Award Date

8-1-2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Life Sciences

First Committee Member

Helen Wing

Second Committee Member

Eduardo Robleto

Third Committee Member

Corrella Detweiler

Fourth Committee Member

Laurel Raftery

Fifth Committee Member

Ronald Gary

Number of Pages

170

Abstract

Shigella species, the causal agents of bacillary dysentery, use a type III secretion system (T3SS) to inject two waves of virulence proteins, known as effectors, into the human colonic epithelium to subvert host cell machinery. Transcriptional regulation of these virulence genes is controlled by the three-tiered VirF/VirB/MxiE signaling cascade. Of these, VirB has the largest regulon (~50 genes); however, VirB regulatory properties are poorly understood. To date, VirB is known to function to counter or 'anti-silence' transcriptional silencing mediated by the histone-like nucleoid structuring protein H-NS. To better understand VirB regulation, my colleagues and I chose to better define the biologically relevant DNA-binding site for VirB by engineering a molecular tool to capture protein-DNA interactions in vivo. Equipped with this tool, we found that a sequence organized as a near-perfect inverted repeat is required for VirB to bind DNA in vivo, thus, challenging the previously proposed VirB-binding site. Using our newly defined site, I identified and characterized two VirB regulatory sites upstream of the VirB-dependent ospD1. While OspD1 is an important T3SS effector and temporal regulator of other effectors, the regulation of ospD1 is not well understood. Thus, I identified the ospD1 transcription start site (+1) and showed that the ospD1 promoter is remotely regulated by both VirB and H-NS. This validated our proposed VirB-binding site and provided evidence that cis-acting regulatory sites for transcriptional anti-silencers and silencers, like VirB and H-NS, can lie far upstream of the canonical bacterial promoter region (i.e., -250 to +1).

While investigating the ospD1 promoter, I found a putative MxiE DNA-binding site. This observation led to the discovery of a novel regulatory activity for MxiE and its chaperone IpgC that reveal a negative feedback loop in the VirF/VirB/MxiE cascade. I show that MxiE and IpgC negatively feedback at the virB promoter, thus, decreasing VirB- dependent promoter activity like that at ospD1. Unlike other proteins in the AraC family, of which VirF and MxiE belong, the switch between positive and negative MxiE- and IpgC- dependent regulation does not require a stimulus. Rather, overlapping regions required for negative MxiE- and IpgC-dependent regulation and positive VirF regulation of the virB promoter suggest competitive binding. This novel negative regulation is likely specific to the virB promoter since it was not observed at a different VirF-dependent promoter.

Lastly, I investigated VirB-dependent regulation at the promoters of nine genes previously suggested to be VirB-regulated. My data show that ospC2 and ospC4 are likely directly regulated by both VirB and H-NS. Activity from the remaining promoters were dependent on H-NS (i.e., ipgD and virB), MxiE/IpgC (i.e., ospF), or both (i.e., virA, spa15, and ospC1) making this the first observation of regulatory inputs by both H-NS and MxiE/IpgC at a single gene. Thus, this suggests that the large VirB regulon may be comprised of both directly and indirectly VirB regulated loci. In sum, my dissertation work significantly increases the resolution of complex transcriptional regulatory networks that govern virulence gene expression in the bacterial pathogen Shigella flexneri.

Keywords

MxiE; Shigella; Transcriptional regulation; Type III Secretion System; VirB; VirF

Disciplines

Biology | Microbiology | Molecular Biology

File Format

pdf

File Size

4700 KB

Degree Grantor

University of Nevada, Las Vegas

Language

English

Rights

IN COPYRIGHT. For more information about this rights statement, please visit http://rightsstatements.org/vocab/InC/1.0/

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