Mycobacterium tuberculosis ESX-5 Paralogs Modulate Macrophage Responses and Bacterial Metal Homeostasis

Abstract

University of Washington Abstract Mycobacterium tuberculosis ESX-5 Paralogs Modulate Macrophage Responses and Bacterial Metal Homeostasis Austin Michael Haynes Chair of the Supervisory Committee:Thomas R. Hawn Department of Medicine, Division of Allergy and Infectious Diseases and Department of Global Health Extended co-evolutionary history of humans and Mycobacterium tuberculosis (Mtb), the causative agent of TB disease, has led to an evolutionary arms race between host and pathogen. The processes by which the immune system recognizes and destroys invading pathogens has been extensively co-opted and subverted by Mtb via the evolution of specially adapted virulence effectors. Through a tightly orchestrated bacterial response, Mtb moves these effectors out of the bacterium and into the host cell interface. Traditional bacterial transport systems such as the generalized secretion (SEC) and twin arginine transport (TAT) systems move many effectors, however in Mtb, Type-7 or "ESX" systems play an essential role in virulence processes. Of the five ESX systems in Mtb, some have experimentally defined roles in virulence. ESX-1 is essential for phagosome antagonism while ESX-3 sequesters iron from the host, an essential micronutrient. ESX-5 in contrast is poorly resolved despite secreting a large number of effectors (5-10% of Mtb coding capacity). Based on prior clinical, genetic, and lab-based studies, we hypothesize ESX-5 may play roles in virulence and nutrient acquisition. To date, limited functional work has been performed to fully elucidate functional roles for many ESX-5 secreted putative virulence factors. In complement, the human immune system is persistently evolving in the face of extensive pathogen pressure. The historic and current burden of Mtb infection across modern hominids has pushed human populations to evolve strategies countering pathogens such as Mtb. While humans evolve substantially slower than their mycobacterial counterparts, there is evidence to suggest human populations are actively evolving mechanisms to counter Mtb infection resulting in resistance. Clinically, populations who resist TB infection via persistently negative testing (RSTRs) are being extensively studied to understand the complex genetic interplay resulting in presumed enhanced control of Mtb during early exposure. However, given the complexity of human genetics, the interplay of multiple genes and cell types, and the heterogeneity of Mtb infection itself, we still don't mechanistically understand how RSTRs are able to remain Mtb negative compared to their susceptible counterparts (LTBI). As such we aimed to understand how the genetics of Mtb resistant populations (RSTRs) differ from susceptible individuals (LTBI), affording possible functional insight into resistance processes. Ultimately, we aim to gain functional insight into both human and bacterial genetics to more clearly resolve early Mtb-Host interactions during early infection. Initially, we performed a brief transcriptomic screen of RSTR and LTBI alveolar macrophages to explore the role of human genetics in altering transcriptional responses to early Mtb infection. We demonstrated that alveolar macrophages from these populations display similar but distinct responses. These transcriptional responses in RSTRs are marked by an increased inflammatory response to interferons relative to LTBI counterparts, which could possibly lead to enhanced control of Mtb. Subsequently we investigated the function of specific Mtb gene clusters during early primary macrophage infection. We independently deleted ESX-5a, ESX-5b, and ESX-5c, each containing a PE/PPE heterodimer and Esx heterodimer from the H37Rv strain of Mtb. We then examined the functional consequences of these deletions in macrophages, mice, and axenic bacterial culture. We initially observed that deletion of these gene clusters significantly alters cytokine levels of TNF, IL-6, and IL-1β in primary macrophages. Further analysis revealed this may be due to differential post-transcriptional or translational regulation of target cytokines, resulting in divergent cytokine profiles within infected macrophages. We next examined if this impact observed in human macrophages would impact the fitness of these strains in vivo where we observed early time point growth defects of our mutant strains tested in C57BL/6 mice. Subsequent transcriptome analysis revealed these gene clusters likely play a role in heavy metal response or homeostasis. Indeed, we demonstrated these gene clusters are metal responsive and that their level of expression is correlated to cytokine response levels in primary human macrophages. Together, these observations indicate that both human and bacterial genetics play a role in the outcome of early disease. Not only are these ESX-5 gene clusters involved in stimulating a cellular response in vitro and in vivo, but that these paralogs likely also play a dual role in heavy metal response. These suggest novel function for these previously undefined paralogs and highlight the importance of studying both bacteria and host concurrently to gain novel insight into host-pathogen interactions.