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The ferric uptake regulator Fur is a potential regulator of Campylobacter jejuni acid survival and cross-protection against other stresses

ABSTRACT: Campylobacter jejuni is a prevalent cause of bacterial gastroenteritis in humans worldwide. The mechanism by which C. jejuni survives stomach acidity remains unknown. Herein, we have demonstrated that C. jejuni with a fur deletion was more sensitive to acid than the wild-type strain. Profiling the acid stimulon of the C. jejuni ∆fur mutant allowed us to uncover Fur-regulated genes under acidic conditions. The up-regulation of heat shock genes and the down-regulation of genes involved in flagellar and cell envelope biogenesis in the fur mutant highlight the importance of Fur in Campylobacter acid survival. Interestingly, prior acid exposure of C. jejuni cross-protected the bacterium against oxidative stress. Western-blot analysis and real-time qRT-PCR revealed an increased expression of the catalase KatA in acid-stressed C. jejuni relative to unstressed bacteria. The enhanced survival of C. jejuni to oxidative stress was shown to be Fur-dependent through the regulation of katA expression. Electrophoretic mobility shift assay (EMSA) demonstrated that the binding affinity between Fur and katA is reduced under low pH allowing for higher expression of katA and the defense against oxidative stress. Strikingly, the ∆fur mutant exhibited a reduced virulence capacity in both human epithelial cells and G. mellonella infection model as compared to C. jejuni wild-type. Altogether, this is the first study showing that in addition to its role in iron metabolism, Fur is an important regulator of C. jejuni acid response and cross-protection against other stresses. Moreover, our results clearly demonstrate that Fur plays a substantial role in C. jejuni host pathogenesis. Campylobacter jejuni is a prevalent cause of bacterial gastroenteritis in humans worldwide. The mechanism by which C. jejuni survives stomach acidity remains unknown. Herein, we have demonstrated that C. jejuni with a fur deletion was more sensitive to acid than the wild-type strain. Profiling the acid stimulon of the C. jejuni ∆fur mutant allowed us to uncover Fur-regulated genes under acidic conditions. The up-regulation of heat shock genes and the down-regulation of genes involved in flagellar and cell envelope biogenesis in the fur mutant highlight the importance of Fur in Campylobacter acid survival. Interestingly, prior acid exposure of C. jejuni cross-protected the bacterium against oxidative stress. Western-blot analysis and real-time qRT-PCR revealed an increased expression of the catalase KatA in acid-stressed C. jejuni relative to unstressed bacteria. The enhanced survival of C. jejuni to oxidative stress was shown to be Fur-dependent through the regulation of katA expression. Electrophoretic mobility shift assay (EMSA) demonstrated that the binding affinity between Fur and katA is reduced under low pH allowing for higher expression of katA and the defense against oxidative stress. Strikingly, the ∆fur mutant exhibited a reduced virulence capacity in both human epithelial cells and G. mellonella infection model as compared to C. jejuni wild-type. Altogether, this is the first study showing that in addition to its role in iron metabolism, Fur is an important regulator of C. jejuni acid response and cross-protection against other stresses. Moreover, our results clearly demonstrate that Fur plays a substantial role in C. jejuni host pathogenesis.

ORGANISM(S): Campylobacter jejuni

PROVIDER: GSE73796 | GEO | 2016/03/17

SECONDARY ACCESSION(S): PRJNA297903

REPOSITORIES: GEO

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Project description:Campylobacter jejuni is the prevalent cause of bacterial gastroenteritis in human worldwide. The ability to survive stomach acidity is a fundamental requirement for C. jejuni to colonize the host and cause disease. However, the mechanism of C. jejuni acid survival is still unknown. Herein, we demonstrated that C. jejuni is able to survive acidic conditions at pH 4 up to 8 min without a drop in viability. The acid stimulon of C. jejuni 81-176 revealed the up-regulation of many genes important for Campylobacter acid survival such as heat shock genes and genes involved in energy metabolism. On the other hand, the repression of ribosomal genes highlights the ability of C. jejuni to direct its machinery to survive stressful conditions. Prior acid exposure cross-protected C. jejuni against oxidative stress suggesting an overlap in C. jejuni’s responses to various stresses. Interestingly, the induced expression of virulence genes in C. jejuni upon acid exposure such as the Campylobacter invasion antigen (ciaB) indicates that acid stress plays a role in C. jejuni host pathogenesis. Acid exposure significantly enhanced C. jejuni pathogenesis in both eukaryotic cells and G. melonella. To the best of our knowledge, this is the first study characterizes the influence of acid stress on C. jejuni pathogenesis in an infection model. Altogether, this study uncovers the transcriptional profile of C. jejuni in response to acidic conditions as those encountered in the stomach. In addition, our results demonstrate that acid stress jump-starts C. jejuni for efficient gut colonization and host pathogenesis. Campylobacter jejuni is the prevalent cause of bacterial gastroenteritis in human worldwide. The ability to survive stomach acidity is a fundamental requirement for C. jejuni to colonize the host and cause disease. However, the mechanism of C. jejuni acid survival is still unknown. Herein, we demonstrated that C. jejuni is able to survive acidic conditions at pH 4 up to 8 min without a drop in viability. The acid stimulon of C. jejuni 81-176 revealed the up-regulation of many genes important for Campylobacter acid survival such as heat shock genes and genes involved in energy metabolism. On the other hand, the repression of ribosomal genes highlights the ability of C. jejuni to direct its machinery to survive stressful conditions. Prior acid exposure cross-protected C. jejuni against oxidative stress suggesting an overlap in C. jejuni’s responses to various stresses. Interestingly, the induced expression of virulence genes in C. jejuni upon acid exposure such as the Campylobacter invasion antigen (ciaB) indicates that acid stress plays a role in C. jejuni host pathogenesis. Acid exposure significantly enhanced C. jejuni pathogenesis in both eukaryotic cells and G. melonella. To the best of our knowledge, this is the first study characterizes the influence of acid stress on C. jejuni pathogenesis in an infection model. Altogether, this study uncovers the transcriptional profile of C. jejuni in response to acidic conditions as those encountered in the stomach. In addition, our results demonstrate that acid stress jump-starts C. jejuni for efficient gut colonization and host pathogenesis.

Project description:Background: During gut colonization, the enteric pathogen C. jejuni has to surmount the toxic effects of reactive oxygen species produced by its own metabolism, by the host immune system and by the intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology. Results: The mechanisms of oxidative stress defenses in Campylobacter jejuni were characterized by transcriptional profiling, genes mutagenesis, and phenotypic analysis. The transcriptome changes, in response to H2O2, cumene hydroperoxide, or menadione exposure, were found to be oxidant specific and revealed the differential expression of genes belonging to a variety of biological pathways, from the classical oxidative stress defense systems, to the heat shock response, DNA repair and metabolism, fatty acid and capsule biosynthesis, and multidrug efflux pumps. To define the peroxide sensing regulator PerR, an isogenic mutant was constructed and its transcriptome profile compared to the wild-type strain. Sixty-six genes were found to belong to the PerR regulon. PerR appear to regulate gene expression both dependently and independently of the presence of iron and/or H2O2. The perR mutant was affected in its motility and attenuated in the chick colonization model. Mutagenic and phenotypic studies of the superoxide disumutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed their role in oxidative stress defenses and chick gut colonization. Conclusion: This study reveals the interplay between PerR, the iron metabolism and the oxidative stress defenses and highlights their role in the colonization and/or survival of C. jejuni in the chick cecum. Keywords: Transcriptional response to 3 oxidants (H2O2, menadione and cumene hydroperoxide) and characterization of the perR regulon (comparison of the transciptomes from the wild-type and perR mutant). To investigate the transcriptional responses of C. jejuni to oxidant exposure, hydrogen peroxide (H2O2), cumene hydroperoxide (CHP), or menadione sodium bisulfite (MND) was added to the 50 ml broth at a final concentration of 1 mM. The same amount of water or DMSO was added to the bacterial culture that served as reference samples for the transcriptional profile study in response to H2O2, MND or CHP. Furthermore, to investigate the transcriptional response of C. jejuni to H2O2 exposure in the presence of excess iron, ferrous sulfate was added to the bacterial culture at a final concentration of 40 µM, 15 min prior to H2O2 exposure. Ten minutes after the addition of the oxidant, total RNA was extracted and processed for microarray hybridization. To identify the PerR regulon, the wild-type strain C. jejuni NCTC 11168 and the perR mutant were grown in 500 ml flasks containing 250 ml of MEMα medium. At mid-log phase, 50 ml of the cultures were transferred to 100 ml flasks and ferrous sulfate and/or H2O2 were added . Ten minutes following the addition of H2O2 the cells were collected and the total RNA extracted.

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The ferric uptake regulator Fur is a potential regulator of Campylobacter jejuni acid survival and cross-protection against other stresses

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