Importance of two Enterococcus faecium loci encoding Gls-like proteins for in vitro bile salts stress response and virulence

Abstract

General stress proteins, Gls24 and GlsB, were previously shown to be involved in bile salts resistance of Enterococcus faecalis and in virulence. Here, we identified 2 gene clusters in Enterococcus faecium each encoding a homolog of Gls24 (Gls33 and Gls20; designated on the basis of their predicted sizes) and of GlsB (GlsB and GlsB1). The sequences of the gls33 and gls20 gene clusters from available genomes indicate distinct lineages, with those of hospital-associated CC17 isolates differing from non-CC17 by ∼7% and ∼3.5%, respectively. Deletion of an individual locus did not have a significant effect on virulence in a mouse peritonitis model, whereas a double-deletion mutant was highly attenuated (P<.004) versus wild-type. However, mutants lacking either gls33-glsB, gls20-glsB1, or both all exhibited increased sensitivity to bile salts. These results suggest that gls-encoded loci may be important for adaptation to the intestinal environment, in addition to being important for virulence functions.

Figure 1.

Organization of the 2 gls loci of Enterococcus faecium and their transcriptional and protein expression analysis. A, Schematic representation of these loci in E. faecium TX16 and TX1330. The annotations were predicted from bioinformatics analysis for orf1 to orf7 and orfA to orfB, which are shown in Supplementary Table 2. Predicted transcriptional terminators downstream of glsB and glsB1 are indicated with lollypops. Arrows above each of the 2 gls clusters of TX1330 indicate messenger RNA (mRNA) co-transcripts. Location of each intergenic primer pair for reverse transcriptase polymerase chain reaction (RT-PCR) is shown with arrow heads (see panel B below). B, RT-PCR analysis of the 2 gls clusters of TX1330. Top gel, RT-PCR with DNase-treated total RNA (30 ng) isolated from mid-exponential cells; middle gel, control reaction of the same RNA preparation amplified without reverse transcriptase; bottom gel, control reaction amplified with genomic TX1330 DNA. An intragenic region of the gyrase gene was used as an additional control. Numbering and lettering of the lanes corresponds to the intergenic primer pairs shown in panel A. M, molecular weight marker. C, Western blot analysis of Zwittergent whole-cell extracts from E. faecium TX1330, its 3 gls deletion mutants, and complementation derivatives. The membrane was probed with affinity-purified anti-Gls24 (E. faecalis) antibodies [26], which cross-react with both Gls33 and Gls20. Locations of bands corresponding to Gls33 and Gls20 are indicated with arrows.

Figure 2.

Comparison of three gls deletion mutants and complementation constructs versus wild-type (WT) and vector alone controls for survival after an exposure to bile salts. The mean and SD for combined results are shown from ≥5 independent experiments. Survival rates of WT versus the 3 gls deletion mutants were compared by analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons, and complementation constructs versus the vector only controls by the unpaired t test.

Figure 3.

Effect of the gls deletions on Enterococcus faecium virulence in a mouse peritonitis model. Kaplan-Meier survival curves after injecting 2 different inocula of (A) wild-type (WT) and TX6060 (TX1330Δgls33-glsB), (B) WT and TX6066 (TX1330Δgls20-glsB1), and (C) WT and TX6067 (TX1330Δgls33-glsBΔgls20-glsB1). A log-rank test was used for comparisons of survival rates. Survival rates of each single deletion mutant at 2 inocula shown above were nonsignificant versus WT, with P values .1 to .7.