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ABSTRACTShigella flexneriis a Gram-negative pathogen that invades the colonic epithelium and causes millions of cases of watery diarrhea or bacillary dysentery predominately in children under the age of 5 years in developing countries. The effector Shigellaenterotoxin 2 (ShET2), or OspD3, is encoded by the senor ospD3gene on the virulence plasmid. Previous literature has suggested that ospD3is in an operon downstream of the ospC1gene, and expression of both genes is controlled by a promoter upstream of ospC1. Since the intergenic region is 328 bases in length and contains several putative promoter regions, we hypothesized the genes are independently expressed. Here we provide data that ospD3and ospC1are not co-transcribed and that OspC1 is not required for OspD3/ShET2 function. Most importantly, we identified strong promoter activity in the intergenic region and demonstrate that OspD3/ShET2 can be expressed and secreted independently of OspC1. This work increases our understanding of the synthesis of a unique virulence factor and provides further insights into Shigellapathogenesis.

ABSTRACTShigellaspecies Gram-negative bacteria which cause a diarrheal disease, known as shigellosis, by invading and destroying the colonic mucosa and inducing a robust inflammatory response. With no vaccine available, shigellosis annually kills over 600,000 children in developing countries. This study demonstrates the utility of combining high-throughput bioinformatic methods with in vitroand in vivoassays to provide new insights into pathogenesis. Comparisons of in vivoand in vitrogene expression identified genes associated with intracellular growth. Additional bioinformatics analyses identified genes that are present in S. flexneriisolates but not in the three other Shigellaspecies. Comparison of these two analyses revealed nine genes that are differentially expressed during invasion and that are specific to S. flexneri. One gene, a DeoR family transcriptional regulator with decreased expression during invasion, was further characterized and is now designated icgR, for intracellular growth regulator. Deletion of icgRcaused no difference in growth in vitrobut resulted in increased intracellular replication in HCT-8 cells. Further in vitroand in vivostudies using high-throughput sequencing of RNA transcripts (RNA-seq) of an isogenic ?icgRmutant identified 34 genes that were upregulated under both growth conditions. This combined informatics and functional approach has allowed the characterization of a gene and pathway previously unknown in Shigellapathogenesis and provides a framework for further identification of novel virulence factors and regulatory pathways.