Mycobacteria-macrophage interactions
The receptor/ligand interactions mediating uptake of M. tuberculosis by host cell macrophages are being characterised. Current studies are directed at understanding the role of CD43 (sialophorin) and CD11b/CD18 (complement receptor 3) in the binding of TB to macrophages and at determining whether or not the survival and replication of M. tuberculosis within macrophages is influenced by the receptor and/or the type of macrophage which mediates the uptake of the mycobacterium. Knowledge about this interaction should help us to design novel strategies and therapeutics that will subvert the ability of the bacteria to survive in the host cell. I am also investigating the identity of the bacterial cell wall ligands that bind to these receptors.

The role of the mycobacterial capsule
I have shown that the capsule, or Outer Layer of the Cell Wall Envelope (OL-CWE), of M. tuberculosis acts to limit its interaction with macrophages and that the OL-CWE of opportunistic pathogens (e.g. M. avium) and saprophytes (e.g. M. smegmatis) does not have a similar function. I hypothesize that the OL-CWE of M. tuberculosis acts to limit the interaction of the bacteria with macrophages in a similar fashion to that of the capsule present on other microbial pathogens. I am currently investigating the role that the OL-CWE of M. tuberculosis plays in the pathogenesis of the bacteria. The specific objectives of this study are : 1) To characterize the OL-CWE of intracellular and axenically grown M. tuberculosis; 2) To identify and characterize the component(s) within the OL-CWE of M. tuberculosis that mediate the observed inhibition of the association of M. tuberculosis with macrophages; 3) To construct and characterize OL-CWE mutants.

Molecular definition of mycobacterial virulence factors
We are attempting to identify genes and their products that enable mycobacteria to survive and multiply within their host. This will be achieved by identifying genes that are only found in virulent isolates of mycobacteria but are absent in avirulent isolates. Using virulent and isogenic avirulent mutant MAC strains, genes conferring virulence are being identified using a number of approaches (proteomics, 2 dimensional DNA analysis, characterization of transposon mutants). Once these genes have been identified and characterised, we hope to be able to design novel strategies to nullify these genes or their products and thus compromise the survival of the mycobacteria. In addition, array analysis of a virulent and avirulent pair of TB strains is being used to identify expression differences within the bacteria during their interaction with macrophages.

Stokes, R.W., Norris-Jones, R., Brooks, D.E., Beveridge, T.J. , Doxsee, D. and Thorson, L.M.,“The glycan rich outer layer of the cell wall envelope of Mycobacterium tuberculosis acts as an anti-phagocytic capsule limiting the association of the bacterium with macrophages.” Infection and Immunity, 72 : 5676-5686 (2004).

Wang, J., Thorson, L., Stokes, R.W., Santosuosso, M., Huygen, K., Zganiacz, A., Hitt, M., and Xing, Z. “Single Mucosal, but not Parenteral, Immunization with Recombinant Adenoviral-Based Vaccine Provides Potent Protection from Pulmonary Tuberculosis ” Journal of Immunology, 173 : 6357-6365 (2004).

Randhawa, A.K., Ziltener, H.J. and Stokes, R.W. “Characterization of the interaction of macrophage CD43 with Mycobacterium tuberculosis " Canadian Journal of Clinical Pharmacology 10: 99-100 (2003).

Malloff, C.A, Dullaghan, E.M., Li, A,H., Stokes, R.W., Fernandez, R.C., and Lam, W.L. “ Two-dimensional DNA displays for comparisons of bacterial genomes. Biological Procedures Online 5 , 143-152. (2003)

Dullaghan, EM, Malloff , CA , Li, AH, Lam, WL and Stokes, RW "Two-Dimensional Bacterial Genome Display: A Method For The Genomic Analysis Of Mycobacteria" Microbiology 148: 3111-3117 (2002) * highlighted in "Hot of the Press" in Microbiology Today 29, 210 Nov 2002.

Thorson, LM, Doxsee, D, Scott, M, Wheeler, P and Stokes, RW. "The effect of mycobacterial phospholipids on the interaction of Mycobacterium tuberculosis with macrophages" Infection and Immunity 69: 2172-2179 (2001).

Melo , MD , Catchpole, IR, Haggar, G. and Stokes, RW. "Utilization of CD11b knockout mice to characterize the role of complement receptor 3 (CR3, CD11b/CD18) in the pathogenesis of Mycobacterium tuberculosis." Cellular Immunology 205: 13-23 (2000).

Melo, MD Stokes, RW "Interaction of Mycobacterium tuberculosis with MH-S, an immortalized murine alveolar macrophage cell line: a comparison with primary murine macrophages." Tubercle 80: 35-46 (2000).

Lisa Thorson - Lab manager & technician
Amanda Rooyakkers – Technician

Jennifer Lynett – Graduate student

Alice Li – Graduate student

April Randhawa – Graduate student

Tyler Hickey – Graduate student