Name: Campylobacter jejuni virulence factors identified by modulating their sCampylobacter jejuni virulence factors identified by modulating their synthesis on ribosomes with altered rRNA methylationynthesis on ribosomes with altered rRNA methylation
Keywords: Campylobacter

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Campylobacter jejuni virulence factors identified by modulating their sCampylobacter jejuni virulence factors identified by modulating their synthesis on ribosomes with altered rRNA methylationynthesis on ribosomes with altered rRNA methylation

ABSTRACT: Campylobacter jejuni is a major cause of food poisoning worldwide, and remains the main infective agent in gastroenteritis and related intestinal disorders in Europe and the USA. As with all bacterial infections, the stages of adhesion to host tissue, survival in the host and eliciting disease all require the synthesis of proteinaceous virulence factors on the ribosomes of the pathogen. Here, we describe how C. jejuni virulence is attenuated by altering the methylation of its ribosomes to disrupt the composition of its proteome, and how this in turn provides a means of identifying factors that are essential for infection and pathogenesis. Specifically, inactivation of the C. jejuni Cj0588/TlyA methyltransferase prevents methylation of nucleotide C1920 in the 23S rRNA of its ribosomes and reduces the pathogen’s ability to form biofilms, to attach, invade and survive in host cells, and to provoke the innate immune response. Mass spectrometric analyses of C. jejuni TlyA-minus strains revealed an array of subtle changes in the proteome composition. These included reduced amounts of the cytolethal distending toxin (CdtC) and the MlaEFD proteins connected with outer membrane vesicle (OMV) production. Inactivation of the cdtC and mlaEFD genes confirms the importance of the toxin and OMVs in establishing infection. Collectively, the data identify a collection of genes required for the onset of human campylobacteriosis, and serve as a proof of principle for use of this approach in detecting proteins involved in bacterial pathogenesis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Campylobacter Jejuni Subsp. Jejuni Bacteria

TISSUE(S): Cell Culture

DISEASE(S): Diarrhea

SUBMITTER: Agnieszka Sałamaszyńska-Guz

LAB HEAD: Agnieszka Sałamaszyńska-Guz

PROVIDER: PXD030125 | Pride | 2022-01-26

REPOSITORIES: Pride

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Publications

Campylobacter jejuni Virulence Factors Identified by Modulating Their Synthesis on Ribosomes With Altered rRNA Methylation.

Sałamaszyńska-Guz Agnieszka A Rasmussen Pernille Kronholm PK Murawska Małgorzata M Douthwaite Stephen S

Frontiers in cellular and infection microbiology 20220113

Campylobacter jejuni is a major cause of food poisoning worldwide, and remains the main infective agent in gastroenteritis and related intestinal disorders in Europe and the USA. As with all bacterial infections, the stages of adhesion to host tissue, survival in the host and eliciting disease all require the synthesis of proteinaceous virulence factors on the ribosomes of the pathogen. Here, we describe how C. jejuni virulence is attenuated by altering the methylation of its ribos ...[more]

PMID: 35096652

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Project description:Campylobacter jejuni is a highly prevalent food-borne pathogen that causes diarrhoeal disease in humans. A natural zoonotic, it must overcome significant stresses both in vivo and during transmission despite the absence of several traditional stress response genes. Its ability to interact with and invade human intestinal epithelial cells closely correlates with virulence. A C. jejuni microarray-based screen revealed that several known virulence genes and several uncharacterized genes, including spoT, were rapidly upregulated during infection of human epithelial cells. spoT and its homologue relA have been shown in other bacteria to regulate the stringent response, an important stress response that to date had not been demonstrated for C. jejuni. We have found that C. jejuni mounts a stringent response that is regulated by spoT. Detailed analyses of a C. jejuni delta spoT mutant revealed that the stringent response is required for several specific stress, transmission and antibiotic resistance-related phenotypes. These include stationary phase survival, growth and survival under low CO2/high O2 conditions, and rifampicin resistance. A secondary suppressor strain that specifically rescues the low CO2 growth defect of the delta spoT mutant was also isolated. The stringent response additionally proved to be required for the virulence-related phenotypes of adherence, invasion, and intracellular survival in two human epithelial cell culture models of infection; spoT is the first C. jejuni gene shown to participate in longer term survival in epithelial cells. Comparing wt to the delta spoT mutant also revealed a strong correlation between gene expression profiles and phenotype differences observed. Together, these data demonstrate a critical role for the C. jejuni stringent response in multiple aspects of C. jejuni biology and pathogenesis. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set

Project description:C. jejuni, a spiral-shaped gram-negative bacterium, is a leading bacterial cause of human foodborne illness. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Further, maximal host cell invasion requires the secretion of proteins termed Campylobacter invasion antigens (Cia). As bile acids are known to alter the pathogenic behavior of other gastrointestinal pathogens, we hypothesized that the virulence potential of Campylobacter may be triggered by the bile acid deoxycholate (DOC). In support of this hypothesis, culturing C. jejuni with a physiologically relevant concentration of DOC significantly altered the kinetics of cell invasion as evidenced by gentamicin-protection assays. In contrast to C. jejuni harvested from Mueller-Hinton (MH) agar plates, C. jejuni harvested from MH agar plates supplemented with DOC demonstrated Cia secretion as judged by metabolic labeling experiments. DOC was also found to induce the expression of the ciaB gene as judged by B-galactosidase reporter assays and real-time RT-PCR. Microarray analysis revealed that DOC induced the expression of virulence genes (i.e., ciaB, cmeABC, dccR, and tlyA). In summary, we demonstrate that it is possible to enhance the pathogenic behavior of C. jejuni by modifying the culture conditions. These results provide a foundation to identify genes expressed by C. jejuni in response to in vivo-like culture conditions. Keywords: Stress response For the expression profiling arrays, an indirect comparison of gene expression was performed, where the expression profile of the C. jejuni F38011 cultured in the presence and absence of DOC was measured separately on different slides as described previously (26). Briefly, Cy5 labeled reference DNA from the C. jejuni F38011 strain was mixed with Cy3 labeled test cDNA (C. jejuni F38011 cultured in the presence or absence of DOC) and hybridized to the Campylobacter cDNA array (26) on separate slides. DNA microarrays were scanned using an Axon GenePix 4000B microarray laser scanner (Axon Instruments, Union City, CA) and the data for spot and background intensities were processed using the GenePix 4.0 software. To compensate for any effect of the amount of template and uneven Cy-dye incorporation, data normalization was performed as previously described (26). For the comparison of genes differentially expressed in the presence and absence of DOC,six hybridization measurements were generated per biological experiment (three technical replicate arrays and two replicate features per array).

Dataset Information

Campylobacter jejuni virulence factors identified by modulating their sCampylobacter jejuni virulence factors identified by modulating their synthesis on ribosomes with altered rRNA methylationynthesis on ribosomes with altered rRNA methylation

Publications

<i>Campylobacter jejuni</i> Virulence Factors Identified by Modulating Their Synthesis on Ribosomes With Altered rRNA Methylation.

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