Campylobacter jejuni , a Gram-negative microaerophilic bacterium, is a predominant cause of bacterial foodborne gastroenteritis in humans. Despite its importance as a major foodborne pathogen, our understanding of the molecular mechanisms underlying C. jejuni colonization and pathogenesis in disparate hosts and environments is limited. C. jejuni is ubiquitously found in the intestinal tract of warm blooded animals but chickens are the natural reservoirs. Most campylobacteriosis outbreaks in humans are attributed to consumption of contaminated, raw and undercooked poultry meat. Stress response mechanisms of C. jejuni help its survival in adverse conditions and resist stresses encountered by the enteric pathogen in the environment, and inside the gastrointestinal tract of its hosts. The work in this dissertation is broadly divided into two sections: i) contribution of Transducer like proteins (Tlps) to C. jejuni colonization, ii) Polyphosphate Kinases (PPKs) role in stress survival.
Persistent colonization of the avian gut by C. jejuni depends on multiple factors. Of particular importance is chemotaxis, which mediates directional motility towards a favorable chemical gradient in the environment. Chemotaxis system in C. jejuni consists of core signal transduction proteins and Methyl Accepting domain containing Transducer like proteins (Tlps). Tlps act as nutrient sensors for chemoeffectors (ligands); attractants or repellents. Tlp-ligand binding relays a signal to chemotaxis proteins in the cytoplasm thus initiating a signal transduction cascade, which culminates into a directional flagellar movement. C. jejuni genome encodes ten chemoreceptors or Tlps, which are either internal membrane proteins or cytoplasmic proteins. Though several Tlps have been associated with different ligands, the role of Tlp in mediating substrate specific chemotaxis, physiology, virulence and tissue specific colonization is still not very clear. We therefore hypothesize that Tlps facilitate substrate-specific chemotaxis in C. jejuni , which plays an important role in C. jejuni virulence associated traits and colonization of the chicken gastrointestinal tract. Findings from this study will have implications for developing strategies to control Campylobacter through identification of new targets for vaccines and antimicrobials.
In the first study, we investigated the role of Tlps in C. jejuni chemotaxis, motility, pathogenesis and colonization in the chicken host, using a deletion mutagenesis approach. We identified potential ligands for the many uncharacterized Tlps. Our findings reveal that more than one Tlp is involved in mediating chemotaxis towards the same nutrient. The deletion of tlps affected virulence-associated phenotypes such as motility, biofilm, aerobic survival, and invasion and intracellular survival in human intestinal epithelial cells (INT 407). Two tlp mutants (Δtlp3 and 8 ) displayed increased motility in soft agar, and the Δtlp8 also exhibited decreased biofilm formation and a defect in aerobic stress survival. We found that Tlps are important for colonization of the different segments of the chicken gastrointestinal tract with the Δtlp10 mutant being defective in colonization of the chicken proximal and distal gastrointestinal tract. Taken together, our results highlight the importance of Tlps in C. jejuni adaptation and pathobiology.
In the second study, we investigated the contribution of Tlp2 to C. jejuni chemotaxis, in-vitro virulence and colonization in the chicken gastrointestinal tract. Our findings revealed that the tlp2 deletion mutant showed decreased chemotaxis towards aspartate, pyruvate, inorganic phosphate (Pi) and iron (FeSO4). Promoter fusion reporter assays revealed that the tlp2 promoter (Ptlp2) was induced by iron, both in the ferrous (Fe2+) and ferric form (Fe3+) and by Pi. Interestingly, phoX gene (encoding alkaline phosphatase), located downstream of tlp2, is found to be co-transcribed with tlp2. The alkaline phosphatase activity in the Δtlp2 mutant was found to be higher than the activity in wild type. The Δtlp2 mutant showed nutrient survival defect in the stationary phase and also demonstrated an invasion defect in INT 407 cells as well as a decreased colonization of the chicken gastrointestinal tract. Collectively, our findings describe an iron-mediated regulation of a chemotaxis gene, tlp2, which plays a role in C. jejuni pathogenesis and colonization in the chicken host.
The success of C. jejuni as a foodborne pathogen depends upon its ability to adapt to various stresses encountered in diverse niches inhabited by this enteric pathogen. In the absence of an RpoS-mediated classical stress response mechanism, C. jejuni employs alternative regulators to mediate stress survival. Previous studies have demonstrated the role of poly P as a key regulator of stress responses and virulence in C. jejuni , however; the precise molecular mechanisms involved in poly P -mediated stress tolerance and role in C. jejuni pathogenesis remain unidentified till date. Therefore, the third study was focused on understanding how poly-P associated enzymes, PPK1 and PPK2 regulate various cellular processes, including stress response and survival in C. jejuni . Here, we investigated the ppk1 and ppk2 mediated regulation of genes in C. jejuni using high throughput RNA seq transcriptome analysis. PPK-1 and PPK-2 are essential for metabolism of polyphosphate, a key stress regulator in C. jejuni . We hypothesize that ppk1 and ppk2 mediate various cellular processes that provide stress survival advantages to C. jejuni . Our findings revealed that both ppk1 and ppk2 regulate different sets of genes in the exponential and stationary phase of growth. However, it was in the stationary phase of growth, that we observed maximum number of differentially expressed genes in the ppk1 mutant, compared to the log phase. Most of the differentially regulated genes in the ppk1 mutant, in the stationary phase were genes involved in motility, transport and stress survival. However, in the ppk2 mutant, we noticed that most differentially expressed genes were those involved in C. jejuni metabolism and protein metabolism. Taken together, this study revealed that PPK1 and PPK2 modulate cellular processes; including stress response by regulating the transcription of C. jejuni genes involved in metabolism, motility, transport and virulence.