Modulators of Enterococcus faecalis Cell Envelope Integrity and Antimicrobial Resistance Influence Stable Colonization of the Mammalian Gastrointestinal Tract

Abstract

The Gram-positive bacterium Enterococcus faecalis is both a colonizer of the gastrointestinal tract (GIT) and an agent of serious nosocomial infections. Although it is typically required for pathogenesis, GIT colonization by E. faecalis is poorly understood. E. faecalis tolerates high concentrations of GIT antimicrobials, like cholate and lysozyme, leading us to hypothesize that resistance to intestinal antimicrobials is essential for long-term GIT colonization. Analyses of E. faecalis mutants exhibiting defects in antimicrobial resistance revealed that IreK, a determinant of envelope integrity and antimicrobial resistance, is required for long-term GIT colonization. IreK is a member of the PASTA kinase protein family, bacterial transmembrane signaling proteins implicated in the regulation of cell wall homeostasis. Among several determinants of cholate and lysozyme resistance in E. faecalis, IreK was the only one found to be required for intestinal colonization, emphasizing the importance of this protein to enterococcal adaptation to the GIT. By studying ΔireK suppressor mutants that recovered the ability to colonize the GIT, we identified two conserved enterococcal proteins (OG1RF_11271 and OG1RF_11272) that function antagonistically to IreK and interfere with cell envelope integrity, antimicrobial resistance, and GIT colonization. Our data suggest that IreK, through its kinase activity, inhibits the actions of these proteins. IreK, OG1RF_11271, and OG1RF_11272 are found in all enterococci, suggesting that their effect on GIT colonization is universal across enterococci. Thus, we have defined conserved genes in the enterococcal core genome that influence GIT colonization through their effect on enterococcal envelope integrity and antimicrobial resistance.

Keywords: Enterococcus; IreK; antimicrobial resistance; cell envelope integrity; colonization.

FIG 1

Loss of cholate resistance alone does not cause the ΔireK colonization defect. (A) Cholate resistance was determined for wild-type (WT) E. faecalis (OG1RF) and cholate-sensitive mutant ΔireK (CK119), as well as the brp/blh (23J13) and ispA (28M17) transposon mutants. The MICs reported represent the median values from three independent biological replicates. (B) CPRG hydrolysis was measured for the strains listed in panel A. CPRG hydrolysis in cultures (representative cultures shown directly above bar for each indicated strain) was quantified by measuring the absorbance at 570 nm after the removal of bacteria by centrifugation and normalization to the absorbance at 630 nm. The reported measurements represent averages from three independent cultures, and error bars represent standard deviations. Statistical significance was evaluated by t test. ****, P < 0.0001 versus WT. (C to F) Groups of mice (5 per group) were colonized with the strains indicated in panels A and B. Bacterial loads were determined by enumerating the enterococci in feces by culture on rifampin-supplemented BHI agar. Dashed lines, the limit of detection. Symbols for mice with undetectable colonization levels were omitted, and instead, the number of mice for which colonization was not detected (ND)/total number of mice tested is shown underneath the dashed lines.

FIG 2

Loss of lysozyme resistance alone does not cause the ΔireK colonization defect. (A) Cholate resistance was determined for the WT E. faecalis strain (OG1RF), the cholate-sensitive ΔireK (CK119) and ΔcroR ΔcroS (SB6) mutants, and the sigV transposon mutant (35H2). The reported MICs represent the median values from three independent biological replicates. (B) CPRG hydrolysis was measured for the strains listed in panel A. The reported measurements represent averages from three independent cultures, and error bars represent standard deviations. Statistical significance was evaluated by t test. ****, P < 0.0001 versus WT. (C and D) Intestinal colonization of the lysozyme-sensitive mutants listed in panel A. Groups of 5 mice were colonized with either the ΔireK mutant or one of the tested lysozyme-sensitive mutants. Colonization levels were determined by enumerating the enterococci in feces by culture on rifampin-supplemented BHI agar. Dashed lines, the limit of detection.

FIG 3

The intestinal environment can select for suppressor mutants that recover envelope integrity, antimicrobial resistance, and the ability to colonize. (A) Groups of mice (5 per group) were colonized with either WT E. faecalis (OG1RF) or the ΔireK mutant (CK119). Colonization levels were determined by enumerating the enterococci in feces by culture on rifampin-supplemented BHI agar. Arrows, mice harboring ΔireK* clones; dashed line, the limit of detection. Symbols for mice with undetectable colonization levels were omitted, and instead, the number of mice for which colonization was not detected (ND)/total number of mice tested is shown underneath the dashed line. (B) CPRG hydrolysis was measured for the ΔireK* suppressor mutant, the WT, and the ΔireK mutant. Reported measurements represent averages from three independent cultures, and error bars represent standard deviations. Statistical significance was evaluated by t test. ****, P < 0.0001 versus WT. (C) The cholate and lysozyme resistance of the strains listed in panel B was determined. The reported MICs represent the median values from three independent biological replicates. (D) Groups of mice (5 per group) were colonized with either WT E. faecalis (OG1RF), the ΔireK mutant (CK119), or the ΔireK* suppressor mutant. Intestinal colonization was assessed as described in the legend to panel A.

FIG 4

Disruption of OG1RF_11271 and OG1RF_11272 drives the phenotype of ΔireK* suppressor mutants. (A) Genetic architecture near the nucleotide variants uncovered by whole-genome sequencing (the drawing is not to scale). The locations of the nucleotide variants are indicated by black arrows. (B) The cholate and lysozyme resistance of the WT E. faecalis strain (OG1RF), the ΔireK mutant (CK119), and the ΔireK* suppressor mutant carrying the empty vector (pJRG9) or expressing wild-type copies of the indicated genes was determined. The reported MICs represent the median values from three independent biological replicates. (C) CPRG hydrolysis was measured for the strains listed in panel B. The reported measurements represent averages from three independent cultures, and error bars represent standard deviations. Statistical significance was evaluated by t test. ****, P < 0.0001.

FIG 5

Deletion of OG1RF_11272 in the ΔireK mutant background is sufficient to elicit the suppressor mutant phenotype. (A) The cholate and lysozyme resistance of the WT E. faecalis strain (OG1RF) and the ΔireK (CK119), ΔireK ΔOG1RF_11271 (IB21), ΔireK ΔOG1RF_11272 (IB22), and ΔireK ΔOG1RF_11271 ΔOG1RF_11272 (IB23) mutants was determined. The reported MICs represent the median values from three independent biological replicates. (B) CPRG hydrolysis was measured for the strains listed in panel A. The reported measurements represent averages from three independent cultures, and error bars represent standard deviations. Statistical significance was evaluated by t test. ****, P < 0.0001. (C) Groups of mice (5 per group) were colonized with the ΔireK (CK119), ΔireK ΔOG1RF_11271 (IB21), or ΔireK ΔOG1RF_11272 (IB22) mutant. Colonization loads were determined by enumerating the enterococci in feces by culture on rifampin-supplemented BHI agar. Dashed line, the limit of detection. Symbols for mice with undetectable colonization levels were omitted, and instead, the number of mice for which colonization was not detected (ND)/total number of mice tested is shown underneath the dotted line.

FIG 6

The inhibitory actions of OG1RF_11271 and OG1RF_11272 are limited in the presence of IreK. (A) The cholate and lysozyme resistance of the WT E. faecalis strain (OG1RF) and the ΔireK (CK119), ΔOG1RF_11271 (IB18), ΔOG1RF_11272 (IB19), and ΔOG1RF_11271 ΔOG1RF_11272 (IB20) mutants was determined. The reported MICs represent the median values from three independent biological replicates. (B) The cholate and lysozyme resistance of the WT E. faecalis strain (OG1RF) and the ΔireK mutant (CK119) carrying the empty vector (pJRG9) or expressing wild-type copies of the indicated genes was determined. (C) CPRG hydrolysis was measured for the strains listed in panel B. The reported measurements represent averages from three independent cultures, and error bars represent standard deviations. Statistical significance was evaluated by t test. ****, P < 0.0001.

FIG 7

Restriction of OG1RF_11271 and OG1RF_11272 actions require IreK's kinase activity. (A) The cholate and lysozyme resistance of a strain with an ireK K41R mutation (BL102) carrying an empty vector as well as an ireK K41R ΔOG1RF_11271 ΔOG1RF_11272 mutant (IB36) carrying an empty vector or complemented with OG1RF_11271-OG1RF_11272 was determined. The reported MICs represent the median values from three independent biological replicates. (B) CPRG hydrolysis was measured for the strains listed in panel A. The reported measurements represent averages from three independent cultures, and error bars represent standard deviations. Statistical significance was evaluated by t test. ****, P < 0.0001. (C) Proposed model for IreK and OG1RF_11271/OG1RF_11272 modulation of cell envelope integrity, antimicrobial resistance, and colonization. (Inset) A nonscaled depiction of the protein domain architecture for OG1RF_11271 and OG1RF_11272.