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Bacterial bunker blueprints: IreK-mediated, cell wall protective phosphorylation in Enterococcus faecalis

ABSTRACT: Enterococcus faecalis is a Gram-positive bacterium that is a major cause of hospital-acquired infections due to its intrinsic resistance to cell wall-active antimicrobials. One critical determinant of this resistance is the transmembrane kinase IreK, which belongs to the PASTA kinase family of bacterial signaling proteins involved with the regulation of cell wall homeostasis. IreK has enhanced activity in response to cell wall stress, but direct substrates of IreK phosphorylation leading to antimicrobial resistance are largely unknown. To better understand stress-modulated phosphorylation events contributing to virulence, wild type E. faecalis treated with cell wall-active chlorhexidine and ceftriaxone were examined via phosphoproteomics. Among the most prominent changes were increased phosphorylation of divisome components after both treatments, implicating cell division proteins in antimicrobial defense signaling. Phosphorylation mediated by IreK was then determined via similar analysis with a E. faecalis ΔireK mutant strain, revealing potential IreK substrates involved with the formation/maintenance of biofilms and within the E. faecalis two-component system, another common signal transduction pathway for antimicrobial resistance. These results reveal critical insights into the biological functions of IreK and the mechanisms of E. faecalis antimicrobial resistance.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Enterococcus Faecalis (streptococcus Faecalis)

SUBMITTER: Leslie Hicks

LAB HEAD: Leslie Hicks

PROVIDER: PXD023265 | Pride | 2021-11-03

REPOSITORIES: Pride

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			Action	DRS
	20200819_AAI_Ef_Phospho_1uL_JL206_1.raw	Raw
	20200819_AAI_Ef_Phospho_1uL_JL206_2.raw	Raw
	20200819_AAI_Ef_Phospho_1uL_JL206_3.raw	Raw
	20200819_AAI_Ef_Phospho_1uL_JL206_4.raw	Raw
	20200819_AAI_Ef_Phospho_1uL_JL206_5.raw	Raw

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Project description:Emerging antibiotic resistance among clinically relevant bacteria, paired with their ability to form biofilms on medical and technical devices, represents a serious problem in terms of effective and long-term decontamination in health care environments and gives rise to an urgent need for new antimicrobial materials. Here we present the first study of the impact of AGXXÂ®, a novel broad-spectrum antimicrobial surface coating consisting of micro galvanic elements formed by silver and ruthenium, on the transcriptome of the nosocomial pathogen Enterococcus faecalis. E. faecalis was subjected to metal stress by growing it for different periods of time in the presence of AGXXÂ® or silver-coated steel meshes. Subsequently, total RNA was isolated and next-generation RNA sequencing was performed to analyze variations in gene expression levels in the presence of the antimicrobial materials with focus on known stress genes. Exposure to AGXXÂ® had a large impact on the transcriptome of E. faecalis. After 24 minutes almost 1/5 of the E. faecalis genome displayed differential expression. At each time-point the cop operon was strongly up-regulated, providing indirect evidence for the presence of free Ag+-ions. Moreover, exposure to AGXXÂ® induced a broad general stress response in E. faecalis. Genes coding for the chaperones GroEL and GroES as well as the Clp proteases ClpE and ClpB were among the top up-regulated heat shock genes. Furthermore, differential expression of genes coding for thioredoxin, superoxide dismutase and glutathione synthetase indicates a high level of oxidative stress. We postulate a mechanism of action where the combination of Ag+-ions and reactive oxygen species generated by AGXXÂ® results in a synergistic antimicrobial effect, which is superior to that of conventional silver coatings. Gene expression analysis of Enterococcus faecalis 12030 either subjected to metal stress by exposure to an antimicrobial AGXXÂ®- or Ag-coated V2A steel mesh or exposed to an uncoated V2A steel mesh or left untreated performing RNA Sequencing with an Ion ProtonTM Sequencer and subsequent data analysis with a T-REx RNA-Sequencing expression analysis pipeline.

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Bacterial bunker blueprints: IreK-mediated, cell wall protective phosphorylation in Enterococcus faecalis

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