Involvement of PhoP-PhoS homologs in Enterococcus faecalis virulence

Abstract

Eleven PhoP-PhoS homolog pairs were identified by searching the Enterococcus faecalis V583 genome sequence database at The Institute for Genomic Research with the Bacillus subtilis PhoP-PhoS sequences. Each pair appears to be a potential two-component system composed of a response regulator and a sensor kinase. Seven of the homologs were disrupted in E. faecalis strain OG1RF. TX10293, a mutant disrupted in one of these genes (etaR, the first gene of the gene pair designated etaRS), showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. The predicted EtaR protein sequence showed greatest similarity to LisR of Listeria monocytogenes (77%) and CsrR of Streptococcus pyogenes (70%); EtaS is 53% similar to LisK and 54% similar to CsrS. When grown in vitro, the TX10293 mutant was more sensitive to low pH (pH 3.4) and more resistant to high temperature (55 degrees C) than wild-type OG1RF. In conclusion, many potential two-component systems are identified for E. faecalis, one of which, EtaRS, was shown to be involved in stress response and virulence.

FIG. 1.

Percentages of surviving mice injected with OG1RF, TX10293, or TX37200 over time. (A) The inoculum size and the number of mice used for each strain were as follows: OG1RF, 5.0 × 10⁷ CFU (six mice); TX10293, 5.0 × 10⁷ CFU (six mice); and TX37200, 8.0 × 10⁷ CFU (six mice). (B) The inoculum size and the number of mice used for each strain were as follows: OG1RF, 2.7 × 10⁷ (12 mice); TX10293, 3.0 × 10⁷ CFU (12 mice); TX37200, 3.2 × 10⁷ CFU (12 mice). The LD₅₀ values were calculated based on this experiment (see the text).

FIG. 2.

(A) Alignment of EtaR, LisR, and CsrR polypeptide sequences. (B) Alignment of EtaS, LisK, and CsrS polypeptide sequences. The Pileup program in the GCG software package (Genetics Computer Group, Madison, Wis.) was used to make the alignment, and the GeneDoc software was used for editing and shading (the bottom line shows the consensus sequence).

FIG. 2.

(A) Alignment of EtaR, LisR, and CsrR polypeptide sequences. (B) Alignment of EtaS, LisK, and CsrS polypeptide sequences. The Pileup program in the GCG software package (Genetics Computer Group, Madison, Wis.) was used to make the alignment, and the GeneDoc software was used for editing and shading (the bottom line shows the consensus sequence).

FIG. 3.

(A) Survival of OG1RF, TX10293, and TX37200 in BHI, pH 3.4, adjusted with lactic acid. (B) Survival of OG1RF, TX10293, and TX37200 in BHI at 55°C. The percent surviving 40 min from time zero is shown (see Materials and Methods for details).