; c, mutant with disruption of fsrC; e, mutant with disruption of gelE; s, mutant with disruption of sprE.

FIG. 4

Northern blot analysis of fsr and sprE mutants. (A, B, C, and D) Northern blots of mutants using fsrB, fsrC, gelE, and sprE probes, respectively. Lanes: o1, mutant with disruption of orf1; o, OG1RF; a, mutant with disruption of fsrA; b, mutant with disruption of fsrB; c, mutant with disruption of fsrC; s, mutant with disruption of sprE.

FIG. 5

Kaplan-Meier survival plots of wild-type OG1RF and fsr and sprE mutants in the mouse peritonitis model. (A) Kaplan-Meier survival plots of OG1RF, the fsrA mutant (TX5240) and an fsrC revertant. (B) OG1RF and the fsrB mutant (TX5241). (C) OG1RF and the sprE mutant (TX5243). Twelve mice were tested with each inoculum of each of the strains shown. The P value refers to mutant versus OG1RF at the same or a smaller inoculum (▴ versus ▵ or ■ versus □).