Date of Award

Summer 8-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Enterotoxigenic Escherichia coli (ETEC) are one of the leading causes of death due to diarrhea in children living in developing countries. ETEC are also the leading cause of diarrhea in travelers to developing countries lacking sanitation and safe drinking water. Unfortunately, there is no broadly protective vaccine available against these important pathogens. In order to cause infection, ETEC colonize the intestinal epithelium and secrete toxins, including heat-labile toxin (LT) and/or heat-stable toxin (ST). Efficient delivery of these toxins to the cognate receptors on target intestinal cells requires direct ETEC-host interactions. Earlier studies demonstrated that ETEC facilitate interactions with host by pathotype specific adhesive pili, termed as colonization factor (CF). However, recent studies demonstrate that in order to establish interactions with host, ETEC engage multiple factors, including number of ETEC-specific virulence factors as well as factors those are highly conserved across different E. coli.

Detection of increased expression of type 1 pili encoding genes from cell adherent ETEC led to the hypotheses that these pili facilitate ETEC adhesion to intestinal epithelial cells and promote delivery of toxins to target cells. Using polarized culture of intestinal cell lines as well as human ileal enteroids, we observe that FimH adhesin of type 1 pili facilitate ETEC adhesion, antibody mediated or chemical inhibition of FimH interaction decrease ETEC adhesion and increased expression of highly mannosylated glycoprotein receptor of FimH enhance ETEC adhesion to host. Additionally, we find that FimH interactions augment delivery of toxins and are required for virulence, as we detect reduced fluid accumulation by fimH mutant in the rabbit ileal loop assays, a standard model of ETEC pathogenesis.

Although earlier studies reported that LT enhances ETEC-host interactions, the underlying enhancement mechanism is undefined. Using RNAseq and mass spectrometric analysis we demonstrate that LT enhances glycoprotein synthesis and increases surface presentation high mannose glycans. Additionally, LT increases expression of CEACAM6 receptor of FimH adhesin on intestinal epithelial cells. Interestingly, deletion of FimH-CEACAM6 interactions decreased ETEC adhesion to host, identifying a novel ETEC-host interaction. However, in contrast to the contribution of FimH in adhesion, deletion of FimH activity increased colonization of mice by ETEC. Interestingly, we detected increased expression of several adhesion related factors, including PqiB, a homologue of Mam7 adhesin, by fimH mutant recovered from mice, signifying the importance of functionally complementary adhesins of ETEC. Together, these findings propose a mechanistic explanation of LT-mediated enhancement of ETEC adhesion, and identify a novel ETEC-host interaction.

Collectively, data presented in the thesis demonstrate the contribution of type 1 pili in ETEC pathogenesis, notice previously unidentified FimH-CEACAM6 interactions of ETEC and identify a highly conserved putative Mam7 adhesin that may facilitate intestinal colonization by ETEC. Overall, these findings expand our understanding of ETEC pathogenesis and could supplement rational approach to broadly protective ETEC vaccine design.

Language

English (en)

Chair and Committee

James M. Fleckenstein

Committee Members

Scott J. Hultgren, Phillip I. Tarr, Thaddeus S. Stappenbeck, Mark J. Miller,

Comments

Permanent URL: https://doi.org/10.7936/K7KD1XBP

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