Vincent Isabella

Award

  • 2010 - Awarded IPNC 2010 Trainee Travel Award from NIH
  • 2010 - Winner of 2010 Melville A. Hare Award for the best Ph.D. thesis in Microbiology and Immunology
  • 2007 - Winner of 2007 Melville A. Hare Award for Excellence in Teaching

Bio

Photo of Vincent Isabella, Ph. D.Vincent graduated from the Rochester Institute of Technology with a Bachelor of Science in Biotechnology in 2005. That same year, Vincent began graduate studies at the University of Rochester in the Department of Microbiology and Immunology. His research interests remained in the field of bacteriology, and he began his professional training in the laboratory of Virginia Clark in 2006. Vincent completed his Ph.D in the fall of 2010 for the study of the pathogenic organism N. gonorrhoeae. During that time, Vincent received awards for both distinction in research and for the teaching of an undergraduate microbiology laboratory course. Vincent has had the opportunity to present the work he completed at the University of Rochester at numerous meetings, including abstract presentations at the American Society for Microbiology (2007), and the International Pathogenic Neisseria Conference (2008 and 2010). Vincent has also mentored numerous undergraduate and graduate rotation students in the laboratory.

Research Description

The ability of Neisseria gonorrhoeae to reduce nitric oxide (NO) may have important immunomodulary effects on the host during infection. Vincent’s work began by deciphering the complex regulatory mechanism of the gonococcal nitric oxide reductase gene (norB). He discovered that NO induces norB gene expression, and that this regulatory control is exerted through nsrR, an Rrf2-type transcriptional repressor. He also determined that Fur, an iron-responsive regulatory protein, controls norB expression by a novel indirect activation method, by preventing the binding of a gonococcal ArsR homolog, a second repressor whose putative binding site overlaps that of the Fur binding site. In addition, NsrR regulates expression of other gonococcal promoters, including aniA (encoding nitrite reductase), dnrN (encoding a reactive oxygen/nitrogen repair protein), and nnrS (function unknown).

Vincent was the first to purify a functional NsrR protein for use in in vitro studies. A highly pure extract of epitope tagged NsrR isolated and mass spectroscopic analysis demonstrated that the protein contained a [2Fe-2S] cluster. Using EMSA analysis, NsrR::FLAG Vincent showed that it interacts with predicted operators in the norB, aniA, and nnrS upstream regions with a Kd of 7 nM, 19 nM, and 35 nM respectively. DNase I footprint analysis on the upstream regions of norB and nnrS, showed that NsrR protects the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97, or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding. Mass spectroscopic analysis of NsrR C90A demonstrated the absence of a [2Fe-2S] cluster, consistent with the presumed role of these cysteine residues in the coordination of a NO-sensitive Fe-S center required for high affinity DNA binding.

Because an anaerobic lifestyle may be important during the course of gonococcal infection, Vincent performed deep sequencing experiments to compare the gonococcal transcriptomes of aerobic and anaerobically grown cells. His work helped to identify the anaerobic stimulon in gonococci, which was found to be larger than expected. He found that 198 chromosomal genes were differentially expressed in response to anaerobic conditions (~10% of the genome), as well as a large induction of genes encoded within a cryptic plasmid, pJD1. Validation of RNA-seq data using translational-lacZ fusions or RT-PCR demonstrated the RNA-seq results to be very reproducible. Surprisingly, many genes of prophage origin were induced anaerobically, as well as several transcriptional regulators previously unknown to be involved in anaerobic growth. This work also helped in the discovery of an oxygen-responsive small regulatory RNA, likely a functional equivalent of fnrS in the Enterobacteriaceae family.

RNA-seq analysis showed that anaerobic growth induced expression of many genes of unknown function. Mutation of one such gene NGO1024, encoding a protein belonging to the 2-nitropropane dioxygenase-like superfamiliy of proteins results in an inability of gonococci to grow anaerobically. Anaerobic growth of an NG1024 mutant was restored upon supplementation with unsaturated fatty acids (UFA), but not with the saturated fatty acid palmitate. Gonococcal fatty acid profiles confirmed that NGO1024 was involved in UFA synthesis anaerobically, but not aerobically, demonstrating that gonococci contain two distinct pathways for the production of UFAs, with a yet unidentified aerobic mechanism, and an anaerobic mechanism involving NGO1024. NGO1024 homologs form a distinct lineage within the 2-nitropropane dioxygenase-like superfamily, and are found in many facultative and obligate anaerobes that produce UFAs but lack any recognizable UFA sysnthesis machinery, suggesting that UfaA is part of a widespread pathway involved in UFA synthesis.

Articles Published

  1. Isabella, V. M., and V. L Clark (2011). Identification of a conserved protein involved in unsaturated fatty acid synthesis in N. gonorrhoeae: implications for facultative and obligate anaerobes that lack FabA. (Submitted)
  2. Spence, S. A., Clark, V. L., and V. M. Isabella (2011). The role of catalase in gonococcal resistance to peroxynitrite. (Submitted).
  3. Isabella, V. M., and V. L. Clark (2011). Deep-sequencing based analysis of the anaerobic stimulon in Neisseria gonorrhoeae. BMC Genomics, 12: 51.
  4. Clark, V. L., Isabella, V. M., Barth, K., and T. Overton (2010). Regulation and function of the Neisserial denitrification pathway: life with limited oxygen. Caroline Genco and Lee Wetzler, editors. Neisseria: ¬Molecular Mechanisms of Pathogenesis. Horizon Scientific Press, Norwich, UK.
  5. Barth, K., Isabella, V. M., and V. L. Clark (2009). Biochemical and genomic analysis of the denitrification pathway within the Neisseria genus. Microbiology. 155: 4093-4103.
  6. Barth, K., Isabella, V. M., Wright, L., and V. L. Clark (2009). Resistance to reactive nitrogen species in Neisseria gonorrhoeae. Microbiology. 155: 2532-2545.
  7. Isabella, V. M., Lapek, J. D., Kennedy, E. M., and V. L. Clark (2009). Functional analysis of NsrR, a nitric oxide-sensing Rrf2 repressor in Neisseria gonorrhoeae. Mol. Micro. 71:227-239.
  8. Isabella, V. M., Wright, L., Barth, K., Spence, J. M., Grogan, S., Genco, C. A., and V. L. Clark (2008). cis- and trans-acting elements involved in regulation of norB (norZ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae. Microbiology. 154: 226-239.
  9. Wagner, V. E., Li, L. L., Isabella, V. M., and B. H. Iglewski (2007). Analysis of the hierarchy of quorum-sensing regulation in Pseudomonas aeruginosa. Anal. Bioanal. Chem. 387:469-479.

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