Dataset Information

Proteomic characterization of persisters in Enterococcus faecium.

ABSTRACT:

Background

Enterococcus faecium is a Gram-positive bacterium, naturally present in the human intestinal microbiota, but is also an opportunistic pathogen responsible for healthcare-associated infections. Persisters are individuals of a subpopulation able to survive by arrest of growth coping with conditions that are lethal for the rest of the population. These persistent cells can grow again when the stress disappears from their environment and can cause relapses.

Results

In this study, we highlighted that ciprofloxacin (10-fold the MIC) led to the formation of persister cells of E. faecium. The kill curve was typically biphasic with an initial drop of survival (more than 2 orders of magnitude reduction) followed by a constant bacterial count. Growth curves and antimicrobial susceptibility tests of these persisters were similar to those of the original cells. In addition, by genomic analyses, we confirmed that the persisters were genotypically identical to the wild type. Comparative proteomic analysis revealed that 56 proteins have significantly different abundances in persisters compared to cells harvested before the addition of stressing agent. Most of them were related to energetic metabolisms, some polypeptides were involved in transcription regulation, and seven were stress proteins like CspA, PrsA, ClpX and particularly enzymes linked to the oxidative stress response.

Conclusions

This work provided evidences that the pathogen E. faecium was able to enter a state of persister that may have an impact in chronic infections and relapses. Moreover, putative key effectors of this phenotypical behavior were identified by proteomic approach.

SUBMITTER: Pont CL

PROVIDER: S-EPMC10765921 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Publications

Proteomic characterization of persisters in Enterococcus faecium.

Pont Charlotte Le CL Bernay Benoît B Gérard Mattéo M Dhalluin Anne A Gravey François F Giard Jean-Christophe JC

BMC microbiology 20240103 1

<h4>Background</h4>Enterococcus faecium is a Gram-positive bacterium, naturally present in the human intestinal microbiota, but is also an opportunistic pathogen responsible for healthcare-associated infections. Persisters are individuals of a subpopulation able to survive by arrest of growth coping with conditions that are lethal for the rest of the population. These persistent cells can grow again when the stress disappears from their environment and can cause relapses.<h4>Results</h4>In this ...[more]

PMID: 38172710

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Project description:BackgroundAll gastrointestinal pathogens, including Enterococcus faecalis and Enterococcus faecium, undergo adaptation processes during colonization and infection. In this study, we investigated by data-independent acquisition mass spectrometry (DIA-MS) two crucial adaptations of these two Enterococcus species at the proteome level. Firstly, we examined the adjustments to cope with bile acid concentrations at 0.05% that the pathogens encounter during a potential gallbladder infection. Therefore, we chose the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) as well as the secondary bile acid deoxycholic acid (DCA), as these are the most prominent bile acids. Secondly, we investigated the adaptations from an aerobic to a microaerophilic environment, as encountered after oral-fecal infection, in the absence and presence of deoxycholic acid (DCA).ResultsOur findings showed similarities, but also species-specific variations in the response to the different bile acids. Both Enterococcus species showed an IC50 in the range of 0.01- 0.023% for DCA and CDCA in growth experiments and both species were resistant towards 0.05% CA. DCA and CDCA had a strong effect on down-expression of proteins involved in translation, transcription and replication in E. faecalis (424 down-expressed proteins with DCA, 376 down-expressed proteins with CDCA) and in E. faecium (362 down-expressed proteins with DCA, 391 down-expressed proteins with CDCA). Proteins commonly significantly altered in their expression in all bile acid treated samples were identified for both species and represent a "general bile acid response". Among these, various subunits of a V-type ATPase, different ABC-transporters, multi-drug transporters and proteins related to cell wall biogenesis were up-expressed in both species and thus seem to play an essential role in bile acid resistance. Most of the differentially expressed proteins were also identified when E. faecalis was incubated with low levels of DCA at microaerophilic conditions instead of aerobic conditions, indicating that adaptations to bile acids and to a microaerophilic atmosphere can occur simultaneously.ConclusionsOverall, these findings provide a detailed insight into the proteomic stress response of two Enterococcus species and help to understand the resistance potential and the stress-coping mechanisms of these important gastrointestinal bacteria.

Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Dataset Information

Proteomic characterization of persisters in Enterococcus faecium.

Background

Results

Conclusions

Publications

Proteomic characterization of persisters in Enterococcus faecium.

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