• Characterization of Enterotoxigenic Escherichia coli Isolates of Varying Clinical Presentations using Comparative Genomics and Transcriptomics

      Sistrunk, Jeticia R.; Rasko, David A.; 0000-0001-9129-6529 (2017)
      Enterotoxigenic Escherichia coli (ETEC) is a non-invasive, diarrheagenic pathovar of E. coli that causes 40 million cases of Travelers' Diarrhea and 500,000 deaths every year. ETEC virulence is characterized by the production of plasmid-encoded enterotoxins that typically lead to a profuse watery diarrheal illness. However, ETEC has been isolated from study participants exhibiting a range of clinical presentations including severe and mild diarrhea, and even asymptomatic colonization. Genomic sequencing of large collections of > 90 ETEC isolates representing diverse toxin and colonization factor genotypes were conducted in order to characterize and identify the genetic features associated with varying clinical presentations seen in ETEC. The comparative genomics methodologies identified distinct sequence types, new putative colonization factor variants, as well as relevant vaccine candidates in these isolates. Additionally, comparative transcriptomics of a diverse set of four ETEC isolates was assessed in response to the host product, bile, in nutrient-rich and nutrient-limited media to characterize variations in transcriptional regulation ETEC virulence genes. RNA-Seq and qRT-PCR on four ETEC isolates identified transcriptomic differences, even amongst two isolates associated with severe disease, which indicated that diverse transcriptional regulatory profiles exist within ETEC. The transcriptomics analysis also revealed bileassociated differential expression of colonization factor, transcriptional regulator, and motility genes within the ETEC isolates. Lastly, a unique AraC-family transcriptional regulator, peaR, was identified in severe, non-prototype ETEC isolate 2729250 as significantly up regulated in response to bile. In silico comparison of PeaR to the canonical ETEC AraC-like regulator, Rns, revealed PeaR has a similar peptide sequence and may have similar functions in regulating colonization factors. Mutagenesis of peaR revealed differences in motility and colonization factor gene expression compared to wild type suggesting that PeaR may be able to regulate the CS5 and/or CS6 colonization factor genes. Collectively, these studies demonstrate the use of next-generation sequencing technologies to characterize pathogenic determinants of ETEC. Future investigation that incorporates proteomic and in vivo analysis of transcriptional regulators, such as peaR, incorporates proteomic and in vivo analysis of transcriptional regulators, such as peaR, may reveal important pathogenic mechanisms that explain the differing clinical presentations seen in ETEC-associated diarrhea.
    • Novel Roles for Two Proteins Required for Type IV Pilus Expression in Enteropathogenic Escherichia coli

      De Masi, Leon; Donnenberg, Michael S. (2012)
      Enteropathogenic Escherichia coli (EPEC) causes diarrhea among infants in developing countries. The Bundle Forming Pilus (BFP) is a type IV pilus found on the surface of EPEC and essential for pathogenesis. The machinery for BFP polymerization is encoded by an operon of 14 genes. To examine the role of BfpI, a putative pilin-like protein, and BfpL, a protein of unknown function, in expression of BFP, deletions of bfpI and bfpL were created in wild type EPEC and a bfpF mutant deficient for an ATPase required for pilus retraction. I found that both bfpI and bfpL mutants were deficient for BFP expression and BFP associated phenotypes in a wild type EPEC background. However a bfpF bfpI double mutant still expressed BFP while a bfpF bfpL double mutant did not. Western blots of sheared pilus preparations did not suggest that BfpL is a component of BFP. Topology studies using C-terminal truncations and a dual-reporter revealed that the majority of BfpL lies in the periplasm. In addition I demonstrated that BfpL interacts with the periplasmic face of BfpC, a bitopic inner membrane protein, by yeast 2 hybrid assays and confirmed this finding by fluorescence anisotropy. I examined the rate of pilus extension and retraction over time using flow cytometry and found that the bfpF/I double mutant expresses more surface bundlin than the bfpF mutant, but when these strains were complemented with a vector containing bfpF and placed in conditions inducing retraction, the double mutant had less surface expression over time than the bfpF mutant. These data indicate that BfpL is absolutely required for bundle-forming pilus biogenesis and associates with the inner membrane subassembly complex, while BfpI is not absolutely required for biogenesis and impedes pilus retraction.