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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Biology of Human Pathogens Associated with Food

Location: Produce Safety and Microbiology Research

Title: The hyperosmotic stress response of Campylobacter jejuni

Authors
item Cameron, Andrew -
item Frirdich, Emilisa -
item Huynh, Steven
item Parker, Craig
item Gaynor, Erin -

Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 31, 2012
Publication Date: October 26, 2012
Repository URL: http://doi:10.1128/JB.01409-12
Citation: Cameron, A., Frirdich, E., Huynh, S., Parker, C., Gaynor, E.C. 2012. The hyperosmotic stress response of Campylobacter jejuni. Journal of Bacteriology. November 2012, 194:(22)6116-6130.

Interpretive Summary: Gastrointestinal pathogens encounter frequent changes in osmotic concentration as they move among environment and host niches. While other enteric bacteria have highly conserved adaptive systems for osmotic changes, C. jejuni encodes no characterized osmoprotectant machinery despite its prevalence and pervasiveness. We hypothesized that C. jejuni has novel mechanisms for responding to hyperosmotic stress with fundamental roles in the pathogenic potential of this enigmatic bacterium. To test this, we altered the salt amount (osmotic concentration) of C. jejuni culture medium which shifted the concentration from conditions similar to the human intestinal tract to those similar to conditions in the chicken cecum and duodenum. Growth analyses revealed early adaptation but late-stage growth defects, severe filamentation and population length heterogeneity, and an MIC of 0.620 osmol/L for both ionic and non-ionic osmotic stressors. Gene expression following exposure to 1.0% NaCl revealed immediate up-regulation of heat shock proteins and enzymes for the synthesis of glutamine and glutamate, and modest, cross-protective up-regulation of oxidative stress response mechanisms. Also up-regulated were genes for ATP synthase (Atp) and the capsule export protein KpsM. A transposon mutant screen likewise identified insertions in 3 capsule genes with 100-fold decreased osmotic tolerance. Microscopy and FACS of wildtype C. jejuni harboring a GFP fusion to the atpF’ promoter showed population bifurcation into GFPhigh (stress-capable) and GFPlow (less-culturable) sub-groups in the presence of NaCl. Interestingly, 7/50 wildtype colonies exhibited spontaneous sensitivity to hyperosmotic stress, further demonstrating C. jejuni population heterogeneity. This sensitivity was stable over multiple generations and was not due to “capsule-off” phase variation. Our experimental data suggest atypical stress management and survival tactics in an organism lacking many hallmark stress response factors. The role of heat shock proteins and the capsule have clear significance for colonization of susceptible hosts. These studies also implicate cryptic genetic mechanisms underlying C. jejuni population heterogeneity, which may reflect an important “bet-hedging” stress-survival mechanism.

Technical Abstract: The hyperosmotic stress response of Campylobacter jejuni: The diarrheal pathogen Campylobacter jejuni and other gastrointestinal bacteria encounter changes in osmolarity in the environment, through exposure to food processing, or upon entering host organisms, where osmotic adaptation can be associated with virulence. In this study, growth profiles, transcriptomics, and phenotypic, mutant, and single-cell analyses were used to explore the effects of hyperosmotic stress exposure on C. jejuni. Increased growth inhibition correlated with increased osmotic concentration, with both ionic and non-ionic stressors inhibiting growth at 0.620 total osmol L-1. C. jejuni adaptation to a range of osmotic stressors and concentrations was accompanied by severe filamentation in subpopulations, with microscopy indicating septum formation and phenotypic diversity between individual cells in a filament. Population heterogeneity was also exemplified by the bifurcation of colony morphology into small and large variants on salt stress plates. Flow cytometry of C. jejuni harboring GFP fused to the ATP synthase promoter likewise revealed bimodal subpopulations under hyperosmotic stress. We also identified frequent hyperosmotic stress-sensitive variants within the clonal wild-type population propagated on standard laboratory medium. Microarray analysis following hyperosmotic upshift revealed enhanced expression of heat shock genes, genes encoding enzymes for synthesis of potential osmoprotectants, and cross-protective induction of oxidative stress genes. The capsule export gene kpsM was also upregulated, and an acapsular mutant was defective for growth under hyperosmotic stress. For C. jejuni, an organism lacking most conventional osmotic response factors, these data suggest an unusual hyperosmotic stress response, including likely 'bet-hedging' survival strategies relying on the presence of stress-fit individuals in a heterogeneous population.

Last Modified: 8/30/2014
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