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Indentification of Novel APEC Virulence Genes via Functional Genomics

Nolan, Lisa
Iowa State University
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Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is a multimillion dollar annual problem for this country s poultry industry. Also, recent recognition that APEC may be a food-borne source of E. coli causing human disease, suggests that control of avian colibacillosis may be highly desirable for reasons of both animal and human health. Unfortunately, control of this disease has been elusive, since the virulence mechanisms used by APEC are poorly understood, hampering identification of rational control targets. Also, evidence suggests that APEC are capable of rapid change, meaning that the effectiveness of current control measures may be fleeting. Recent completion of the first APEC genomic sequence (APEC O1) provides an unprecedented opportunity to explore mechanisms of APEC virulence. Once identified, these mechanisms can be used as the basis of novel control strategies with long-term efficacy. In an initial effort to exploit APEC O1s genomic sequence to better understand APEC virulence, we will test the hypothesis that APEC genes are differentially regulated when APEC is grown under conditions simulating infection or in its natural host. To accomplish this goal, we have constructed the first ever APEC DNA microarray. This array, containing probes for each unique gene of APEC O1, will be used to perform a transcriptome analysis of APEC O1, grown under culture conditions or under conditions simulating infection (here we use chicken body temperature). Identification of genes that undergo differential expression will identify putative targets for future control and develop preliminary data that will make us competitive for federal funding. We feel that these studies have great potential to lessen the impact of colibacillosis and provide a roadmap for others seeking to exploit genomic data to enhance disease control.
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NON-TECHNICAL SUMMARY: A major challenge to all bacteria is adaptation to changing environmental conditions. This adaptation involves regulating gene expression in response to different environmental signals. Expression of genes that are required in a particular situation will be up-regulated, whereas unnecessary functions in the same situation will be down-regulated. We hypothesize that the same principle can also be applied to virulence genes, which should also be subject to regulatory mechanisms that ensure expression under the appropriate conditions. In a first effort to exploit the APEC genome for the benefit of poultry producers, we constructed the first ever APEC pangenomic microarray and will use it to gain insight into APEC virulence. We believe these insights will enable us to reduce the impact of colibacillosis on production birds. This microarray, containing probes for each unique open-reading frame (ORF) of the APEC O1 genome, will be used to perform a transcriptome analysis of APEC O1 under different temperatures including those mimicking host infection. Identification of genes that undergo differential expression under conditions mimicking host infection, as opposed to routine culture, will pinpoint putative targets for future control.

APPROACH: Specifically, to test our hypothesis, we will assess differential regulation of the APEC pangenome under conditions simulating infection using the APEC pangenome array, verify the results using real-time RT- PCR, and assess the contributions of the top-most upregulated genes to virulence using site-directed mutagenesis techniques.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Escherichia coli
Bacterial Pathogens
Viruses and Prions
Meat, Poultry, Game