• 96 plays a role in the virulence of C. jejuni

      Rathbun, Kimberly M; Department of Medicine (2009-05)
      Campylobacter jejuni is a gastrointestinal pathogen of humans but part of the normal flora of poultry. C. jejuni therefore grows well at both 37°C and 42°C. Proteomic studies on temperature regulation in C. jejuni strain 81-176 revealed the upregulation at 37°C of CJ0596, a predicted periplasmic chaperone that is similar to proteins found to be involved in outer membrane protein (OMP) folding and virulence in other bacteria. The cj0596 gene was highly conserved in multiple strains and species of Campylobacter (24 in total), implying the importance of this gene. To study the role CJ0596 plays in Campylobacter pathogenesis, a mutant derivative of strain 81-176 was constructed in which the cj0596 gene was precisely deleted. This mutant was complemented by restoring the gene to its original chromosomal location. The mutant strain demonstrated a decreased growth rate and lower final growth yield, yet was more motile than wild-type. The cj0596 mutant also showed altered levels of several outer membrane proteins (OMPs), and changes in membrane-associated characteristics (antimicrobial sensitivity, autoagglutination, and biofilm formation). In either single or mixed infections, the mutant was less able to colonize mice than wild-type. Purified, recombinant CJ0596 had peptidyl-prolyl cistrans isomerase (PPIase) activitty, but did not functionally complement an E. coli surA mutant. These results suggest that C. jejuni CJ0596 is a PPIase and loss of CJ0596 alters phenotypes that have been shown to be related to the pathogenesis of the bacterium.
    • Regulation of Virulence in the Human Pathogen Campylobacter jejuni by the RNA Binding Protein CsrA

      Fields, Joshua A; Department of Medicine (2011-10)
      Campylobacter jejuni is a leading bacterial cause of gastroenteritis in both the industrialized and developing world and has been associated with the onset of long term, debilitating sequelae such as Guillain-Barre Syndrome and reactive arthritis. The RNA binding protein CsrA (carbon storage regulator A), one of the relatively few regulatory elements in the C. jejuni genome, has been shown to regulate a number of processes in several other bacterial species including metabolism and virulence characteristics. We proposed the hypothesis that CsrA globally regulates C. jejuni pathogenesis via post-transcriptional repression or activation of virulence associated proteins. We created a csrA mutant in the C. jejuni strain 81-176 to investigate the role of CsrA in the virulence and physiology of the organism. In the absence of CsrA, we found that C. jejuni was no longer able to resist oxidative stresses, form biofilms, or adhere to intestinal epithelial cells in vitro in comparison to the wild type. We also found that C. jejuni was less motile than its parent strain and was defective in autoagglutination and fibronectin binding in vitro and mouse colonization in vivo. When we compared the proteome of the mutant strain to that of the wild type, we found that CsrA acted mostly upon the expression of proteins in stationary phase. In the absence of CsrA proteins responsible for various steps in C. jejuni metabolism, motility, oxidative stress responses, and epithelial cell adherence were differentially expressed. Finally, to further understand the molecular mechanisms of C. jejuni CsrA, we expressed it in a csrA mutant strain of E. coli. By heterologously expressing the C. jejuni protein in strain in which CsrA had been thoroughly characterized, we were able to show by complementation that C. jejuni CsrA was capable of both activating and repressing known targets of E. coli CsrA indicating that the molecular mechanisms of the two proteins are inherently the same.