Alterations In Macrophage Motility Underlying Disseminated Mycobacterial Infection

Abstract

Successful intracellular pathogens possess the ability to hijack and exploit host cell machinery to establish persistent infections. Studying the intracellular host-pathogen interface not only enhances our understanding of pathogenicity, but also uncovers roles for host cell proteins that are not apparent in the absence of infection. The work presented here focuses on Mycobacterium tuberculosis, the causative agent of tuberculosis disease and an expert manipulator of host immunity. While studies have established several M. tuberculosis effectors as important for its intracellular lifestyle, identifying host targets has been challenging, and the factors underlying variable disease outcomes during infection with the extensively diverse array of circulating M. tuberculosis strains remain unclear. The introductory chapter of this work will introduce the vast diversity of pathogenic Mycobacterium species to provide context for the analysis of a non-tuberculous mycobacterial outbreak presented in Chapter 2. Next, the introduction will introduce key concepts needed to understand the work presented in Chapter 3 ranging from the variety of circulating tuberculosis strains to the arsenal of effectors used by M. tuberculosis to manipulate host cells. Additionally, this chapter will introduce the host WASH complex and its roles in cytoskeletal remodeling as background for the work presented in Chapter 4. The work presented in Chapter 2 encompasses a bioinformatic analysis of a cluster outbreak of non-tuberculous mycobacteria following hurricane relief efforts. While speciation was not available at the point of care, post-hoc whole genome sequencing revealed that two individuals were infected with distinct, closely related members of the M. terrae complex. A third individual presented with similar symptoms during the study but was infected with a more distantly related species in the M. avium complex. The work in this chapter highlights the importance of next generation sequencing in accurately identifying outbreaks within the rapidly expanding group of non-tuberculous mycobacteria. Chapter 3 describes the analysis of a unique outbreak strain of M. tuberculosis that led to high rates of disseminated skeletal disease. Bioinformatic analysis revealed that the outbreak strain was among the ancestral lineages of M. tuberculosis, and that these lineages secrete a dissemination promoting factor, EsxM. Using the zebrafish-M. marinum model, we show that dissemination is dependent on EsxM in vivo. We also investigated the effects of EsxM on macrophages, a key subcellular niche for M. tuberculosis, and found that EsxM promotes a shift in macrophage migration modality characterized by increased migration velocity and the appearance of spiky projections from the cell surface. We conclude by demonstrating that mycobacterial EsxM may be mediating its effects along the host cytoskeletal axis, and hypothesize that truncation of EsxM in modern M. tuberculosis strains may confer a transmissibility advantage by limiting non-productive dissemination. Next, Chapter 4 addresses the putative host target of EsxM, a member of the WASH complex. The WASH complex is involved in cytoskeletal remodeling of F-actin, with known roles in endocytic recycling. Using two macrophage models, we demonstrated that in the absence of infection, disruption of EsxM target WASH was sufficient to drive changes in cellular morphology similar to those observed in the presence of EsxM. We found that the WASH complex is important for mycobacterial control in a mammalian model of infection, and that in our zebrafish-M. marinum model it has a role in mycobacterial dissemination and macrophage migration. These studies served not only to investigate the putative host target of EsxM, but also to demonstrate novel roles for the WASH complex in macrophages. Finally, Chapter 5 addresses the major conclusions from this work and the questions that remain for future exploration, setting the stage for a deeper understanding of the complex cell biology at the M. tuberculosis host-pathogen interface.