The goal of the Purdy Lab is to further define the dynamic interface between the human pathogen Mycobacterium tuberculosis and the host macrophage. Tuberculosis has re-emerged as a global health concern. The World Health Organization estimates that Mycobacterium tuberculosis (Mtb) infects one third of the world population. The ability to survive and multiply within the host macrophage is key to its pathogenesis. In resting macrophages, the bacterium arrests phagosome maturation and replicates in a compartment that resembles early endosomes. Activated macrophages promote clearance of mycobacteria through both non-oxidative and oxidative mechanisms: In activated macrophages there is increased fusion of the mycobacteria-containing vacuole with the lysosome. We previously showed that Ubiquitin-derived peptides (Ub-peptides) contribute to the bactericidal activity of the lysosome. Activation also triggers the production of reactive oxygen and nitrogen intermediates (ROI and RNI). M. tuberculosis possesses mechanisms to resist environmental stresses imposed by the host immune system. These stress response pathways are necessary for the bacterium to establish and maintain infections. Current projects funded by NIH focus on 1) defining the mechanisms by which mycobacteria response to and resist the antimicrobial repertoire of the macrophage and 2) understanding the biosynthesis and immunomodulatory properties of the Mtb cell wall.
Dr. Purdy is accepting rotation students. Students who join the Purdy laboratory can expect to gain experience in a wide range of techniques. Most research projects will include some or all of the following:
- Genomic analyses We use DNA microarrays and bioinformatic approaches to define mycobacterial stress response regulons.
- Bacterial and molecular genetics Our approaches include transposon mutant screens to identify Mtb mutants with specific phenotypes and site-directed mutagenesis of proteins of interest.
- Biochemistry We routinely perform protein purification and are characterizing the function of several classes of proteins. In addition, we use chromatography, mass spectroscopy and differential scanning calorimetry to better understand the architecture and composition of the mycobacterial cell wall.
- Immunobiology We routinely assess the ability of wild type and mutant Mtb strains to infect macrophages. We use microscopy to define the niche established by the bacterium in these immune cells and we use functional readouts such as cytokine ELISAs to assess the response of macrophages to infection. We use cell biology techniques to study the mycobactericidal properties of macrophages.
- Structural biology Ongoing studies in collaboration with other groups, will determine the structure of host antimicrobial peptides that kill Mtb and other bacterial pathogens.
Activated macrophages kill TB in the lysosome in a process that involves ubiquitin-derived peptides. Click to enlarge.
A major project in the lab focuses on bacterial mutants with altered susceptibility to antimicrobial ubiquitin peptides. This project will elucidate the mode of action of ubiquitin-derived peptides through identification and characterization of mycobacterial hyper-susceptible and hyper-resistant mutants and will define the role of host antimicrobial peptides in M. tuberculosis infection. The characterization of mycobacterial mutants hyper-resistant to the ubiquitin-derived peptide Ub2 will give insight into the bacterial targets of these peptides. Many pathogenic bacteria have developed resistance mechanisms to reduce the affects of cationic antimicrobial peptides, such as modulating the bacterial cell wall and exporting antimicrobial peptides using efflux or ABC transport systems. Isolation and characterization of mycobacterial hyper-susceptible mutants will identify antimicrobial-resistance mechanisms employed by M. tuberculosis.
The mycobactericidal action of macrophages is multifold and includes the generation of reactive oxygen and nitrogen intermediates in addition to non-oxidative processes such as the delivery of the bacterium to the lysosome and the action of ubiqutin-derived peptides. The contribution of antimicrobial susceptibility and resistance factors to M. tuberculosis virulence will be determined through macrophage survival assays and in mouse infection studies.
These studies will further define the mechanism by which ubiquitin-derived peptides are antimycobacterial, identify potential targets of antimycobacterial treatment, and determine the contribution of these factors to mycobacterial virulence. Since antimicrobial peptides are a crucial component of the innate immune response, the project will contribute to our understanding of the pathogenic mechanisms of M. tuberculosis and give insight into the importance of host antimicrobial peptides to the course of disease using the mouse model of infection. It is envisioned that future work will further define bacterial resistance mechanisms through biochemical and molecular approaches.