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In situ Staphylococcus aureus gene expression in a human prosthetic joint infection

ABSTRACT: Little is known about regulation of gene activity of the major pathogen Staphylococcus aureus during actual human infection. Here we characterize the transcriptome using deep RNA sequencing and the metabolome using NMR of S. aureus infected joint fluid derived from an acute human prosthetic joint infection, and compare them with the genome, transcriptome and metabolome of an isolate obtained from the sample grown in vitro (LB medium). The transcriptome indicated that the bacterial infection sustained on a versatile human-cell-based diet consisting of amino acids, glycans and nucleosides, since significant upregulation of genes involved in the catabolic degradation pathways of these compounds were observed in situ. This is consistent with metabolite analysis of the infected joint fluid and of S. aureus culture supernatants where the concentration of most amino acids and some amino sugars were found to be higher in the joint fluid, whereas the concentration of glucose was higher in culture supernatant. Furthermore, presumably because of oxygen limitations in the joint fluid, transcriptomic evidence for fermentation was observed, consistent with the presence of fermentation products (ethanol) in situ. Moreover, many, but not all, of the known virulence factor genes were upregulated in situ as well as the nine genes encoding the iron uptaking siderophore synthesis system.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE62091 | GEO | 2015/10/01

SECONDARY ACCESSION(S): PRJNA263175

REPOSITORIES: GEO

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Project description:Different transcriptome, metabolome, and phosphoproteome studies have already indicated a regulatory role of Ser/Thr phosphorylation in the central metabolism of S. aureus. However, it remains still unknown how this exactly tunes its metabolism. For that reason, Extreme Pathway Analysis was used to understand metabolic flux regulation by Ser/Thr phosphorylation. YANAsquare software and YANAvergence routine were chosen to calculate pathway activities of WT, ΔpknB, Δstp, and double mutant S. aureus NewmanHG strains. Gene expression data of S. aureus NewmanHG were collected taking the wild type and mutations of kinase (pknB), phosphatase (stp) or both (pknB/stp) phenotypes. The resulting flux changes were modelled with YANAvergence taking the different gene expression data and an established genome scale model of S. aureus metabolic modes as basis. The fluxes of the different strains were next calculated, taking the gene expression data into account. A concerted metabolic change of fluxes was observed. In particular the pathways for peptidoglycan, nucleotide, and aromatic amino acid synthesis as well as catabolism involving aspartate transaminase (GOT) changed significantly compared to the wild type. Single mutations of pknB and stp were compared to illustrate their different contribution to the phenotype, e.g. pyrimidine synthesis is dramatically impaired by pknB deletion but much less by loss of stp. In the double knock out strain, there is higher activity of peptidoglycan, purine, and aromatic amino acids synthesis from glucose, but lower activity of pyrimidine synthesis from glucose compared to WT. The results suggest a probable regulatory role of Ser/Thr phosphorylation in amino acid catabolism and the glycolysis/gluconeogenesis switch to provide the cell with sufficient cell wall components, nucleotides, and aromatic amino acids. Yvck is suggested to serve as regulatory protein linking Ser/Thr phosphorylation to the previous results of S. aureus metabolism. Moreover, Yvck seems in particular to be involved in the switch between glycolysis and gluconeogenesis. However, further studies based on experimental approaches are needed to demonstrate that Ser/Thr phosphorylation and Yvck are necessary for a correct cell growth under gluconeogenic conditions. The metabolic model allows not only a comprehensive representation of these changes for all involved pathways in the four strains, it further points out the function of the previously unannotated ycvk operon.. The relevance of these specific metabolic pathway adaptations regarding the pathogenicity of S.aureus and their contributions to the infection process will be analyzed and discussed.

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.

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In situ Staphylococcus aureus gene expression in a human prosthetic joint infection

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