Jeffery Cox - US grants

DESCRIPTION (provided by applicant) Tuberculosis (TB) is a persistent lung infection that has plagued mankind for centuries and ranks as one of the most serious threats to world health today. The 2-3 million deaths attributed yearly to the disease, as well as the emergence of strains resistant to all of the available chemotherapeutic agents, urgently call for the development of new therapies to treat TB. For years, the identification of new drug targets has been hampered by the intractability of the bacillus to genetic analysis. Now with the advent of powerful genetic tools, combined with well-established mouse infection models, we have isolated novel M. tuberculosis mutants with lesions in individual genes that are required for normal growth during acute infection. Our initial results have led us to the hypothesis that M. tuberculosis influences host- pathogen contacts by utilizing the MmpL family of transporters to secrete biologically active lipids to the surface of the mycobacterial cell and ultimately into infected host cells. The studies proposed here give us the opportunity to test this model and thus understand the molecular details host- pathogen interactions critical during this stage of M.tuberculosis infection. Specifically, we will study a subset of Mmpls that are required for disease and identify the host-pathogen interactions mediated by these virulence molecules. We will determine the mechanism of transport of the cell wall lipid phthiocerol dimycocerosate (PDIM) by MmpL7 and seek to understand why this molecule is important for lung specificity of M. tuberculosis. Furthermore, we will identify the molecules transported by the other MmpL proteins identified by our genetic screens and determine their role in pathogenesis. Finally, we will determine if these molecules serve distinct roles in modifying the host for the benefit of the bacterium. Because members of the MmpL family of transporters are highly homologous to one another and to MmpL proteins of other mycobacterial pathogens, understanding the common mechanisms of their function may lead to the development of inhibitors that could be useful for treating a broad range of infectious diseases. The results from these studies will direct our long-term plans to understand the role secreted lipids play in the struggle between M. tuberculosis and the host. Ultimately, by understanding tuberculosis pathogenesis at the molecular level, we hope to aid in the discovery of new therapies to combat and eradicate this persistent infection.

Tuberculosis (TB) is a persistent lung infection that ranks as one of the most serious threats to world health today. The 2-3 million deaths attributed yearly to the disease, as well as the emergence drug-resistant Mycobacterium tuberculosis (MTB) strains, urgently call for the development of new therapies to treat TB. Indeed, the threat of drug-resistant MTB as a bioterrorism agent has led to it's listing as a NIAID Category C Priority Pathogen for biodefense research. We have isolated novel MTB mutants with lesions in individual genes that are required for normal growth during acute infection. In particular, we have identified the first specialized protein secretion system in MTB, which we have named the Snm pathway. Our initial results have led us to the hypothesis that proteins secreted by this pathway interact with macrophages to specifically inhibit innate macrophage response to infection. The studies proposed here give us the opportunity to test this model and thus understand the molecular details of host-pathogen interactions critical during the early stages of MTB infection. Specifically, we will identify all of the substrates secreted by the Snm system and determine their role in virulence. We will study a subset of Snm substrates that are required for manipulating macrophage responses and identify the host-pathogen interactions mediated by these virulence molecules. Finally, we will probe the impact of the Snm pathway on the global transcriptional response of macrophages to MTB infection. Because components of the Snm pathway are conserved in many other bacteria, understanding the common mechanisms of their function may lead to the development of inhibitors that could be useful for treating a broad range of infectious diseases. The results from these studies will direct our long-term plans to understand the role secreted proteins play in the struggle between MTB and the host. Ultimately, by understanding tuberculosis pathogenesis at the molecular level, we hope to aid in the discovery of new therapies to combat and eradicate this persistent infection.

[unreadable] DESCRIPTION (provided by applicant): Tuberculosis (TB) is a persistent lung infection that has plagued mankind for centuries and ranks as one of the most serious threats to world health today. The 2-3 million deaths attributed yearly to the disease, as well as the emergence of strains resistant to all of the available chemotherapeutic agents, urgently call for the development of new therapies to treat TB. For years, the identification of new drug targets has been hampered by the intractability of the bacillus to genetic analysis. Now with the advent of powerful genetic tools, combined with well-established mouse infection models, we have isolated novel M. tuberculosis mutants with lesions in individual genes that are required for normal growth during acute infection. Our results have led us to the hypothesis that M. tuberculosis influences host-pathogen contacts by utilizing two virulence secretion systems: the MmpL family of transporters, which secretes polyketide lipids to the bacterial cell surface, and the Snm secretion system, which secretes proteins into host cells. Both of these secretion pathways are central to M. tuberculosis virulence. The studies proposed here will seek to determine the protein complexes that target and translocate virulence factors across the cell membrane in M. tuberculosis. In the case of MmpL proteins, we will test the hypothesis that interactions between synthase and transporter provide efficiency and specificity to lipid transport. Likewise, we will use our knowledge of some of the proteins that make up the Snm secretion system to identify new components using both genetics and biochemistry. The results from these studies will direct our long-term plans to understand the mechanism by which M. tuberculosis virulence factors are secreted from the bacterium and into the host. Ultimately, by understanding tuberculosis pathogenesis at the molecular level, we hope to aid in the discovery of new therapies to combat and eradicate this persistent infection. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a persistent lung infection that has plagued mankind for centuries and ranks as one of the most serious threats to world health today. The 2-3 million deaths attributed yearly to the disease, as well as the emergence of strains resistant to all of the available chemotherapeutic agents, urgently call for the development of new therapies to treat TB. Furthermore, the threat of drug-resistant TB as a bioterrorism agent has led to it's listing as a NIAID Category C Priority Pathogen for biodefense research. The primary objective of the proposed research is to understand the mechanisms by which this pathogen manipulates its human host to evade killing by the immune system. We showed previously that M. tuberculosis utilizes the ESX-1 protein secretion system to export virulence factors that disarm host macrophages. We found that EspR is a key regulator of ESX-1 that is required for secretion and virulence in mice. EspR activates transcription of an operon that includes three ESX-1 components, whose expression in turn promotes secretion of ESX-1 substrates. Surprisingly, efflux of the DNA-binding regulator itself eventually results in reduced transcription, and thus reduced ESX-1 secretion. Our results reveal a direct negative feedback loop that titrates the activity of a secretion system essential for virulence of a major human pathogen. We hypothesize that such a regulatory scheme provides a timing mechanism that allows for a pulse of virulence factor secretion early after infection, followed by inhibition of secretion. This is an appealing hypothesis as many of the ESX-1 virulence factors are also major antigens recognized by the adaptive immune response. Thus, these studies will test the hypothesis that M. tuberculosis activates this pathway early during infection to deliver virulence factors, but then inactivates the pathway in order to hide from the immune system. Results from these studies may reveal ways to engineer better vaccines to fight TB.

This proposal is Project 2 within a P01 renewal, entitled Intracellular pathogens and innate immunity. M. tuberculosis is an important human pathogen that causes severe morbidity and mortality around the world. The goal of the previous proposal was to identify the mechanisms by which M. tuberculosis triggers and manipulates host responses of its primary host cell, the macrophage. We made the striking discovery that M. tuberculosis, a phagosomal pathogen, activates the host cytosolic surveillance pathway (CSP), an innate signaling pathway that senses bacterial molecules in the cytoplasm. We are interested in host and bacterial factors required for CSP activation, and in elucidating the functional role of the pathway in M. tuberculosis pathogenesis. We have shown that the ESX-1 secretion pathway and the cell wall lipid PDIM are critical for perturbing phagosomal membranes of macrophages, allowing activation of cytosolic DNA receptors. Unexpectedly, cytosolic access also targets bacteria to the autophagy pathway. We hypothesize that intracellular pathogens perturb intracellular membranes to promote virulence, but activation of the CSP allows host cells to discriminate and mount qualitatively different immune responses to pathogens versus non-pathogens. In Aim 1, we propose to elucidate the mechanism by which M. tuberculosis gains access to the cytosol and activates the CSP, building upon our evidence that a single ESX-1 substrate, ESAT-6, functions to permeabilize the phagosomal membrane. We propose to identify the nature of the DNA that is recognized by the host, test the role of putative host receptors responsible for CSP activation, and probe the role of cytosolic signaling in M. tuberculosis infection. In Aim 2, we will examine how cytosolic access leads to targeting of M. tuberculosis to the autophagy pathway. In particular, we will test the role of ubiquitination and ubiquitin-binding adapters in targeting of autophagic vesicles to M. tuberculosis during infection. In the final Aim, We will collaborate extensively with the Portnoy (Project 1) and Vance (Project 3) groups to screen for modulators of M. tuberculosis growth and host innate responses by carrying out a screen in macrophages isolated from ENU mutagenized mice, and by performing an RNAi screen in macrophages.

DESCRIPTION (provided by applicant): This competing IMSD grant renewal from the University of California, San Francisco (UCSF) is designed to continue to provide support for students from underrepresented groups to complete the PhD in biomedical and behavioral research and to advance to competitive postdoctoral and academic positions. The proposed program will increase opportunities for underrepresented students to thrive in the biomedical and behavioral sciences by providing them with the necessary tools, skills, and resources to complete their PhDs at UCSF in a timely fashion and to prepare for productive careers in research. Strong academic and professional mentoring leading to the successful completion of PhDs will increase the proportion of underrepresented students who join the biomedical workforce. Robust implementation and evaluation of measurable objectives, milestones, and outcomes will help create a more diverse population of future research scientists. The overall goal of this program will be achieved by the following specific objectives: 1) To ensure that IMSD Fellows' first-year research rotations and thesis lab placements are with renowned faculty in prestigious research programs who are also proven mentors supportive of new students and sensitive to unique issues facing many minority students. Strong mentorship by established researchers is key to the retention of students in the graduate program as well as for meaningful guidance toward obtaining postdoctoral positions and academic appointments after graduation. 2) To promote sustained success of IMSD students throughout their PhD work by fostering a community network of Fellows. Group learning, social engagement, peer mentoring, group discussions with role models, and participation in community service projects will create a support network to promote the progression of the group as a whole. 3) To accelerate IMSD Fellows' academic achievement by developing strong communication and organizational skills in order to maximize their research career progress and success. Progress can be measured by timely completion of milestones toward the degree; success can be measured by Fellows' presentations of their research in local and national settings, awards of competitive extramural fellowships, and eventual publications in high-impact journals. 4) To facilitate Fellows' ability t take a pro-active approach to career planning, by providing them with opportunities for career exploration and with tools and strategies to be competitive in the academic research job market. 5) To nurture a mindset that will encourage IMSD Fellows to serve as mentors, led by example, and support IMSD and similar programs throughout their academic careers by encouraging Fellows who have moved off IMSD funding to remain engaged with the Program through group activities, mentoring of junior fellows, and participation in campus outreach and diversity efforts With multi-faceted institutional support systems at UCSF, this proposed program will help to foster and sustain diversity in tomorrow's biomedical workforce.

? DESCRIPTION (provided by applicant): Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, remains a major cause of human morbidity and mortality, particularly in the developing world. Chronic M. tuberculosis infection requires long- term interactions between the bacterium and host immune system, and tissue macrophages play key roles in the outcome of infection. Although technologies to monitor global changes in host gene expression have catalyzed our understanding of the important roles for TLR activation and interferon during TB infection, modulation of transcription represents only one of many cellular responses to bacterial infection. Post- translational modification of proteins, such as ubiquitylation, phosphorylation, and acetylation play a role in regulating virtually every cellular process. How these signaling events lead to observed changes in metabolic pathways, autophagy, and vesicular trafficking during bacterial infection remain unknown. Furthermore, how these processes may be manipulated by pathogens is crucial for understanding the pathogenic strategies of microorganisms. This proposal seeks to use powerful new proteomic technologies to globally quantify changes in ubiquitylation in order to identify novel functional macrophage responses to M. tuberculosis infection. Our preliminary experiments utilizing this approach have uncovered profound changes in host protein ubiquitylation in response to intracellular pathogens. These studies have provided the first glimpse into a vast unknown of post-translational modifications during innate immune responses. Our hypothesis is that these changes play fundamental roles in shaping the subsequent innate responses to infection by controlling autophagy, metabolism, protein degradation, and signaling, and may be manipulated by pathogens for their own benefit. Our preliminary genetic work indicates that novel pathways are controlled by ubiquitylation during infection and are capable of restricting M. tuberculosis growth in macrophages, further validating the power of this approach to uncover new biology. Ultimately, our long-term goal is to harness these powerful immune mechanisms for therapeutic purposes.

Project Summary Emerging antibiotic resistance is a global health crisis. From broadly resistant ?superbugs? to extremely drug resistant Mycobacterium tuberculosis (XDR-TB), the specter of untreatable bacterial infection has become an alarming reality. The problem has become so acute that President Barack Obama recently issued an executive order calling multidrug resistant bacteria a national security priority. Likewise, the need to develop better antibiotics that shorten the treatment time to cure persistent bacterial infections also remains a major goal, especially for some of the most notorious pathogens of man, including M. tuberculosis. Due to the slow rate of new antibiotics emerging from the lab, countering antibiotic resistance and persistence by modifying existing drugs or developing inhibitors to new bacterial targets is unlikely to keep up with the increasing demand. The goal of this project is to take a radically different approach to new antibiotic development by identifying small molecules that target powerful host immune pathways of innate immune cells, creating adjunctive therapies that will synergize with conventional antibiotics. This approach is antithetical to traditional antibiotic development programs, which seek to identify molecular inhibitors that target essential pathways of the bacterium but avoid host pathways. The potential impact of ?Host-Directed Therapies? (HDTs) could be dramatic, as they may re-sensitize drug-resistant strains and shorten the time to eradicate chronic infections. In particular, this proposal seeks to translate our understanding of host-pathogen interactions into a novel program for identifying small molecules targeting host processes that will synergize with traditional antibiotics during TB infection. The implications of success may extend beyond bacterial infection, as this approach could have huge implications for a broad range of infectious agents, and even boost vaccine efficacy.

Project Summary/Abstract (Project 2, Cox) Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, remains a major cause of human morbidity and mortality, particularly in the developing world. Chronic M. tuberculosis infection requires long- term interactions between the bacterium and host immune system, and tissue macrophages play key roles in the outcome of infection. The goal of this project is to identify the interactions between M. tuberculosis and macrophages at the molecular level, and to understand how they influence bacterial killing. Our previous work has identified a macrophage pathway activated by M. tuberculosis infection that induces both antibacterial responses of macrophages and, paradoxically, anti-viral responses that inhibit immunity. In this proposal, we seek to dissect these two antagonistic responses activated by this pathway, which requires the host factor STING, with the long-term goal of developing host-directed therapeutics that push the balance in favor of TB immunity. In Aim 1 we investigate the molecular mechanisms by which the STING pathway targets M. tuberculosis to autophagy, a powerful anti-infection pathway of the host. In Aim 2, we explore the role of Galectin-9 in host defense against M. tuberculosis infection, as this carbohydrate-binding protein is a prime candidate for collaborating with STING to promote autophagy. In Aim 3, we seek to understand the mechanism by which STING-activated interferon responses function to promote M. tuberculosis infection. Importantly, both the autophagy targeting and anti-viral signaling pathways are controlled by important kinases, indicating that post-translational modification of host proteins play important roles in mediating these effects. Thus, our proposed use of cutting-edge proteomics in Aim 4 to identify the proteins modified by these enzymes during infection represents a powerful way to uncover mechanisms by which macrophages control infection. Importantly, our work is highly synergistic with the other projects and cores that make up this Program Project, and the comparisons of host responses elicited by M. tuberculosis with those induced by L. monocytogenes and L. pneumophila will allow us to identify pathogen-specific responses as well as common responses induced by diverse intracellular bacterial pathogens. These studies will not only provide deeper mechanistic understanding of how autophagy and anti-viral signaling control infection, but may also uncover novel host pathways that function to eliminate intracellular bacterial pathogens.

PROJECT 2: GENETIC IDENTIFICATION OF HOST INNATE PATHWAYS THAT CONTROL BACTERIAL PATHOGENESIS SUMMARY Phagocytes of the mammalian innate immune system, in particular macrophages and neutrophils, form the first line of defense upon bacterial infection and are armed with powerful mechanisms to limit bacterial growth and eradicate invaders. Bacterial pathogens, however, have evolved mechanisms to thwart these killing mechanisms of phagocytes, and to persist in human tissues. In addition to having direct antimicrobial activity, macrophages and neutrophils also initiate and shape powerful inflammatory responses that dramatically influence disease. Indeed, the inflammatory pathways elicited by each of the pathogens involved in this study - Mycobacterium tuberculosis (Mtb), Staphylococcus aureus (SA), and Chlamydia trachomatis (CT) - play major roles in bacterial persistence in tissues and in promoting disease. Pathogens must subvert these cells in order to grow and persist, but the host genes and cellular pathways that dictate the outcome of infection are not entirely clear. The broad idea of this proposal is to use unbiased, systematic approaches to probe the intimate interactions between pathogen and innate immune cells, and to use this information to make predictions about bacterial infectivity that will be tested in human samples in an iterative fashion to model host response during infection. We propose to systematically identify innate immune gene networks that underlie pathogenesis in clinically relevant model systems of infections with these three important bacterial pathogens. Using powerful CRISPR-based knockout strategies in both ex vivo infections and in mouse models, coupled with innovative approaches to identify functionally relevant polymorphisms in clinical samples, we seek to dramatically increase our understanding of infection biology of humans. We anticipate that these insights, coupled with the other elements of the HPMI Center, will point to novel vulnerabilities with therapeutic relevance.

Hurdles for controlling tuberculosis (TB) include developing a highly efficacious vaccine, preventing transmission and infection in endemic areas, and discovering drug treatment regimens that work rapidly and kill dormant bacilli within macrophages. After exposure to Mycobacterium tuberculosis (Mtb), outcomes vary widely including resistance, asymptomatic latent infection, active pulmonary disease, and disseminated infections including TB meningitis (TBM). This heterogeneity complicates clinical treatment decisions with regards to choosing the number of drugs and duration of treatment. This broad clinical spectrum also presents a unique opportunity for understanding the biological mechanisms that control TB pathogenesis. A major source of heterogeneity is a combination of genetic variation in both humans and Mtb that are evolving under constant selective pressure. Our overall program objective is to use genetic, genomic, proteomic, and bioinformatic strategies to discover host and pathogen variants of genes and gene products that are associated with TB clinical outcomes and to determine how these variants interact to regulate molecular, cellular, and in vivo functions. Our strategy is anchored upon two powerful cohorts in Vietnam and Uganda (Core A) that capture the full spectrum of resistance to traditional LTBI (latent TB infection), LTBI, pulmonary TB disease, and disseminated disease in the form of TBM. Core A examines paired host and Mtb genetic data and the association with these diverse clinical outcomes. In Project 1, we use genetic and new proteomic strategies to examine how the Mtb genes and variants identified by Core A function and how the encoded proteins interact with and regulate macrophage responses. In Project 2, we use human genetic methods along with proteomic strategies in macrophages to uncover regulatory host genes and variants that are associated with resistance to Mtb infection and/or disseminated TB. In Project 3, we examine in vivo mechanisms of transmission and dissemination that are attributed to specific host genes and pathways and Mtb variants, employing a new and powerful mouse model of infection that recapitulates many of the manifestations that occur in human TB. Core B uses pathway-driven and novel bioinformatics approaches to integrate the genetic results from Core A with the multiple large-scale and diverse datasets to dynamically identify and prioritize pathways and protein networks for functional testing. Together, this multidisciplinary program and strategy will enable us to test our overall hypothesis that variants of Mtb and host genes dictate heterogeneous clinical outcomes and encode factors that interact with and alter innate immune cells. We will use genetic, genomic, proteomic, and bioinformatic strategies to examine variation in Mtb and its paired human host to examine mechanisms of resistance and susceptibility to infection and disease with discovery of biomarkers for clinical management and novel immunomodulatory therapies.

Tuberculosis (TB) is a multifaceted disease that has extensive variation in clinical manifestations despite being the product of infection with a single pathogen, Mycobacterium tuberculosis. The extensive genetic diversity in human and Mycobacterium tuberculosis genomes responsible for differences in clinical outcomes represent modifications of host-pathogen interactions that are key to pathogenesis. Understanding the basis for these heterogenous responses will uncover new mechanisms of virulence and resistance and will impact treatment and diagnostics. Unfortunately, the challenges of studying the mechanisms of differential outcomes of infection in humans not only includes identification of correlations between host/pathogen genotypes with phenotypes in human populations, but also the subsequent identification of the mechanisms that are causal for disease which require studying them in the laboratory without the pathogen?s natural host organism. This Program takes advantage of unique, ongoing genome-wide association studies (GWAS) that have identified both human and pathogen variants that are associated with heterogenous clinical responses in two different human populations. To determine the mechanisms underlying these variations, we will employ a powerful set of experimental assays, including new proteomics-based scanning platform to probe host responses during experimental macrophage infection that is orthogonal to traditional mRNA profiling, in order to broadly search for changes in host innate immune pathways that correlate with disease outcomes associated with these clinical strains. Based on our preliminary data, we hypothesize that many of these interactions occur early during infection and are mediated by proteins secreted by M. tuberculosis. In this proposal, we focus primarily on correlations between two unique sets of clinical bacterial variants, strains that are associated greater transmission of pulmonary TB disease and strains that are more prone to dissemination to distal sites in the body. An unexpected theme from both of these sets of strains is the prevalence of TB proteins secreted by the ESX systems expressed in M. tuberculosis. Both the ESX-1 and ESX-5 secretion systems of M. tuberculosis are key virulence determinants required for intracellular growth and for eliciting distinct innate immune responses during macrophage infection. A central hypothesis is that the set of bacterial proteins that influence disease outcomes are enriched for secreted proteins that mediate interactions between pathogen and host macrophages. To test this hypothesis, we will collaborate with Cores A and B to use an integrative approach to combine genetic data from the M. tuberculosis GWAS datasets with genetic and proteomic screen to identify causal genes that mediate interactions with macrophages. We will use these same technologies to collaborate with Projects 2 and 3 to identify proteins/pathways responsible for host resistance and bacterial dissemination.

Project Summary/Abstract (Project 2, Cox) Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, remains a major cause of human morbidity and mortality, particularly in the developing world. The spread of antibiotic resistant strains of M. tuberculosis has increased the urgency to develop new vaccines with greater efficacy than Bacille Calmette- Guerin (BCG), which is ineffective to prevent pulmonary infection in adults, by far the most common form of TB. The ESX-1 secretion system of Mycobacterium tuberculosis is a key virulence determinant that is required for intracellular growth and for eliciting distinct innate immune responses, autophagy and type I interferon (IFN) during infection. BCG is extremely similar to M. tuberculosis but it lacks ESX-1 and thus cannot trigger these powerful innate immune pathways. The overarching goal of this project is to identify the M. tuberculosis factors that specifically activate these responses, and use this to create BCG strains that engage these pathways during vaccination. In Aim 1, we investigate ESX-1 secreted substrates that are required for perforating phagosomal membranes in collaboration with Project 1, and use a new system to express these factors in BCG to restore perforation but without the rest of the ESX-1 secretion system. In Aim 2, we explore two hypotheses for how M. tuberculosis limits targeting to autophagy, a powerful anti-bacterial host defense mechanism. In Aim 3, we will utilize our existing knowledge, as well as information from Aims 1 and 2, to engineer BCG strains to specifically test the role of autophagy and type I IFN in promoting immunity. Moreover, we will collaborate with Projects 1, 3, 4, and Core B, to compare and combine our recombinant BCG strategy with STING-targeted adjuvants (Project 4) and bacterial metabolites (Project 1) to identify synergies between these approaches, and to test their efficacy in novel models of vaccination (Projects 3 and 4). Thus, these studies will not only provide deeper mechanistic understanding of how autophagy and anti-viral signaling are activated, but may lead directly to the creation of new TB vaccines.