ace, Which encodes an adhesin in Enterococcus faecalis, is regulated by Ers and is involved in virulence - PubMed (original) (raw)

ace, Which encodes an adhesin in Enterococcus faecalis, is regulated by Ers and is involved in virulence

Francois Lebreton et al. Infect Immun. 2009 Jul.

Abstract

Enterococcus faecalis is an opportunistic pathogen that causes numerous infectious diseases in humans and is a major agent of nosocomial infections. In this work, we showed that the recently identified transcriptional regulator Ers (PrfA like), known to be involved in the cellular metabolism and the virulence of E. faecalis, acts as a repressor of ace, which encodes a collagen-binding protein. We characterized the promoter region of ace, and transcriptional analysis by reverse transcription-quantitative PCR and mobility shift protein-DNA binding assays revealed that Ers directly regulates the expression of ace. Transcription of ace appeared to be induced by the presence of bile salts, probably via the deregulation of ers. Moreover, with an ace deletion mutant and the complemented strain and by using an insect (Galleria mellonella) virulence model, as well as in vivo-in vitro murine macrophage models, we demonstrated for the first time that Ace can be considered a virulence factor for E. faecalis. Furthermore, animal experiments revealed that Ace is also involved in urinary tract infection by E. faecalis.

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Figures

FIG. 1.

FIG. 1.

(A) Sequence of the ace promoter region. Potential −35 and −10 region and ribosome binding site (RBS) sequences are underlined. The transcriptional start site (+1) is in boldface, and the Ers box is boxed. (B) EMSA with the promoter region of the ace gene and different concentrations (2 to 0.2 μg) of His6-ErsHTH protein. (C) EMSA of His6-ErsHTH binding to the D4-labeled DNA fragment containing the ace regulatory region. Amplifications were performed with D4-Pu and D4-Pr (Table 2). Crude cell extracts prepared from an E. faecalis Δ_ers_ mutant strain (0.1 μg protein) were added to all of the reaction mixtures. Shown are the labeled ace promoter without protein (lane 1), with His6-ErsHTH (1 μg protein) (lane 2), with His6-ErsHTH and an unlabeled competitor (lane 3), and with His6-ErsHTH and a nonspecific DNA fragment (internal fragment of the ef1843 gene) (lane 4).

FIG. 2.

FIG. 2.

Expression of ers and ace determined by Western blot or RT-qPCR analysis. (A) Western blot analysis (with antiserum against His-tagged Ers) of E. faecalis protein extracts (40 μg) from strain JH2-2 grown at 37°C (lane 2), at 46°C (lane 3), or in the presence of bile salts (lane 4) and from strain JH2-2/p3535_ers_ (lane 5). A 0.1-μg sample of purified His6-Ers protein was loaded into lane 1. (B) RT-qPCRs experiments with the ers and ace genes and primers described in Table 2. RNAs were extracted from cells cultured under the same conditions used for protein extracts. Values that are significantly different are >2 (P < 0.05).

FIG. 3.

FIG. 3.

Effect of ace inactivation on virulence. Percent survival of G. mellonella larvae at 16, 20, and 24 h after infection with L. lactis IL1403 (black bar), E. faecalis JH2-2 (gray bar), the JH2-2 Δ_ace_ mutant (hatched bar), and the complemented Δ_ace_ mutant strain (white bar). We used 6 × 106 CFU counted on an agar plate per injection. Experiments were repeated at least three times, and the results represent the mean ± standard deviation of live larvae.

FIG. 4.

FIG. 4.

Time course of intracellular survival of E. faecalis JH2-2 (squares) and the Δ_ace_ mutant (triangles) within murine peritoneal macrophages. The results shown represent the mean number ± the standard deviation of viable intracellular bacteria per 105 macrophages of three independent experiments with three wells.

FIG. 5.

FIG. 5.

Infection with 104 cells of wild-type E. faecalis JH2-2 (•) and its isogenic Δ_ace_ mutant strain (▪). Kidney pair homogenates were obtained from groups of 15 mice that were sacrificed and necropsied 48 h after a transurethral challenge. Results are expressed as log10 CFU per gram of tissue. A value of 0 was assigned to uninfected kidneys. Horizontal bars represent the geometric means.

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