THE ROLE AND MECHANISM OF PARAHYDROXYBENZOIC ACID DERIVATIVES IN BOVINE TB PATHOGENESIS

Bovine tuberculosis (bTB), caused by Mycobacterium bovis imposes a significant financial burden on the global cattle industry. Although considerable effort is being spent developing vaccines for bTB disease prevention and control, a licensed vaccine for use in cattle has yet to emerge. Mycobacterial cell envelope antigens are known to interact with the host immune system and contribute to TB pathogenesis. Thus, a better understanding of the effects of M. bovis cell envelope antigens on the bovine immune system will aid the rational development of effective bTB vaccines. In this thesis, I first report a comparative analysis of the immune-stimulatory effects of various fractions of the M. bovis cell envelope on bovine dendritic cells (bDCs), which are key immune cells required for TB control by the host. I found that the cell surface sugar extract (CSSE) fraction is the least immune-stimulatory suggesting that this fraction might contain an immunosuppressive molecule. Given that CSSE fractions of the M. tuberculosis complex are enriched in phenolic glycans, including para-hydroxy benzoic acid derivatives (p-HBADs), which are known to have immunosuppressive properties, I then examined the immunomodulatory effects of p-HBAD-1, the major p-HBAD made by M. bovis on bDCs. I found that p-HBAD-1 has opposing effects in non-primed and IFN-γ primed bDCs in vitro. In non-primed bDCs, p-HBAD-1 induces a tolerogenic response, while a pro-inflammatory response is observed with IFN-γ primed bDCs. These findings suggest that M. bovis p-HBAD-1 is an immune-regulatory molecule that might have a dual function in bTB pathogenesis. Finally, I successfully prepared plasmid constructs required to disrupt the gene encoding a key enzyme involved in p-HBAD synthesis in M. bovis. These will be used in future efforts to generate an M. bovis mutant deficient in p-HBAD-1 for detailed studies in animal models of TB

Leishmania major-derived lipophosphoglycan influences the host’s early immune response by inducing platelet activation and DKK1 production via TLR1/2

BackgroundPlatelets are rapidly deployed to infection sites and respond to pathogenic molecules via pattern recognition receptors (TLR, NLRP). Dickkopf1 (DKK1) is a quintessential Wnt antagonist produced by a variety of cell types including platelets, endothelial cells, and is known to modulate pro-inflammatory responses in infectious diseases and cancer. Moreover, DKK1 is critical for forming leukocyte-platelet aggregates and induction of type 2 cell-mediated immune responses. Our previous publication showed activated platelets release DKK1 following Leishmania major recognition.ResultsHere we probed the role of the key surface virulence glycoconjugate lipophosphoglycan (LPG), on DKK1 production using null mutants deficient in LPG synthesis (Δlpg1- and Δlpg2-). Leishmania-induced DKK1 production was reduced to control levels in the absence of LPG in both mutants and was restored upon re-expression of the cognate LPG1 or LPG2 genes. Furthermore, the formation of leukocyte-platelet aggregates was dependent on LPG. LPG mediated platelet activation and DKK1 production occurs through TLR1/2.ConclusionThus, LPG is a key virulence factor that induces DKK1 production from activated platelets, and the circulating DKK1 promotes Th2 cell polarization. This suggests that LPG-activated platelets can drive innate and adaptive immune responses to Leishmania infection