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Specific Aim 1: Mapping the in vivo transcriptome and identification of regulatory elements active during colonization of the chicken intestinal tract. With the paucity of putative transcription factors encoded in the C. jejuni genome, and the high degree of genetic regulation, we hypothesize that C. jejuni encodes many undiscovered regulatory elements. The transcriptome of C. jejuni during colonization of the chicken cecum will be determined and compared to the transcriptome of laboratory grown cultures. With this approach, new regulatory elements will be identified, and differences in gene expression profiles of cells grown in the laboratory and in chickens will be determined. Novel regulatory mechanisms will be characterized to determine a role in regulating specific loci as well as to understand mechanisms of regulation during in vivo colonization. Once this methodology has been mastered, transcriptomes of C. jejuni colonizing other animal models and transcriptomes of the host organism in response infection or colonization may also be explored. <P>Specific Aim 2: Characterizing the regulatory mechanisms of zinc transport during in vitro and in vivo growth conditions. The ability to study in vitro gene regulation for C. jejuni has been well characterized, yet little has been done to discover novel regulatory pathways in vivo. As a result, we set out to study in vivo regulation in this pathogen utilizing zinc transport, with its three transporters, znuABC, zntA and zupT, as our model system. Existing molecular methods to identify transcription factors will be employed to determine the regulatory mechanism of these loci in vitro.
Non-Technical Summary:<br/>
The goal of this project is to identify the mechanisms by which Campylobacter jejuni regulates gene expression to survive in diverse ecological niches, most notably in the chicken cecum. C. jejuni is a major human pathogen and a leading cause of bacterial food-derived gastroenteritis world-wide. Preventative measures that limit human exposure from contaminated poultry have been the focus of much research, with much attention invested in identifying genes required for chicken colonization. Most work on C. jejuni has centered on in vitro studies with little knowledge regarding the function or expression of genes during colonization of relevant animal models. C. jejuni regulates gene expression in response to various environmental conditions and stresses; however, based on analysis of its complete chromosome sequence, it encodes few transcription factors and lacks key canonical regulators. Thus, we hypothesize that C. jejuni regulates gene expression using uncharacterized mechanisms including small RNAs, riboswitches and antisense transcripts. To address this hypothesis, we propose to identify all of the RNAs produced by in vivo grown C. jejuni and develop tools to study regulation during colonization. These experiments will aid in identification of colonization factors and enable the study of their regulatory mechanisms. Uncovering mechanisms of gene expression during colonization is an effort at instituting a one-health approach to studying this pathogen. New transcription and colonization factors identified in this study can be used to develop anti-campylobacter strategies to prevent spread of this pathogen from the environment to animals, thereby limiting human exposure.
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Approach:<br/>
Emerging molecular biology techniques will be used to understand the in vivo regulatory mechanisms of C. jejuni. Unbiased massively parallel sequencing approaches (RNAseq) will be used to map the transcriptomes of in vivo and in vitro C. jejuni. RNA will be isolated from C. jejuni grown in laboratory cultures as well as C. jejuni isolated directly from cecal contents of chickens. In vivo RNA will be isolated from the day-of-hatch chicken model, 7-days post inoculation. cDNA libraries will be constructed based of the Illumina TruSeq methods and sequenced on the Illumina HiSeq2000 platform. Sequences will be mapped back to the C. jejuni 81-176 published genome and bioinformatic analysis will be employed to determine differences in gene expression as well as the identification of novel regulatory elements, including small RNAs and riboswitches. We will also characterize regulatory mechanisms utilizing traditional molecular biology techniques including reporter fusions and qRT-PCR. Luciferase reporter constructs will be used in live animal imaging (IVIS) experiments to determine when and where specific genes are expressed during colonization of the chicken. We will colonize day-of-hatch chicks with strains in which specific promoters have been engineered to control expression of the luxCDABE operon. Luciferase expression will be monitored at specific time points during colonization using Xenogen IVIS Spectrum. These reporter constructs will also be used to identify regulatory mechanisms of the thee zinc transport systems through random transposon mutagenesis. Putative regulatory factors will be identified and the mechanisms of regulation will be characterized using qRT-PCR, EMSA and reporter fusions.