Microbiology

Diaz successfully defends PhD thesis

Diaz Cake

Manisha Diaz successfully defended her PhD thesis, "Regulation of virulence gene expression by Rsm homologs in Pseudomonas aeruginosa," on Friday, April 18, 2014. Diaz is pictured here with her mentor, Timothy Yahr, PhD.

Pseudomonas aeruginosa RsmA belongs to the CsrA family of RNA binding proteins. CsrA family members are post-transcriptional regulators of global gene expression. Previous work from our lab determined that RsmA is required for maximal T3SS gene expression in P. aeruginosa strain PA103. Nevertheless, the molecular mechanism underlying the RsmA-mediated control of T3SS gene expression was unknown. My work shows that RsmA directly binds and stimulates ExsA expression at the post-transcriptional level.

Most Pseudomonas species carry at least two homologs of CsrA on the chromosome, but only one copy had been identified in P. aeruginosa. In collaboration with the Wolfgang lab, we identified a second CsrA homolog, RsmF in P. aeruginosa. I examined the role of RsmF in regulating RsmA-controlled processes associated with acute (T3SS) and chronic (T6SS and biofilm formation) infection. I discovered that while an rsmF mutant alone does not exhibit a phenotype, simultaneous deletion of both rsmA and rsmF significantly accentuates the phenotypes exhibited by an rsmA mutant alone. I also found that RsmA directly binds and represses RsmF translation and that the small regulatory RNAs RsmZ and RsmY do not significantly modulate RsmF activity. Site-directed mutagenesis revealed that Arg 62, located in the β1-β5 fold, is essential for biological activity in vivo and RNA-binding in vitro suggesting a conserved mechanism of RNA recognition maintained across all CsrA family members. Finally, I show that RsmF binds to only a subset of RsmA targets and is not involved in the regulation of all RsmA-controlled processes.

My thesis work also identified high-affinity RNA ligands from a chemically synthesized oligonucleotide library using systematic evolution of ligands by exponential enrichment (SELEX) and high-throughput sequencing. From preliminary analyses of high-throughput sequencing data, the RsmF-binding consensus was determined as 5’-RUACARGGAC-3’, with the ARGGA motif being 95% conserved. Collectively, this work shows that Rsm homologs play important roles in regulating virulence gene expression in P. aeruginosa.

ABOUT MANISHA

Manisha was born and raised in Bangalore, India, by her loving parents, Stephen and Fatima. After graduating from Army Public School (Bangalore) in 2003, she enrolled at the St. Joseph’s College (Arts and Science), Bangalore, and made the extremely strenuous move 3 miles down the road. As an undergraduate, Manisha enjoyed a part-time career in Zoology dissecting leeches, frogs, grasshoppers, and roaches before she decided a life filled with formaldehyde was not what she had envisioned and answered her true calling of being a Microbiologist. With encouragement from her parents, Manisha pursued a master’s degree at Bowling Green State University in Dr. Carol A. Heckman’s lab where she investigated protein-protein interactions involved in the dynamic assembly and disassembly of focal contacts.

After a brief detour in Cell & Molecular Biology, she entered the Microbiology PhD program at The University of Iowa in Fall 2009. She joined Tim Yahr’s lab in the Summer of 2010 and began her research on RsmA, the global post-transcriptional regulator in Pseudomonas aeruginosa. Manisha has made contributions to a review article and research publications. In addition, she has presented her work at multiple conferences including the Midwest Microbial Pathogenesis Meetings in St. Louis, MO, and Milwaukee, WI, and the ASM General Meetings in New Orleans, LA, and Denver, CO.

Outside of lab, Manisha is an avid supporter of the extremely glamorous Indian national cricket team. Manisha is known for her love of spicy food and her talent at game of chance. She loves spending time with her husband Prasath cooking and travelling. She is also an amateur kayaker and hiker.