From mycobacterial native protein method development to graduate student professional development

Abstract

Mycobacterium tuberculosis (Mtb) is a major global health concern, and is currently the leading cause of death due to an infectious disease. Among the challenges impeding the control of tuberculosis (TB) disease are the need for improved diagnostics, vaccines, as well as therapeutics. Research aimed at gaining a better understanding of the basic biology of the organism can aid in the development of these critical control mechanisms. Over the years, a wide array of methods have been developed for the isolation of individual bacterial components, including subcellular fractions, lipids, and native proteins. These isolated biomolecules have contributed to our overall understanding of the bacteria and its interactions with the host. These discoveries have brought the research community that much closer to reaching the goal of ending the TB pandemic. As part of our long-standing collaboration with BEI Resources, our lab has a history of isolating and distributing mycobacterial reagents for the research community at large. In chapter 1 of this dissertation, I will explore the isolation of biomolecules from mycobacterial cells and their utilization for global research, and discuss the management and scientific impact of this reagent program. I will also describe how the isolation of biomolecules such as native proteins has contributed to the understanding of the basic biology of mycobacteria, with examples pulled from my own publication history. Chapter 2 explores various novel methods for the screening and purification of native proteins. We hypothesize that we can exploit a priori knowledge of antigen targets, combined with targeted mass spectrometry, to inform and accelerate the development of purification methods for native proteins. Methods explored in this chapter include the use of multiple reaction monitoring mass spectrometry (MRM-MS) for the screening of protein fractions. It also describes a purification method which exploits protein-protein interaction for the isolation of native proteins, here used for purification of ESAT6 (Rv3875), CFP10 (Rv3874), and EspC (Rv3615c). Chapter 3 of this dissertation will explore characterization of the heparin binding hemagglutinin protein (HBHA) of Mtb. HBHA is a post-translationally modified, multi-functional bacterial adhesin, found to have roles both on the mycobacterial cell surface, as well as intracellularly, and it has exciting potential as a biomarker of latent infection when used in an interferon gamma release assay. In our lab, we observe that Mtb has as many as four bands identified by western blot analysis when probed with anti-HBHA antibody. A comparison of subcellular fractions and HBHA-enriched material used for native protein purification shows that the banding pattern varies among different fractions, and that these banding patterns are stable and reproducible. Some, but not all, of these protein bands exhibit evidence of methylation when probed using an anti-methylated lysine antibody. Therefore, we hypothesize that there are four distinct proteoforms of HBHA with differences in their hemagglutination activity, a measure of the protein's ability to function as an adhesin. To further explore these observations, efforts are underway to chromatographically separate the four proteoforms in order to look at differences in their biological activity as measured by hemagglutination assay, and analyze them by mass spectrometry to identify potential differences in post-translational modification. Finally, Chapter 4 of this dissertation chronicles a very different endeavor undertaken during my PhD, which was the creation of a course-based professional development class for graduate students in the Department of Microbiology, Immunology, and Pathology. Scientific training and expertise are only part of the skill set needed for a career in science. This is particularly true post-graduate school, where PhD level scientists are expected to be leaders in their respective fields. However, the demands of graduate school make it challenging for students to seek out and engage in extracurricular professional development opportunities to learn about other aspects of scientific careers, such as laboratory funding and time management. To address this need, we've created an experimental professional development course where we've brought together resources and expertise from across CSU's campus into a modular series, consisting of three one-credit modules, each of which focuses on the needs of students at different stages of their graduate education. The goal is to provide a setting where students can learn about, discuss, and practice relevant professional skills, and to encourage participation in these activities by making it a credit-bearing course that will contribute to their degree.