Heme Oxygenase-1 Regulates Inflammation and Mycobacterial Survival in Human Macrophages during Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for 1.5 million deaths annually. We previously showed that M. tuberculosis infection in mice induces expression of the CO-producing enzyme heme oxygenase (HO1) and that CO is sensed by M. tuberculosis to initiate a dormancy program. Further, mice deficient in HO1 succumb to M. tuberculosis infection more readily than do wild-type mice. Although mouse macrophages control intracellular M. tuberculosis infection through several mechanisms, such as NO synthase, the respiratory burst, acidification, and autophagy, how human macrophages control M. tuberculosis infection remains less well understood. In this article, we show that M. tuberculosis induces and colocalizes with HO1 in both mouse and human tuberculosis lesions in vivo, and that M. tuberculosis induces and colocalizes with HO1 during primary human macrophage infection in vitro. Surprisingly, we find that chemical inhibition of HO1 both reduces inflammatory cytokine production by human macrophages and restricts intracellular growth of mycobacteria. Thus, induction of HO1 by M. tuberculosis infection may be a mycobacterial virulence mechanism to enhance inflammation and bacterial growth.

Identification of scavenger receptor B1 as the airway microfold cell receptor for Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) can enter the body through multiple routes, including via specialized transcytotic cells called microfold cells (M cell). However, the mechanistic basis for M cell entry remains undefined. Here, we show that M cell transcytosis depends on the Mtb Type VII secretion machine and its major virulence factor EsxA. We identify scavenger receptor B1 (SR-B1) as an EsxA receptor on airway M cells. SR-B1 is required for Mtb binding to and translocation across M cells in mouse and human tissue. Together, our data demonstrate a previously undescribed role for Mtb EsxA in mucosal invasion and identify SR-B1 as the airway M cell receptor for Mtb.

Mouse Models to Study Natural Killer Cell-Mediated Immunosurveillance and Metastatic Latency.

Metastatic latency is a major concern in the clinic, yet how these disseminated cancer cells survive and initiate metastases is unknown (Massagué and Obenauf, Nature 529:298-306, 2016). Here, we describe an approach to isolate latency competent cancer (LCC) cells from early stage human lung and breast carcinoma cell lines using mouse xenograft models (Malladi, Cell 165:45-60, 2016). Cancer cell lines labeled with GFP-luciferase and antibiotic selection markers were injected intracardially into athymic mice. Three months, post-injection, LCC cells were identified in situ and isolated. Upon reinjection, LCC cells retain their tumorigenic potential, enter a slow-cycling or quiescent state, and evade NK cell-mediated innate immune surveillance.

Screening Mycobacterium tuberculosis Secreted Proteins Identifies Mpt64 as a Eukaryotic Membrane-Binding Bacterial Effector.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.IMPORTANCE Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.

A baculovirus-conjugated mimotope vaccine targeting Mycobacterium tuberculosis lipoarabinomannan.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a major cause of morbidity and mortality worldwide. However, an effective vaccine for M. tuberculosis is lacking. We panned a phage display library using monoclonal antibodies against M. tuberculosis liporabinomannan (LAM), an important component of the M. tuberculosis cell wall, and identified two peptide sequences, HSFKWLDSPRLR or SGVYKVAYDWQH, with high antibody affinity after multiple rounds of panning. Only the HSFKWLDSPRLR peptide induced an anti-LAM response when conjugated to either keyhole limpet hemocyanin (KLH) or to the baculovirus Autographa californica multicapsid nucleopolyherovirus (AcMNPV) when introduced into mice by injection or via intranasal inoculation, respectively. Vaccination with AcMNPV conjugated HSFKWLDSPRLR peptide delayed mortality in a mouse model of tuberculosis. Thus, we report a proof of principle M. tuberculosis vaccination strategy combining an anti-LAM mimotope with a baculovirus delivery system.

The Ubiquitin Ligase Smurf1 Functions in Selective Autophagy of Mycobacterium tuberculosis and Anti-tuberculous Host Defense.

During antibacterial autophagy, ubiquitination of intracellular bacteria recruits proteins that mediate bacterial delivery to the lysosome for degradation. Smurf1 is an E3 ubiquitin ligase whose role in selective bacterial autophagy is unknown. We show that Smurf1 facilitates selective autophagy of the human pathogen Mycobacterium tuberculosis (Mtb). Smurf1-/- macrophages are defective in recruiting polyubiquitin, the proteasome, the ubiquitin-binding autophagy adaptor NBR1, the autophagy protein LC3, and the lysosomal marker LAMP1 to Mtb-associated structures and are more permissive for Mtb growth. This function of Smurf1 requires both its ubiquitin-ligase and C2 phospholipid-binding domains, and involves K48- rather than K63-linked ubiquitination. Chronically infected Smurf1-/- mice have increased bacterial load, increased lung inflammation, and accelerated mortality. SMURF1 controls Mtb replication in human macrophages and associates with bacteria in lungs of patients with pulmonary tuberculosis. Thus, Smurf1 is required for selective autophagy of Mtb and host defense against tuberculosis infection.

Microfold Cells Actively Translocate Mycobacterium tuberculosis to Initiate Infection.

The prevailing paradigm is that tuberculosis infection is initiated when patrolling alveolar macrophages and dendritic cells within the terminal alveolus ingest inhaled Mycobacterium tuberculosis (Mtb). However, definitive data for this model are lacking. Among the epithelial cells of the upper airway, a specialized epithelial cell known as a microfold cell (M cell) overlies various components of mucosa-associated lymphatic tissue. Here, using multiple mouse models, we show that Mtb invades via M cells to initiate infection. Intranasal Mtb infection in mice lacking M cells either genetically or by antibody depletion resulted in reduced invasion and dissemination to draining lymph nodes. M cell-depleted mice infected via aerosol also had delayed dissemination to lymph nodes and reduced mortality. Translocation of Mtb across two M cell transwell models was rapid and transcellular. Thus, M cell translocation is a vital entry mechanism that contributes to the pathogenesis of Mtb.

Cyclic GMP-AMP Synthase Is an Innate Immune DNA Sensor for Mycobacterium tuberculosis.

Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection.

cor, a novel carbon monoxide resistance gene, is essential for Mycobacterium tuberculosis pathogenesis.

UNLABELLED: Tuberculosis, caused by Mycobacterium tuberculosis, remains a devastating human infectious disease, causing two million deaths annually. We previously demonstrated that M. tuberculosis induces an enzyme, heme oxygenase (HO1), that produces carbon monoxide (CO) gas and that M. tuberculosis adapts its transcriptome during CO exposure. We now demonstrate that M. tuberculosis carries a novel resistance gene to combat CO toxicity. We screened an M. tuberculosis transposon library for CO-susceptible mutants and found that disruption of Rv1829 (carbon monoxide resistance, Cor) leads to marked CO sensitivity. Heterologous expression of Cor in Escherichia coli rescued it from CO toxicity. Importantly, the virulence of the cor mutant is attenuated in a mouse model of tuberculosis. Thus, Cor is necessary and sufficient to protect bacteria from host-derived CO. Taken together, this represents the first report of a role for HO1-derived CO in controlling infection of an intracellular pathogen and the first identification of a CO resistance gene in a pathogenic organism. IMPORTANCE: Macrophages produce a variety of antimicrobial molecules, including nitric oxide (NO), hydrogen peroxide (H2O2), and acid (H+), that serve to kill engulfed bacteria. In addition to these molecules, human and mouse macrophages also produce carbon monoxide (CO) gas by the heme oxygenase (HO1) enzyme. We observed that, in contrast to other bacteria, mycobacteria are resistant to CO, suggesting that this might be an evolutionary adaptation of mycobacteria for survival within macrophages. We screened a panel of ~2,500 M. tuberculosis mutants to determine which genes are required for survival of M. tuberculosis in the presence of CO. Within this panel, we identified one such gene, cor, that specifically confers CO resistance. Importantly, we found that the ability of M. tuberculosis cells carrying a mutated copy of this gene to cause tuberculosis in a mouse disease model is significantly attenuated. This indicates that CO resistance is essential for mycobacterial survival in vivo.