Growth and Laboratory Maintenance of Campylobacter jejuni

Campylobacter jejuni is a fastidious organism, growing in microaerophilic conditions with a temperature range between 37° and 42°C. Multiple types of media can be used to cultivate it; however, Mueller Hinton broth and agar support the best C. jejuni growth. Optimum atmosphere for C. jejuni is 85% N2, 10% CO2, and 5% O2. Curr. Protoc. Microbiol. 10:8A.1.1‐8A.1.7. © 2008 by John Wiley & Sons, Inc.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154622/1/cpmc08a01.pd

Bacterial-associated cholera toxin and GM 1 binding are required for transcytosis of classical biotype Vibrio cholerae through an in vitro M cell model system

To elucidate mechanisms involved in M cell uptake and transcytosis of Vibrio cholerae , we used an in vitro model of human M-like cells in a Caco-2 monolayer. Interspersed among the epithelial monolayer of Caco-2 cells we detect cells that display M-like features with or without prior lymphocyte treatment and we have established key parameters for V. cholerae transcytosis in this model. Cholera toxin (CT) mutants lacking the A subunit alone or both the A and B subunits were deficient for transcytosis. We explored this finding further and showed that expression of both subunits is required for binding by whole V. cholerae to immobilized CT receptor, the glycosphingolipid GM 1 . Confocal microscopy showed CT associated with transcytosing bacteria, and transcytosis was inhibited by pre-incubation with GM 1 before infection. Finally, heat treatment of the bacterial cells caused a loss of binding to GM 1 that was correlated with a significant decrease in uptake and transcytosis by the monolayer. Our data support a model in which the ability of bacteria to interact with GM 1 in a CT-dependent fashion plays a critical role in transcytosis of V. cholerae by M cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74960/1/j.1462-5822.2005.00681.x.pd

Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans in developed countries throughout the world. This bacterium frequently promotes a commensal lifestyle in the gastrointestinal tracts of many animals including birds and consumption or handling of poultry meats is a prevalent source of C. jejuni for infection in humans. To understand how the bacterium promotes commensalism, we used signature-tagged transposon mutagenesis and identified 29 mutants representing 22 different genes of C. jejuni strain 81–176 involved in colonization of the chick gastrointestinal tract. Among the determinants identified were two adjacent genes, one encoding a methyl-accepting chemotaxis protein (MCP), presumably required for proper chemotaxis to a specific environmental component, and another gene encoding a putative cytochrome c peroxidase that may function to reduce periplasmic hydrogen peroxide stress during in vivo growth. Deletion of either gene resulted in attenuation for growth throughout the gastrointestinal tract. Further examination of 10 other putative MCPs or MCP-domain containing proteins of C. jejuni revealed one other required for wild-type levels of caecal colonization. This study represents one of the first genetic screens focusing on the bacterial requirements necessary for promoting commensalism in a vertebrate host.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72403/1/j.1365-2958.2004.03988.x.pd

Membrane localization of the ToxR winged-helix domain is required for TcpP-mediated virulence gene activation in Vibrio cholerae

ToxR is a bitopic membrane protein that controls virulence gene expression in Vibrio cholerae . Its cytoplasmic domain is homologous to the winged helix–turn–helix (‘winged helix’) DNA-binding/transcription activation domain found in a variety of prokaryotic and eukaryotic regulators, whereas its periplasmic domain is of ill-defined function. Several genes in V. cholerae are regulated by ToxR, but by apparently different mechanisms. Whereas ToxR directly controls the transcription of genes encoding two outer membrane proteins, OmpU and OmpT, it co-operates with a second membrane-localized transcription factor called TcpP to activate transcription of the gene encoding ToxT, which regulates transcription of cholera toxin ( ctxAB ) and the toxin-co-regulated pilus ( tcp ). To determine the requirements for gene activation by ToxR, different domains of the protein were analysed for their ability to control expression of toxT , ompU and ompT . Soluble forms of the cytoplasmic winged-helix domain regulated ompU and ompT gene expression properly but did not activate toxT transcription. Membrane localization of the winged helix was sufficient for both omp gene regulation and TcpP-dependent toxT transcription, irrespective of the type of periplasmic domain or even the presence of a periplasmic domain. These results suggest that (i) the major function for membrane localization of ToxR is for its winged-helix domain to co-operate with TcpP to activate transcription; (ii) the periplasmic domain of ToxR is not required for TcpP-dependent activation of toxT transcription; and (iii) membrane localization is not a strict requirement for DNA binding and transcription activation by ToxR.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73850/1/j.1365-2958.2003.03398.x.pd

Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73261/1/j.1365-2958.2001.02376.x.pd

The PAS Domain-Containing Protein HeuR Regulates Heme Uptake in Campylobacter jejuni

Campylobacter jejuni is a leading cause of bacterially derived gastroenteritis. A previous mutant screen demonstrated that the heme uptake system (Chu) is required for full colonization of the chicken gastrointestinal tract. Subsequent work identified a PAS domain-containing regulator, termed HeuR, as being required for chicken colonization. Here we confirm that both the heme uptake system and HeuR are required for full chicken gastrointestinal tract colonization, with the hour mutant being particularly affected during competition with wild-type C. jejuni. Transcriptomic analysis identified the chu genes—and those encoding other iron uptake systems—as regulatory targets of HeuR. Purified HeuR bound the chuZA promoter region in electrophoretic mobility shift assays. Consistent with a role for HeuR in chu expression, heuR mutants were unable to efficiently use heme as a source of iron under iron-limiting conditions, and mutants exhibited decreased levels of cell-associated iron by mass spectrometry. Finally, we demonstrate that an heuR mutant of C. jejuni is resistant to hydrogen peroxide and that this resistance correlates to elevated levels of catalase activity. These results indicate that HeuR directly and positively regulates iron acquisition from heme and negatively impacts catalase activity by an as yet unidentified mechanism in C. jejuni

Differential Expression of the ToxR Regulon in Classical and E1 Tor Biotypes of Vibrio cholerae is Due to Biotype-Specific Control over toxT Expression.

The two major disease-causing biotypes of Vibrio cholerae, classical and El Tor, exhibit differences in their epidemic nature. Their behavior in the laboratory also differs in that El Tor strains produce two major virulence factors, cholera toxin (CT) and the toxin coregulated pilus (TCP), only under very restricted growth conditions, whereas classical strains do so in standard laboratory medium. Expression of toxin and TCP is controlled by two activator proteins, ToxR and ToxT, that operate in cascade fashion with ToxR controlling the synthesis of ToxT. Both biotypes express equivalent levels of ToxR, but only classical strains appear to express ToxT when grown in standard medium. In this report we show that restrictive expression of CT and TCP can be overcome in El Tor strains by expressing ToxT independently of ToxR. An El Tor strain lacking functional ToxT does not express CT or TCP, ruling out existence of a cryptic pathway for virulence regulation in this biotype. These results may have implications for understanding the evolution of El Tor strains toward reduced virulence with respect to classical strains

Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni

Campylobacter jejuni is naturally transformable but is selective in the DNA used in transformation. Natural transformation allows for rapid adaptation and provides a means for DNA repair, both of which enhance bacterial fitness. Our work demonstrates that the methylation status of C. jejuni DNA is critical for transformation; methylation of a single 6-bp sequence in proximity to the transforming DNA is sufficient to allow nontransforming DNA to transform C. jejuni. Our data argue against previously described means for DNA discrimination; specifically, there is no evidence that a restriction enzyme recognizes the unmodified 6-bp sequence, and no evidence of a typical DNA uptake sequence. Therefore, we have identified a distinct DNA discrimination mechanism in Campylobacter

Single‐molecule tracking in live Vibrio cholerae reveals that ToxR recruits the membrane‐bound virulence regulator TcpP to the toxT promoter

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110815/1/mmi12834.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110815/2/mmi12834-sup-0001-si.pd