Student Theses and Dissertations

Date of Award

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Stebbins Laboratory

Abstract

Salmonella enterica continues to be a significant public health concern, causing an estimated 93.8 million cases of non-typhoidal salmonellosis and 21 million cases of typhoid fever worldwide each year. There are thousands of Salmonella enterica serovars, some with a very specific host set, and others that cause disease in rodents, birds, livestock, domestic fowl, and humans alike. In recent years, there has been much progress in the delineation of Salmonella infection, with the goal of understanding Salmonella pathogenesis at the molecular level. Salmonella produces many different effector proteins capable of interacting with and altering numerous biological pathways in the host – enabling host invasion and intracellular survival as well as dissemination and transmission. We used X-ray crystallography to characterize Gifsy-2 Gene E (GtgE), an effector protease from broad-host serovars of Salmonella, that affords these serovars, at least in part, with the ability to maintain a diverse host repertoire. GtgE modulates vesicular trafficking of the Salmonella-containing vacuole by cleaving Rab GTPases, Rab29, Rab32, and Rab38, thereby preventing the delivery of antimicrobial products to the vacuole. In order to gain an understanding of GtgE’s proteolytic mechanism, we determined the structure of GtgE to 1.65Å using single-wavelength anomalous diffraction, and through structure-based mutagenesis and in vitro activity assays, we established the catalytic triad of GtgE, Cys45-His151-Asp169. We also examined a panel of cysteine protease inhibitors and found that N-ethylmaleimide, chymostatin, and antipain were capable of inhibiting GtgE activity in vitro. Furthermore, through work with the catalytically inactive mutant of GtgE (GtgE-C45A), we were able to identify the conditions necessary to form a stable complex between GtgE and Rab38, which may prove useful for further structural work and reveal the nature of GtgE’s interaction with its Rab GTPase substrates. Additionally, we investigated SipC, a Salmonella translocase protein with two effector domains – an N-terminal actin bundling domain and a C-terminal actin nucleation domain. We sought to define minimal constructs of these effector domains for crystallization studies, and obtained needle-like spherulites with the C-terminal domain. We also showed that mouse Exo70 is able to pulldown the C-terminal domain of SipC from cell lysate. Finally, we examined Salmonella effector AvrA, which has been attributed with having deubiquitinase and acetyltransferase activity, although its role in Salmonella pathogenesis remains poorly understood. We determined a minimal construct of AvrA that contains the proposed catalytic triad, and through a yeast two-hybrid experiment, identified ARFGEF2 as a potential interacting partner for AvrA.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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