STARTRAC analyses of scRNAseq data from tumor models reveal T cell dynamics and therapeutic targets.

Single-cell RNA sequencing is a powerful tool to examine cellular heterogeneity, novel markers and target genes, and therapeutic mechanisms in human cancers and animal models. Here, we analyzed single-cell RNA sequencing data of T cells obtained from multiple mouse tumor models by PCA-based subclustering coupled with TCR tracking using the STARTRAC algorithm. This approach revealed various differentiated T cell subsets and activation states, and a correspondence of T cell subsets between human and mouse tumors. STARTRAC analyses demonstrated peripheral T cell subsets that were developmentally connected with tumor-infiltrating CD8+ cells, CD4+ Th1 cells, and T reg cells. In addition, large amounts of paired TCRα/ß sequences enabled us to identify a specific enrichment of paired public TCR clones in tumor. Finally, we identified CCR8 as a tumor-associated T reg cell marker that could preferentially deplete tumor-associated T reg cells. We showed that CCR8-depleting antibody treatment provided therapeutic benefit in CT26 tumors and synergized with anti-PD-1 treatment in MC38 and B16F10 tumor models.

Correction: Crystal structures of human procathepsin H.

[This corrects the article DOI: 10.1371/journal.pone.0200374.].

Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells.

The emergence of Mycobacterium tuberculosis (MTB) strains that are resistant to most or all available antibiotics has created a severe problem for treating tuberculosis and has spurred a quest for new antibiotic targets. Here, we demonstrate that trans-translation is essential for growth of MTB and is a viable target for development of antituberculosis drugs. We also show that an inhibitor of trans-translation, KKL-35, is bactericidal against MTB under both aerobic and anoxic conditions. Biochemical experiments show that this compound targets helix 89 of the 23S rRNA. In silico molecular docking predicts a binding pocket for KKL-35 adjacent to the peptidyl-transfer center in a region not targeted by conventional antibiotics. Computational solvent mapping suggests that this pocket is a druggable hot spot for small molecule binding. Collectively, our findings reveal a new target for antituberculosis drug development and provide critical insight on the mechanism of antibacterial action for KKL-35 and related 1,3,4-oxadiazole benzamides.

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.

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.

Salmonella require the fatty acid regulator PPARδ for the establishment of a metabolic environment essential for long-term persistence.

Host-adapted Salmonella strains are responsible for a number of disease manifestations in mammals, including an asymptomatic chronic infection in which bacteria survive within macrophages located in systemic sites. However, the host cell physiology and metabolic requirements supporting bacterial persistence are poorly understood. In a mouse model of long-term infection, we found that S. typhimurium preferentially associates with anti-inflammatory/M2 macrophages at later stages of infection. Further, PPARδ, a eukaryotic transcription factor involved in sustaining fatty acid metabolism, is upregulated in Salmonella-infected macrophages. PPARδ deficiency dramatically inhibits Salmonella replication, which is linked to the metabolic state of macrophages and the level of intracellular glucose available to bacteria. Pharmacological activation of PPARδ increases glucose availability and enhances bacterial replication in macrophages and mice, while Salmonella fail to persist in Pparδ null mice. These data suggest that M2 macrophages represent a unique niche for long-term intracellular bacterial survival and link the PPARδ-regulated metabolic state of the host cell to persistent bacterial infection.

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.

Mycobacterium tuberculosis activates the DNA-dependent cytosolic surveillance pathway within macrophages.

Cytosolic bacterial pathogens activate the cytosolic surveillance pathway (CSP) and induce innate immune responses, but how the host detects vacuolar pathogens like Mycobacterium tuberculosis is poorly understood. We show that M. tuberculosis also initiates the CSP upon macrophage infection via limited perforation of the phagosome membrane mediated by the ESX-1 secretion system. Although the bacterium remains within the phagosome, this permeabilization results in phagosomal and cytoplasmic mixing and allows extracellular mycobacterial DNA to access host cytosolic receptors, thus blurring the distinction between "vacuolar" and "cytosolic" pathogens. Activation of cytosolic receptors induces signaling through the Sting/Tbk1/Irf3 axis, resulting in IFN-ß production. Surprisingly, Irf3(-/-) mice, which cannot respond to cytosolic DNA, are resistant to long-term M. tuberculosis infection, suggesting that the CSP promotes M. tuberculosis infection. Thus, cytosolic sensing of mycobacterial DNA plays a key role in M. tuberculosis pathogenesis and likely contributes to the high type I IFN signature in tuberculosis.