UCSF

Mycobacterium tuberculosis, an acid-fast bacillus, is a highly successful intracellular pathogen that is estimated to infect approximately one third of the world population. A keystone feature of M. tuberculosis pathogenesis is the parasitism of host macrophages following phagocytic entry, which is thought to be facilitated by the bacteria's ability to prevent the maturation of the phagosome into an acidic, hydrolytic compartment. While M. tuberculosis-mediated phagosome maturation arrest (PMA) is a well-documented phenomenon, bacterial factors responsible for altering phagosome trafficking are not well understood. The overall goal of the work presented here is to decipher the molecular mechanisms employed by M. tuberculosis to mediate PMA during macrophage infection. The first part of this thesis describes a genetic screen that identified several M. tuberculosis mutants defective for PMA. Interestingly, a subset of the trafficking mutants were also defective for Esx-1 secretion, establishing a role for this specialized protein secretion system in mediating PMA. We show that mutants for two known substrates of the Esx-1 pathway did not exhibit trafficking defects, leading us to hypothesize that other effectors of the system may be involved in mediating PMA. These studies reveal a previously unappreciated phenotype of Esx-1 mutants and suggest a novel pathogenic function for this secretion pathway. The second part of this thesis describes our efforts to characterize one of the genes required for PMA, espC, which we show is required for Esx-1 secretion. We demonstrate that, in addition to being required for Esx-1 secretion, espC encodes a substrate of the system, consistent with the mutually-dependent secretion observed with all characterized Esx-1 substrates. We show that secreted EspC associates in a high molecular weight complex that exhibits sphingomyelinase activity. We propose that a protein complex containing EspC may be involved in targeting a sphingomyelinase activity to phagosomal membranes during host infection. By generating ceramide on host membranes, such a sphingomyelinase activity represents an intriguing host-pathogen interaction with the potential to modulate the host response to infection.