Immune modulation in tuberculosis: Insights from the role of a Mycobacterium tuberculosis protease Hip1 and its substrate Open Access

Georgieva, Maria Georgieva (2015)

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Pathogenic microorganisms have evolved to respond rapidly during infection in order to evade host immunity and cause disease. Mycobacterium tuberculosis employs multiple strategies to evade host immune responses, persist within host phagocytic cells and promote pathogenesis. While substantial evidence points to the importance of hydrolytic enzymes in M. tuberculosis pathogenesis, the molecular and biochemical basis for their function remain largely uncharacterized. We have previously shown that the cell envelope-associated M. tuberculosis serine hydrolase, Hip1 (Hydrolase important for pathogenesis 1), prevents robust macrophage activation and interferes with dendritic cell functions, allowing M. tuberculosis to manipulate the host immune response and promote disease progression. However, the molecular basis for the immunomodulatory function of Hip1 remained unclear. In this dissertation work, we provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Additionally, we show that the M. tuberculosis GroEL2 protein is a direct substrate of Hip1 protease activity. Further, enzymatic studies demonstrate that serine protease inhibitors specifically inhibit cleavage of GroEL2. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during M. tuberculosis growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our investigations also reveal that M. tuberculosis modulates dendritic cell (DC) responses through Hip1-mediated proteolysis of GroEL2. Full length GroEL2 protein induced DC maturation, production of T helper 1 (Th1)-polarizing cytokines and promoted antigen presentation to CD4+ T cells. In contrast, cleavage of GroEL2 abrogated these functions. In summary, our work has identified Hip1-dependent proteolysis of GroEL2 as a novel mechanism of immune modulation in M. tuberculosis. Importantly, our findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against M. tuberculosis.

Table of Contents

Chapter 1 Introduction...p. 1

Chapter 2 Mycobacterium tuberculosis Hip1 modulates macrophage responses through proteolysis of GroEL2

Abstract...p. 31

Introduction...p. 32

Results...p. 35

Discussion...p. 45

Materials and Methods...p. 53

Figures...p. 70

Chapter 3 Modulation of dendritic cell responses through cleavage of Mycobacterium tuberculosisGroEL2

Abstract...p. 87

Introduction...p. 88

Results...p. 91

Discussion...p. 95

Materials and Methods...p. 104

Figures...p. 110

Chapter 4 Discussion...p. 115

Chapter 5 Bibliography...p. 125

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