Sandeel Ahmed

Document Type

Thesis

Date of Award

5-2015

School/College

College of Science, Engineering, and Technology (COSET)

Degree Name

MS in Biology

First Advisor

Professor Jason A. Rosenzweig

Abstract

Yerslnia -pestis, a Gram-negative bacterium, has been responsible for widespread devastation such as the Black Death plague. To escape from phagocytic killing of host cells, Y pestis delivers a set of virulence proteins, called Yersinia outer membrane proteins (Yops), across the cell membrane. Yops are at the core . of the Yersinia pathogenicity arsenal. A set of Yop secretion genes encode the proteins of a type III secretion (T3SS) apparatus. Translocated Yops inhibit phagocytosis by disturbing events in the host cell response such as actin polymerization and apoptosis. Previous studies have indicated that ribonucleases are virulence-associated factors in several notable Gram-negative pathogens including yersiniae, salmonellae, Helicobacter pylori, Shigella flexneri and Aeromonas hydrophila. Studies have also shown that several ribonucleases playa pivotal part in the cold shock response whereby they regulate the changing transcript landscape in response to the stress, as well as during acclimation and post-stress subsequent genetic re-programming. The objectives of this research include determining whether RNase II and/or RNase R are required for cold growth, for oxidative stress responses (as well as for coupled oxidative and cold stress response), and for 1 2 optimal virulence. The purpose of this study was to determine if RNase II and/or RNase R playa role in either yersiniae virulence or various stresses responses. To achieve this, Y pestis cultures were grown to saturation and used to infect cultured mammalian cells. "'- These studies employed the biosafety level 2 (BSL-2) exempt and avirulent pgm- YP KIMD27sm wild type strain and its various isogenic mutants generated by either the lamb􁪽a red system or by a suicide plasmid vector; the genes encoding YopB, PNPase, RNase R, and RNase II were deleted using a recombination-based method, thus creating the isogenic mutant strains Syopli, Spnp, Srnr, and Srnb, respectively. Strains were exposed to Hydrogen Peroxide-based oxidative stress tests, proliferations assays, and cold growth assays. RNase II was shown to be required for cold stress response but appeared dispensable for oxidative stress response and bacterial virulence. RNase R, however, did appear to play a role in bacterial virulence, but did not appear to be a requirement for either cold growth or resistance to oxidative stress.

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