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S. aureus exoproteome in the context of genetic competence development

ABSTRACT: To perform natural transformation, one of the three main Horizontal Gene Transfer mechanisms, bacteria need to enter a physiological differentiated state called genetic competence. Interestingly, new bacteria displaying such aptitude are often discovered, and one of the latest is the human pathogen Staphylococcus aureus. Here, we developed a new protocol, based on planktonic cells cultures, leading to a large percentage of the population activating competence development and a significant improvement of S. aureus natural transformation efficiencies. Taking advantage of these new conditions, we performed transcriptomics analyses to characterize each central competence regulators regulon. SigH and ComK1 were both found essential for activating natural transformation genes but also important for activation or repression of peripheral functions. Even though ComK2 was not found important for the control of transformation genes, its regulon showed an important overlap with that of SigH and ComK1. Finally, we showed how microaerobic conditions, sensed by the SrrAB two-component system, was key to activate competence in S. aureus.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Staphylococcus Aureus

TISSUE(S): Cell Suspension Culture

SUBMITTER: David Cornu

LAB HEAD: David Cornu

PROVIDER: PXD040550 | Pride | 2023-04-26

REPOSITORIES: Pride

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			Action	DRS
	DC_MIROUZE_12R1_4ul_020223_Slot2-10_1_7801.d.7z	Other
	DC_MIROUZE_12R1_4ul_020223_Slot2-10_1_7801_5.3.556.mgf	Mgf
	DC_MIROUZE_12R2_4ul_020223_Slot2-11_1_7802.d.7z	Other
	DC_MIROUZE_12R2_4ul_020223_Slot2-11_1_7802_5.3.556.mgf	Mgf
	DC_MIROUZE_12R3_4ul_020223_Slot2-12_1_7803.d.7z	Other

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Project description:Pathogens that grow during infections must cope with the reactive oxygen species (ROS) released by host immune cells. Among the different strategies to prevent oxidative damage during infections, pathogenic bacteria have evolved mechanisms to reduce respiration and other cellular processes that are particularly sensitive to free radicals, obtaining energy preferentially by undergoing to fermentative metabolism. As fermentation produces fewer ATP per glucose than respiration, bacteria ensure an appropriate supply of energy by increasing the glycolytic flux. The opportunistic human pathogen Staphylococcus aureus is one such microbe but the underlying mechanism that enables S. aureus to induce fermentation during infections is still unclear. Here we show that the ComK-like regulator of natural competence that is present in many gram-positive bacteria is crucial to redirect S. aureus metabolism to fermentation during infection. ComK is cryptic in laboratory conditions but highly induced during infections or in response to infection-related cues, such as ROS. ComK induces the glycolytic flux and glucose consumption rate, a key step to redirect ATP production to fermentation. This licenses fermenting S. aureus to reduce oxidative damage while increasing DNA uptake by natural transformation. As a consequence, a comK-defective mutant shows an accumulation of ROS as well as DNA mutations that lower bacterial survival. This mutant shows no distinctive phenotype in laboratory conditions but is unable to cause infection in vertebrates or invertebrate infection models. ComK-mediated synchronization of natural transformation to fermentative metabolism may allow S. aureus, and probably other gram-positive bacteria, to use the fermentation acid end products to eliminate bacterial competitors while assimilating their DNA. Assimilated DNA may serve as a source of nucleotides for DNA repair or to promote genetic variability, thereby enabling a successful host colonization of this bacterium.

Project description:We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner-transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counter-selection of mutants in 21 different genes during the challenge. Eight of the counter-selected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants in which seven of the eight remaining genes, i.e., spr0459/spr0460, spr0777, spr0838, spr1259/spr1260, and spr1357, were deleted, displayed reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation of chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and DNA damage repair in S. pneumoniae. Keywords: GAF competence Selection for genes essential for transformation. Aliquots of a marinerT7 transposon library generated in S. pneumoniae R6 containing approximately 40,000 independent mutants were transformed with an antibiotic resistance marker and grown without (initial library) or with (challenged library) antibiotics for 25 generation until DNA extraction.

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S. aureus exoproteome in the context of genetic competence development

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