Tolerating the Unwelcome Guest; How the Host Withstands Persistent Mycobacterium tuberculosis

Our understanding of the host response to infections has historically focused on resistance mechanisms that directly control pathogen replication. However, both pathogen effectors and antimicrobial immune pathways have the capacity to damage host tissue, and the ability to tolerate these insults can also be critical for host survival. These tolerance mechanisms may be equally as important as resistance to prevent disease in the context of a persistent infection, such as tuberculosis, when resistance mechanisms are ineffective and the pathogen persists in the tissue for long periods. Host tolerance encompasses a wide range of strategies, many of which involve regulation of the inflammatory response. Here we will examine general strategies used by macrophages and T cells to promote tolerance in the context of tuberculosis, and focus on pathways, such as regulation of inflammasome activation, that are emerging as common mediators of tolerance

M. tuberculosis Reprograms Hematopoietic Stem Cells to Limit Myelopoiesis and Impair Trained Immunity

A greater understanding of hematopoietic stem cell (HSC) regulation is required for dissecting protective versus detrimental immunity to pathogens that cause chronic infections such as Mycobacterium tuberculosis (Mtb). We have shown that systemic administration of Bacille Calmette-Guerin (BCG) or beta-glucan reprograms HSCs in the bone marrow (BM) via a type II interferon (IFN-II) or interleukin-1 (IL1) response, respectively, which confers protective trained immunity against Mtb. Here, we demonstrate that, unlike BCG or beta-glucan, Mtb reprograms HSCs via an IFN-I response that suppresses myelopoiesis and impairs development of protective trained immunity to Mtb. Mechanistically, IFN-I signaling dysregulates iron metabolism, depolarizes mitochondrial membrane potential, and induces cell death specifically in myeloid progenitors. Additionally, activation of the IFN-I/iron axis in HSCs impairs trained immunity to Mtb infection. These results identify an unanticipated immune evasion strategy of Mtb in the BM that controls the magnitude and intrinsic anti-microbial capacity of innate immunity to infection

Cell cycle-associated expression patterns predict gene function in mycobacteria [preprint]

While the major events in prokaryotic cell cycle progression are likely to be coordinated with transcriptional and metabolic changes, these processes remain poorly characterized. Unlike many rapidly-growing bacteria, DNA replication and cell division are temporally-resolved in mycobacteria, making these slow-growing organisms a potentially useful system to investigate the prokaryotic cell cycle. To determine if cell-cycle dependent gene regulation occurs in mycobacteria, we characterized the temporal changes in the transcriptome of synchronously replicating populations of Mycobacterium tuberculosis (Mtb). By enriching for genes that display a sinusoidal expression pattern, we discover 485 genes that oscillate with a period consistent with the cell cycle. During cytokinesis, the timing of gene induction could be used to predict the timing of gene function, as mRNA abundance was found to correlate with the order in which proteins were recruited to the developing septum. Similarly, the expression pattern of primary metabolic genes could be used to predict the relative importance of these pathways for different cell cycle processes. Pyrimidine synthetic genes peaked during DNA replication and their depletion caused a filamentation phenotype that phenocopied defects in this process. In contrast, the IMP dehydrogenase guaB2 dedicated to guanosine synthesis displayed the opposite expression pattern and its depletion perturbed septation. Together, these data imply obligate coordination between primary metabolism and cell division, and identify periodically regulated genes that can be related to specific cell biological functions

The Phagocyte Oxidase Controls Tolerance to Mycobacterium tuberculosis infection. [preprint]

Protection from infectious disease relies on two distinct mechanisms. \u27Antimicrobial resistance\u27 directly inhibits pathogen growth, whereas \u27infection tolerance\u27 controls tissue damage. A single immune-mediator can differentially contribute to these mechanisms in distinct contexts, confounding our understanding of protection to different pathogens. For example, the NADPH-dependent phagocyte oxidase complex (Phox) produces anti-microbial superoxides and protects from tuberculosis in humans. However, Phox-deficient mice do not display the expected defect in resistance to M. tuberculosis leaving the role of this complex unclear. We re-examined the mechanisms by which Phox contributes to protection from TB and found that mice lacking the Cybb subunit of Phox suffered from a specific defect in tolerance, which was due to unregulated Caspase1 activation, IL-1β production, and neutrophil influx into the lung. These studies demonstrate that Phox-derived superoxide protect against TB by promoting tolerance to persistent infection, and highlight a central role for Caspase1 in regulating TB disease progression

xCT increases tuberculosis susceptibility by regulating antimicrobial function and inflammation

The physiological functions of macrophage, which plays a central role in the pathogenesis of tuberculosis, depend on its redox state. System xc-, a cystine-glutamate transporter, which consists of xCT and CD98, influences many ROS-dependent pathways by regulating the production of the antioxidant glutathione. xCT\u27s ability to alter this critical host redox balance by increasing the glutathione synthesis aspect of phagocyte physiology suggested that it might influence tuberculosis pathogenesis. In this study, we found that the xCT expression was increased in peripheral blood monocyte of active tuberculosis. xCT expression in macrophage was induced by Mycobacterium tuberculosis (Mtb) through TLR2/Akt- and p38-dependent signaling pathway. Importantly, xCT deficiency conferred protection against tuberculosis, as xCT knock out mice displayed increased Mtb load and reduced pulmonary pathology in lung compared to wild type mice. xCT disruption enhanced the mycobateriacidal activity of macrophage through increasing the mycothiol oxidation. Importantly, chemical inhibition of xCT with sulfasalazine, a specific xCT inhibitor that is already approved by the FDA for treatment of inflammatory bowel disease, produces similar protective effects in vivo and in vitro, indicating xCT might be a novel and useful target for host-directed TB treatment strategy

Phosphorylation on PstP controls cell wall metabolism and antibiotic tolerance in Mycobacterium smegmatis [preprint]

The mycobacterial cell wall is a dynamic structure that protects Mycobacterium tuberculosis and its relatives from environmental stresses. Modulation of cell wall metabolism under stress is thought to be responsible for decreased cell wall permeability and increased tolerance to antibiotics. The signaling pathways that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the signaling capacity of a cell wall master regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We studied how interference with a regulatory phosphorylation site on PstP affects growth, cell wall metabolism and antibiotic tolerance. We find that a phospho-mimetic mutation, pstP T171E, slows growth, misregulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP controls its substrate specificity and is important in the transition between growth and stasis

Mycobacterium tuberculosis evasion of Guanylate Binding Protein-mediated host defense in mice requires the ESX1 secretion system [preprint]

Cell-intrinsic immune mechanisms control intracellular pathogens that infect eukaryotes. The intracellular pathogen Mycobacterium tuberculosis (Mtb) evolved to withstand cell-autonomous immunity to cause persistent infections and disease. A potent inducer of cell-autonomous immunity is the lymphocyte-derived cytokine IFNγ. While the production of IFNγ by T cells is essential to protect against Mtb, it is not capable of fully eradicating Mtb infection. This suggests that Mtb evades a subset of IFNγ-mediated antimicrobial responses, yet what mechanisms Mtb resists remains unclear. The IFNγ-inducible Guanylate binding proteins (GBPs) are key host defense proteins able to control infections with intracellular pathogens. GBPs were previously shown to directly restrict Mycobacterium bovis BCG yet their role during Mtb infection has remained unknown. Here, we examine the importance of a cluster of five GBPs on mouse chromosome 3 in controlling Mycobacterial infection. While M. bovis BCG is directly restricted by GBPs, we find that the GBPs on chromosome 3 do not contribute to the control of Mtb replication or the associated host response to infection. The differential effects of GBPs during Mtb versus M. bovis BCG infection is at least partially explained by the absence of the ESX1 secretion system from M. bovis BCG, since Mtb mutants lacking the ESX1 secretion system become similarly susceptible to GBP-mediated immune defense. Therefore, this specific genetic interaction between the murine host and Mycobacteria reveals a novel function for the ESX1 virulence system in the evasion of GBP-mediated immunity

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research

Mitochondrial respiration contributes to the interferon gamma response in antigen presenting cells [preprint]

The immunological synapse allows antigen presenting cells (APC) to convey a wide array of functionally distinct signals to T cells, which ultimately shape the immune response. The relative effect of stimulatory and inhibitory signals is influenced by the activation state of the APC, which is determined by an interplay between signal transduction and metabolic pathways. While toll-like receptor ligation relies on glycolytic metabolism for the proper expression of inflammatory mediators, little is known about the metabolic dependencies of other critical signals such as interferon gamma (IFNγ). Using CRISPR-Cas9, we performed a series of genome-wide knockout screens in macrophages to identify the regulators of IFNγ-inducible T cell stimulatory or inhibitory proteins MHCII, CD40, and PD-L1. Our multi-screen approach enabled us to identify novel pathways that control these functionally distinct markers. Further integration of these screening data implicated complex I of the mitochondrial respiratory chain in the expression of all three markers, and by extension the IFNγ signaling pathway. We report that the IFNγ response requires mitochondrial respiration, and APCs are unable to activate T cells upon genetic or chemical inhibition of complex I. These findings suggest a dichotomous metabolic dependency between IFNγ and toll-like receptor signaling, implicating mitochondrial function as a fulcrum of innate immunity

Cyclic AMP Regulation of Protein Lysine Acetylation in Mycobacterium Tuberculosis

Protein lysine acetylation networks can regulate central processes such as carbon metabolism and gene expression in bacteria. In Escherichia coli, cyclic-AMP (cAMP) regulates protein lysine acetyltransferase (PAT) activity at the transcriptional level, but in Mycobacterium tuberculosis, fusion of a cyclic-nucleotide binding domain to a Gcn5-like PAT domain enables direct cAMP control of protein acetylation. Here we describe the allosteric activation mechanism of M. tuberculosis PAT. The crystal structures of the auto-inhibited and cAMP-activated PAT reveal that cAMP binds to a cryptic site in the regulatory domain over 32 Å from the catalytic site. An extensive conformational rearrangement relieves auto-inhibition by a substrate-mimicking lid that covers the protein-substrate binding surface. A steric double latch couples the domains by harnessing a classic, cAMP-mediated, conformational switch. The structures suggest general features that enable the evolution of long-range communication between linked domains