Paolo Manzanillo

Extracellular M. tuberculosis DNA Targets Bacteria for Autophagy by Activating the Host DNA-Sensing Pathway

Eukaryotic cells sterilize the cytosol by using autophagy to route invading bacterial pathogens to the lysosome. During macrophage infection with Mycobacterium tuberculosis, a vacuolar pathogen, exogenous induction of autophagy can limit replication, but the mechanism of autophagy targeting and its role in natural infection remain unclear. Here we show that phagosomal permeabilization mediated by the bacterial ESX-1 secretion system allows cytosolic components of the ubiquitin-mediated autophagy pathway access to phagosomal M. tuberculosis. Recognition of extracelluar bacterial DNA by the STING-dependent cytosolic pathway is required for marking bacteria with ubiquitin, and delivery of bacilli to autophagosomes requires the ubiquitin-autophagy receptors p62 and NDP52 and the DNA-responsive kinase TBK1. Remarkably, mice with monocytes incapable of delivering bacilli to the autophagy pathway are extremely susceptible to infection. Our results reveal an unexpected link between DNA sensing, innate immunity, and autophagy and indicate a major role for this autophagy pathway in resistance to M. tuberculosis infection.

The Type I IFN Response to Infection with Mycobacterium tuberculosis Requires ESX-1-Mediated Secretion and Contributes to Pathogenesis

The ESX-1 secretion system is a major determinant of Mycobacterium tuberculosis virulence, although the pathogenic mechanisms resulting from ESX-1-mediated transport remain unclear. By global transcriptional profiling of tissues from mice infected with either wild-type or ESX-1 mutant bacilli, we found that host genes controlled by ESX-1 in vivo are predominantly IFN regulated. ESX-1-mediated secretion is required for the production of host type I IFNs during infection in vivo and in macrophages in vitro. The macrophage signaling pathway leading to the production of type I IFN required the host kinase TANK-binding kinase 1 and occurs independently of TLR signaling. Importantly, the induction of type I IFNs during M. tuberculosis infection is a pathogenic mechanism as mice lacking the type I IFNR were more restrictive for bacterial growth in the spleen than wild-type mice, although growth in the lung was unaffected. We propose that the ESX-1 secretion system secretes effectors into the cytosol of infected macrophages, thereby triggering the type I IFN response for the manipulation of host immunity.

The ubiquitin ligase parkin mediates resistance to intracellular pathogens

Ubiquitin-mediated targeting of intracellular bacteria to the autophagy pathway is a key innate defence mechanism against invading microbes, including the important human pathogen Mycobacterium tuberculosis. However, the ubiquitin ligases responsible for catalysing ubiquitin chains that surround intracellular bacteria are poorly understood. The parkin protein is a ubiquitin ligase with a well-established role in mitophagy, and mutations in the parkin gene (PARK2) lead to increased susceptibility to Parkinson’s disease. Surprisingly, genetic polymorphisms in the PARK2 regulatory region are also associated with increased susceptibility to intracellular bacterial pathogens in humans, including Mycobacterium leprae and Salmonella enterica serovar Typhi, but the function of parkin in immunity has remained unexplored. Here we show that parkin has a role in ubiquitin-mediated autophagy of M. tuberculosis. Both parkin-deficient mice and flies are sensitive to various intracellular bacterial infections, indicating parkin has a conserved role in metazoan innate defence. Moreover, our work reveals an unexpected functional link between mitophagy and infectious disease.

Secreted transcription factor controls Mycobacterium tuberculosis virulence

Bacterial pathogens trigger specialized virulence factor secretion systems on encountering host cells. The ESX-1 protein secretion system of Mycobacterium tuberculosis—the causative agent of the human disease tuberculosis—delivers bacterial proteins into host cells during infection and is critical for virulence, but how it is regulated is unknown. Here we show that EspR (also known as Rv3849) 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, Rv3616c–Rv3614c, whose expression in turn promotes secretion of ESX-1 substrates. EspR directly binds to and activates the Rv3616c–Rv3614c promoter and, unexpectedly, is itself secreted from the bacterial cell by the ESX-1 system that it regulates. Efflux of the DNA-binding regulator results in reduced Rv3616c–Rv3614c transcription, and thus reduced ESX-1 secretion. Our results reveal a direct negative feedback loop that regulates the activity of a secretion system essential for virulence. As the virulence factors secreted by the ESX-1 system are highly antigenic, fine control of secretion may be critical to successful infection.

Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes

The connection between an altered gut microbiota and metabolic disorders such as obesity, diabetes, and cardiovascular disease is well established. Defects in preserving the integrity of the mucosal barriers can result in systemic endotoxaemia that contributes to chronic low-grade inflammation, which further promotes the development of metabolic syndrome. Interleukin (IL)-22 exerts essential roles in eliciting antimicrobial immunity and maintaining mucosal barrier integrity within the intestine. Here we investigate the connection between IL-22 and metabolic disorders. We find that the induction of IL-22 from innate lymphoid cells and CD4+ T cells is impaired in obese mice under various immune challenges, especially in the colon during infection with Citrobacter rodentium. While innate lymphoid cell populations are largely intact in obese mice, the upregulation of IL-23, a cytokine upstream of IL-22, is compromised during the infection. Consequently, these mice are susceptible to C. rodentium infection, and both exogenous IL-22 and IL-23 are able to restore the mucosal host defence. Importantly, we further unveil unexpected functions of IL-22 in regulating metabolism. Mice deficient in IL-22 receptor and fed with high-fat diet are prone to developing metabolic disorders. Strikingly, administration of exogenous IL-22 in genetically obese leptin-receptor-deficient (db/db) mice and mice fed with high-fat diet reverses many of the metabolic symptoms, including hyperglycaemia and insulin resistance. IL-22 shows diverse metabolic benefits, as it improves insulin sensitivity, preserves gut mucosal barrier and endocrine functions, decreases endotoxaemia and chronic inflammation, and regulates lipid metabolism in liver and adipose tissues. In summary, we identify the IL-22 pathway as a novel target for therapeutic intervention in metabolic diseases.

Mycobacterium tuberculosis Senses Host-Derived Carbon Monoxide during Macrophage Infection

Mycobacterium tuberculosis (MTB) expresses a set of genes known as the dormancy regulon in vivo. These genes are expressed in vitro in response to nitric oxide (NO) or hypoxia, conditions used to model MTB persistence in latent infection. Although NO, a macrophage product that inhibits respiration, and hypoxia are likely triggers in vivo, additional cues could activate the dormancy regulon during infection. Here, we show that MTB infection stimulates expression of heme oxygenase (HO-1) by macrophages and that the gaseous product of this enzyme, carbon monoxide (CO), activates expression of the dormancy regulon. Deletion of macrophage HO-1 reduced expression of the dormancy regulon. Furthermore, we show that the MTB DosS/DosT/DosR two-component sensory relay system is required for the response to CO. Together, these findings demonstrate that MTB senses CO during macrophage infection. CO may represent a general cue used by pathogens to sense and adapt to the host environment.

ESX‐1 secreted virulence factors are recognized by multiple cytosolic AAA ATPases in pathogenic mycobacteria

The ESX‐1 secretion system of Mycobacterium tuberculosis delivers bacterial virulence factors to host cells during infection. The most abundant factor, the ESAT‐6/CFP‐10 dimer, is targeted for secretion via a C‐terminal signal sequence on CFP‐10 that is recognized by the cytosolic ATPase, Rv3871. However, the selection determinants for other ESX‐1 substrates appear to be more complex. Some substrates, such as ESAT‐6, are secreted despite lacking signal sequences. Furthermore, all substrates require targeting of the other ESX‐1 secreted proteins, a distinguishing feature of this system. How ESX‐1 substrates are selected and the basis for co‐dependent secretion is unknown. Here we show that the EspC substrate interacts with Rv3868, a cytosolic AAA ATPase, through its C‐terminus. Swapping the C‐termini of EspC and CFP‐10 revealed that these signals are functionally distinct, suggesting that the proteins are targeted via interactions with different ATPases. Surprisingly, biochemical purification experiments demonstrate that these substrates and ATPases form multi‐protein complexes inside the cell and identified a new secreted substrate. By interfering with this protein interaction network, we have partially uncoupled co‐dependent substrate secretion. Our results suggest that proper functioning of the ESX‐1 pathway requires the interaction of multiple ESX‐1 substrates and components prior to their secretion. Ultimately, understanding the details of ESX‐1 targeting may allow for engineering of better vaccines to prevent tuberculosis.

cor, a Novel Carbon Monoxide Resistance Gene, Is Essential for Mycobacterium tuberculosis Pathogenesis

ABSTRACT 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. 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.

Mice deficient in NRROS show abnormal microglial development and neurological disorders

Microglia and other tissue-resident macrophages within the central nervous system (CNS) have essential roles in neural development, inflammation and homeostasis. However, the molecular pathways underlying their development and function remain poorly understood. Here we report that mice deficient in NRROS, a myeloid-expressed transmembrane protein in the endoplasmic reticulum, develop spontaneous neurological disorders. NRROS-deficient (Nrros−/−) mice show defects in motor functions and die before 6 months of age. Nrros−/− mice display astrogliosis and lack normal CD11bhiCD45lo microglia, but they show no detectable demyelination or neuronal loss. Instead, perivascular macrophage-like myeloid cells populate the Nrros−/− CNS. Cx3cr1-driven deletion of Nrros shows its crucial role in microglial establishment during early embryonic stages. NRROS is required for normal expression of Sall1 and other microglial genes that are important for microglial development and function. Our study reveals a NRROS-mediated pathway that controls CNS-resident macrophage development and affects neurological function.