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The proteomic response of Clostridioides difficile to the atypical tetracycline amidochelocardin and concomitant dissipation of its proton motive force

ABSTRACT: Amidochelocardin is an atypical tetracycline which was previously shown to target the bacterial cell membrane although its detailed mode-of-action is still unknown. To gain insight into Clostridioides difficile’s response to membrane targeting antibiotics and to further study the mode-of-action of Amidochelocardin, the stress response of C. difficile 630 to sublethal doses of amidochelocardin were studied on the proteome level. The results and results from downstream experiments, such as quantification of the membrane potential, cellular localization assays and transmission electron microscopy analysis, suggest that amidochelocardin kills bacteria such as C. difficile by dissipating their membrane potential. Furthermore, the data suggest that C. difficile responds to dissipation of the membrane potential by induction of biosynthesis of an hitherto unknown aromatic compound.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Peptoclostridium Difficile (strain 630) (clostridium Difficile)

SUBMITTER: Daniela Zuehlke

LAB HEAD: Susanne Sievers

PROVIDER: PXD029250 | Pride | 2022-08-04

REPOSITORIES: Pride

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Project description:Bacteroides fragilis is an anaerobic commensal in the human gut which can act as opportunistic pathogen when leaving its intestinal niche to reach other body sites. Bacteroides infections have a high lethality and must be treated by antimicrobial chemotherapy. Metronidazole is one of the most frequently administered antimicrobials in the treatment of Bacteroides infections and is highly reliable. However, metronidazole resistance does occur, favoring fatal disease outcomes. Most of the resistant isolates harbor a nim gene (12 are currently known, i.e. nimA to nimL), a transferable resistance determinant for metronidazole. Previous research suggested that Nim proteins might affect the cellular physiology by changing the activity of key enzymes like pyruvate:ferredoxin oxidoreductase (PFOR). In this study we wanted to assess the impact of the nimA gene on protein expression in a standard B. fragilis isolate, 638R, and compared overall protein expression in 638R with and without a nim gene and with the nimA gene in a proteomic study. Further, high-level metronidazole resistance was induced in both strains and the protein expression profiles of resulting resistant daughter strains were also compared with their respective parent strains. We found that comparably few proteins displayed altered expression in 638R with the nimA gene, but flavodiiron protein FprA was repeatedly found upregulated. FprA is often found in anaerobes and reduces molecular oxygen to water and/or nitric oxide to nitrous oxide. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter the observed changes were not only less numerous but also less specific. Based on the results of this study we present a novel hypothetic model of metronidazole resistance and Nim function.

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The proteomic response of Clostridioides difficile to the atypical tetracycline amidochelocardin and concomitant dissipation of its proton motive force

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