Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system.

Mycobacterium tuberculosis PtpA, a secreted tyrosine phosphatase essential for tuberculosis pathogenicity, could be an ideal target for a drug against tuberculosis, but its active-site inhibitors lack selectivity over human phosphatases. Here we found that PtpA suppressed innate immunity dependent on pathways of the kinases Jnk and p38 and the transcription factor NF-κB by exploiting host ubiquitin. Binding of PtpA to ubiquitin via a region with no homology to human proteins activated it to dephosphorylate phosphorylated Jnk and p38, leading to suppression of innate immunity. Furthermore, the host adaptor TAB3 mediated NF-κB signaling by sensing ubiquitin chains, and PtpA blocked this process by competitively binding the ubiquitin-interacting domain of TAB3. Our findings reveal how pathogens subvert innate immunity by coopting host ubiquitin and suggest a potential tuberculosis treatment via targeting of ubiquitin-PtpA interfaces.

Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity.

Upon Mycobacterium tuberculosis (Mtb) infection, protein kinase G (PknG), a eukaryotic-type serine-threonine protein kinase (STPK), is secreted into host macrophages to promote intracellular survival of the pathogen. However, the mechanisms underlying this PknG-host interaction remain unclear. Here, we demonstrate that PknG serves both as a ubiquitin-activating enzyme (E1) and a ubiquitin ligase (E3) to trigger the ubiquitination and degradation of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TGF-ß-activated kinase 1 (TAK1), thereby inhibiting the activation of NF-κB signaling and host innate responses. PknG promotes the attachment of ubiquitin (Ub) to the ubiquitin-conjugating enzyme (E2) UbcH7 via an isopeptide bond (UbcH7 K82-Ub), rather than the usual C86-Ub thiol-ester bond. PknG induces the discharge of Ub from UbcH7 by acting as an isopeptidase, before attaching Ub to its substrates. These results demonstrate that PknG acts as an unusual ubiquitinating enzyme to remove key components of the innate immunity system, thus providing a potential target for tuberculosis treatment.

Simulated microgravity suppresses MAPK pathway-mediated innate immune response to bacterial infection and induces gut microbiota dysbiosis.

During spaceflight, astronauts are subjected to various physical stressors including microgravity, which could cause immune dysfunction and thus potentially predispose astronauts to infections and illness. However, the mechanisms by which microgravity affects innate immunity remain largely unclear. In this study, we conducted RNA-sequencing analysis to show that simulated microgravity (SMG) suppresses the production of inflammatory cytokines including tumor necrosis factor (TNF) and interleukin-6 (IL-6) as well as the activation of the innate immune signaling pathways including the p38 mitogen-activated protein kinase (MAPK) and the Erk1/2 MAPK pathways in the Enteropathogenic escherichia coli (EPEC)-infected macrophage cells. We then adopted hindlimb-unloading (HU) mice, a model mimicking the microgravity of a spaceflight environment, to demonstrate that microgravity suppresses proinflammatory cytokine-mediated intestinal immunity to Citrobacter rodentium infection and induces the disturbance of gut microbiota, both of which phenotypes could be largely corrected by the introduction of VSL#3, a high-concentration probiotic preparation of eight live freeze-dried bacterial species. Taken together, our study provides new insights into microgravity-mediated innate immune suppression and intestinal microbiota disturbance, and suggests that probiotic VSL#3 has great potential as a dietary supplement in protecting individuals from spaceflight mission-associated infections and gut microbiota dysbiosis.

LILRB1-HLA-G axis defines a checkpoint driving natural killer cell exhaustion in tuberculosis.

Chronic infections, including Mycobacterium tuberculosis (Mtb)-caused tuberculosis (TB), can induce host immune exhaustion. However, the key checkpoint molecules involved in this process and the underlying regulatory mechanisms remain largely undefined, which impede the application of checkpoint-based immunotherapy in infectious diseases. Here, through adopting time-of-flight mass cytometry and transcriptional profiling to systematically analyze natural killer (NK) cell surface receptors, we identify leukocyte immunoglobulin like receptor B1 (LILRB1) as a critical checkpoint receptor that defines a TB-associated cell subset (LILRB1+ NK cells) and drives NK cell exhaustion in TB. Mechanistically, Mtb-infected macrophages display high expression of human leukocyte antigen-G (HLA-G), which upregulates and activates LILRB1 on NK cells to impair their functions by inhibiting mitogen-activated protein kinase (MAPK) signaling via tyrosine phosphatases SHP1/2. Furthermore, LILRB1 blockade restores NK cell-dependent anti-Mtb immunity in immuno-humanized mice. Thus, LILRB1-HLA-G axis constitutes a NK cell immune checkpoint in TB and serves as a promising immunotherapy target.

TRIM27 elicits protective immunity against tuberculosis by activating TFEB-mediated autophagy flux.

Infectious diseases, such as Mycobacterium tuberculosis (Mtb)-caused tuberculosis (TB), remain a global threat exacerbated by increasing drug resistance. Host-directed therapy (HDT) is a promising strategy for infection treatment through targeting host immunity. However, the limited understanding of the function and regulatory mechanism of host factors involved in immune defense against infections has impeded HDT development. Here, we identify the ubiquitin ligase (E3) TRIM27 (tripartite motif-containing 27) as a host protective factor against Mtb by enhancing host macroautophagy/autophagy flux in an E3 ligase activity-independent manner. Mechanistically, upon Mtb infection, nuclear-localized TRIM27 increases and functions as a transcription activator of TFEB (transcription factor EB). Specifically, TRIM27 binds to the TFEB promoter and the TFEB transcription factor CREB1 (cAMP responsive element binding protein 1), thus enhancing CREB1-TFEB promoter binding affinity and promoting CREB1 transcription activity toward TFEB, eventually inducing autophagy-related gene expression as well as autophagy flux activation to clear the pathogen. Furthermore, TFEB activator 1 can rescue TRIM27 deficiency-caused decreased autophagy-related gene transcription and attenuated autophagy flux, and accordingly suppressed the intracellular survival of Mtb in cell and mouse models. Taken together, our data reveal that TRIM27 is a host defense factor against Mtb, and the TRIM27-CREB1-TFEB axis is a potential HDT-based TB target that can enhance host autophagy flux.Abbreviations: ATG5: autophagy related 5; BMDMs: bone marrow-derived macrophages; CFU: colony-forming unit; ChIP-seq: chromatin immunoprecipitation followed by sequencing; CREB1: cAMP responsive element binding protein 1; CTSB: cathepsin B; E3: ubiquitin ligase; EMSA: electrophoretic mobility shift assay; HC: healthy control; HDT: host-directed therapy; LAMP: lysosomal associated membrane protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1: mucolipin TPR cation channel 1; Mtb: Mycobacterium tuberculosis; NLS: nuclear localization signal; PBMCs: peripheral blood mononuclear cells; PRKA/PKA: protein kinase cAMP-activated; qRT-PCR: quantitative real-time PCR; RFP: RET finger protein; TB: tuberculosis; TBK1: TANK binding kinase 1; TFEB: transcription factor EB; TRIM: tripartite motif; TSS: transcription start site; ULK1: unc-51 like autophagy activating kinase 1.

TRIM27 maintains gut homeostasis by promoting intestinal stem cell self-renewal.

Dysregulation of gut homeostasis is associated with irritable bowel syndrome (IBS), a chronic functional gastrointestinal disorder affecting approximately 11.2% of the global population. The poorly understood pathogenesis of IBS has impeded its treatment. Here, we report that the E3 ubiquitin ligase tripartite motif-containing 27 (TRIM27) is weakly expressed in IBS but highly expressed in inflammatory bowel disease (IBD), a frequent chronic organic gastrointestinal disorder. Accordingly, knockout of Trim27 in mice causes spontaneously occurring IBS-like symptoms, including increased visceral hyperalgesia and abnormal stool features, as observed in IBS patients. Mechanistically, TRIM27 stabilizes ß-catenin and thus activates Wnt/ß-catenin signaling to promote intestinal stem cell (ISC) self-renewal. Consistent with these findings, Trim27 deficiency disrupts organoid formation, which is rescued by reintroducing TRIM27 or ß-catenin. Furthermore, Wnt/ß-catenin signaling activator treatment ameliorates IBS symptoms by promoting ISC self-renewal. Taken together, these data indicate that TRIM27 is critical for maintaining gut homeostasis, suggesting that targeting the TRIM27/Wnt/ß-catenin axis could be a potential treatment strategy for IBS. Our study also indicates that TRIM27 might serve as a potential biomarker for differentiating IBS from IBD.

A mycobacterial effector promotes ferroptosis-dependent pathogenicity and dissemination.

Ferroptosis is a lipid peroxidation-driven and iron-dependent programmed cell death involved in multiple physical processes and various diseases. Emerging evidence suggests that several pathogens manipulate ferroptosis for their pathogenicity and dissemination, but the underlying molecular mechanisms remain elusive. Here, we identify that protein tyrosine phosphatase A (PtpA), an effector secreted by tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis (Mtb), triggers ferroptosis to promote Mtb pathogenicity and dissemination. Mechanistically, PtpA, through its Cys11 site, interacts with host RanGDP to enter host cell nucleus. Then, the nuclear PtpA enhances asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a) via targeting protein arginine methyltransferase 6 (PRMT6), thus inhibiting glutathione peroxidase 4 (GPX4) expression, eventually inducing ferroptosis to promote Mtb pathogenicity and dissemination. Taken together, our findings provide insights into molecular mechanisms of pathogen-induced ferroptosis, indicating a potential TB treatment via blocking Mtb PtpA-host PRMT6 interface to target GPX4-dependent ferroptosis.

A Golgi-resident GPR108 cooperates with E3 ubiquitin ligase Smurf1 to suppress antiviral innate immunity.

The regulation of antiviral immunity is crucial in maintaining host immune homeostasis, a process that involves dynamic modulations of host organelles. The Golgi apparatus is increasingly perceived as a host organelle functioning as a critical platform for innate immunity, but the detailed mechanism by which it regulates antiviral immunity remains elusive. Here, we identify the Golgi-localized G protein-coupled receptor 108 (GPR108) as a regulator of type Ι interferon responses by targeting interferon regulatory factor 3 (IRF3). Mechanistically, GPR108 enhances the ubiquitin ligase Smad ubiquitylation regulatory factor 1 (Smurf1)-mediated K63-linked polyubiquitination of phosphorylated IRF3 for nuclear dot 10 protein 52 (NDP52)-dependent autophagic degradation, leading to suppression of antiviral immune responses against DNA or RNA viruses. Taken together, our study provides insight into the crosstalk between the Golgi apparatus and antiviral immunity via a dynamic and spatiotemporal regulation of GPR108-Smurf1 axis, thereby indicating a potential target for treating viral infection.

M. tuberculosis PknG manipulates host autophagy flux to promote pathogen intracellular survival.

The eukaryotic-type protein kinase G (PknG), one of the eleven eukaryotic type serine-threonine protein kinase (STPK) in Mycobacterium tuberculosis (Mtb), is involved in mycobacterial survival within macrophages, presumably by suppressing phagosome and autophagosome maturation, which makes PknG an attractive drug target. However, the exact mechanism by which PknG inhibits pathogen clearance during mycobacterial infection remains largely unknown. Here, we show that PknG promotes macroautophagy/autophagy induction but inhibits autophagosome maturation, causing an overall effect of blocked autophagy flux and enhanced pathogen intracellular survival. PknG prevents the activation of AKT (AKT serine/threonine kinase) via competitively binding to its pleckstrin homology (PH) domain, leading to autophagy induction. Remarkably, PknG could also inhibit autophagosome maturation to block autophagy flux via targeting host small GTPase RAB14. Specifically, PknG directly interacts with RAB14 to block RAB14-GTP hydrolysis. Furthermore, PknG phosphorylates TBC1D4/AS160 (TBC1 domain family member 4) to suppress its GTPase-activating protein (GAP) activity toward RAB14. In macrophages and in vivo, PknG promotes Mtb intracellular survival through blocking autophagy flux, which is dependent on RAB14. Taken together, our data unveil a dual-functional bacterial effector that tightly regulates host autophagy flux to benefit pathogen intracellular survival.Abbreviations: AKT: AKT serine/threonine kinase; ATG5: autophagy related 5; BMDMs: bone marrow-derived macrophages; DTT: dithiothreitol; FBS: fetal calf serum; GAP: GTPase-activating protein; MOI: multiplicity of infection; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; OADC: oleic acid-albumin-dextrose-catalase; PC, phosphatidylcholine; PH: pleckstrin homology; PI3K: phosphoinositide 3-kinase; PknG: protein kinase G; PtdIns(3,4,5)P3: phosphatidylinositol(3,4,5)-trisphosphate; SQSTM1: sequestosome 1; STPK: serine-threonine protein kinase; TB: tuberculosis; TBC1D4: TBC1 domain family member 4; TPR: tetratricopeptide repeat; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

A Novel Aquaporin Subfamily Imports Oxygen and Contributes to Pneumococcal Virulence by Controlling the Production and Release of Virulence Factors.

Aquaporins, integral membrane proteins widely distributed in organisms, facilitate the transport of water, glycerol, and other small uncharged solutes across cellular membranes and play important physiological roles in eukaryotes. However, characterizations and physiological functions of the prokaryotic aquaporins remain largely unknown. Here, we report that Streptococcus pneumoniae (pneumococcus) AqpC (Pn-AqpC), representing a new aquaporin subfamily possessing a distinct substrate-selective channel, functions as an oxygen porin by facilitating oxygen movement across the cell membrane and contributes significantly to pneumococcal virulence. The use of a phosphorescent oxygen probe showed that Pn-AqpC facilitates oxygen permeation into pneumococcal and Pn-AqpC-expressing yeast cells. Reconstituting Pn-AqpC into liposomes prepared with pneumococcal and Escherichia coli cellular membranes further verified that Pn-AqpC transports O2 but not water or glycerol. Alanine substitution showed that Pro232 in the substrate channel is key for Pn-AqpC in O2 transport. The deletion of Pn-aqpC significantly reduced H2O2 production and resistance to H2O2 and NO of pneumococci, whereas low-H2O2 treatment helped the ΔPn-aqpC mutant resist higher levels of H2O2 and even NO, indicating that Pn-AqpC-facilitated O2 permeation contributes to pneumococcal resistance to H2O2 and NO. Remarkably, the lack of Pn-aqpC alleviated cell autolysis, thus reducing pneumolysin (Ply) release and decreasing the hemolysis of pneumococci. Accordingly, the ΔPn-aqpC mutant markedly reduced survival in macrophages, decreased damage to macrophages, and significantly reduced lethality in mice. Therefore, the oxygen porin Pn-AqpC, through modulating H2O2 production and pneumolysin release, the two major pneumococcal virulence factors, controls the virulence of pneumococcus. Pn-AqpC orthologs are widely distributed in various pneumococcal serotypes, highlighting that the oxygen porin is important for pneumococcal pathogenicity. IMPORTANCE Pneumococcus is the leading cause of community-acquired pneumonia, bacteremia, and meningitis. This work reports that a novel aquaporin subfamily represented by pneumococcal Pn-AqpC functions as an oxygen porin facilitating O2 influx into cells. Importantly, by mediating O2 influx, Pn-AqpC controls the production and release of H2O2 and Ply, the two major pneumococcal virulence factors. Moreover, by enhancing endogenous H2O2 production, Pn-AqpC significantly increases pneumococcal resistance to H2O2 and even NO, the major bactericidal chemical produced by macrophages. Consequently, the deletion of Pn-aqpC markedly decreased pneumococcal survival in macrophages and reduced damage to macrophages. Accordingly, the ΔPn-aqpC mutant displays significantly attenuated virulence in a murine pneumonia model. Given that Pn-AqpC orthologs are widely distributed in all pneumococcal serotypes, this new subfamily of aquaporins is identified as novel virulence-related proteins.

Mycobacterium tuberculosis Mce2E suppresses the macrophage innate immune response and promotes epithelial cell proliferation.

The intracellular pathogen Mycobacterium tuberculosis (Mtb) can survive in the host and cause disease by interfering with a variety of cellular functions. The mammalian cell entry 2 (mce2) operon of Mtb has been shown to contribute to tuberculosis pathogenicity. However, little is known about the regulatory roles of Mtb Mce2 family proteins towards host cellular functions. Here we show that the Mce2 family protein Mce2E suppressed the macrophage innate immune response and promoted epithelial cell proliferation. Mce2E inhibited activation of the extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling pathways in a non-canonical D motif (a MAPK-docking motif)-dependent manner, leading to reduced expression of TNF and IL-6 in macrophages. Furthermore, Mce2E promoted proliferation of human lung epithelium-derived lung adenoma A549 cells by inhibiting K48-linked polyubiquitination of eEF1A1 in a ß strand region-dependent manner. In summary, Mce2E is a novel multifunctional Mtb virulence factor that regulates host cellular functions in a niche-dependent manner. Our data suggest a potential novel target for TB therapy.

A Mycobacterium tuberculosis surface protein recruits ubiquitin to trigger host xenophagy.

Ubiquitin-mediated xenophagy, a type of selective autophagy, plays crucial roles in host defense against intracellular pathogens including Mycobacterium tuberculosis (Mtb). However, the exact mechanism by which host ubiquitin targets invaded microbes to trigger xenophagy remains obscure. Here we show that ubiquitin could recognize Mtb surface protein Rv1468c, a previously unidentified ubiquitin-binding protein containing a eukaryotic-like ubiquitin-associated (UBA) domain. The UBA-mediated direct binding of ubiquitin to, but not E3 ubiquitin ligases-mediated ubiquitination of, Rv1468c recruits autophagy receptor p62 to deliver mycobacteria into LC3-associated autophagosomes. Disruption of Rv1468c-ubiquitin interaction attenuates xenophagic clearance of Mtb in macrophages, and increases bacterial loads in mice with elevated inflammatory responses. Together, our findings reveal a unique mechanism of host xenophagy triggered by direct binding of ubiquitin to the pathogen surface protein, and indicate a diplomatic strategy adopted by Mtb to benefit its persistent intracellular infection through controlling intracellular bacterial loads and restricting host inflammatory responses.

Gut microbiota and intestinal FXR mediate the clinical benefits of metformin.

The anti-hyperglycemic effect of metformin is believed to be caused by its direct action on signaling processes in hepatocytes, leading to lower hepatic gluconeogenesis. Recently, metformin was reported to alter the gut microbiota community in humans, suggesting that the hyperglycemia-lowering action of the drug could be the result of modulating the population of gut microbiota. However, the critical microbial signaling metabolites and the host targets associated with the metabolic benefits of metformin remained elusive. Here, we performed metagenomic and metabolomic analysis of samples from individuals with newly diagnosed type 2 diabetes (T2D) naively treated with metformin for 3 d, which revealed that Bacteroides fragilis was decreased and the bile acid glycoursodeoxycholic acid (GUDCA) was increased in the gut. These changes were accompanied by inhibition of intestinal farnesoid X receptor (FXR) signaling. We further found that high-fat-diet (HFD)-fed mice colonized with B. fragilis were predisposed to more severe glucose intolerance, and the metabolic benefits of metformin treatment on glucose intolerance were abrogated. GUDCA was further identified as an intestinal FXR antagonist that improved various metabolic endpoints in mice with established obesity. Thus, we conclude that metformin acts in part through a B. fragilis-GUDCA-intestinal FXR axis to improve metabolic dysfunction, including hyperglycemia.

The mycobacterial phosphatase PtpA regulates the expression of host genes and promotes cell proliferation.

Mycobacterium tuberculosis PtpA is a secreted effector protein that dephosphorylates several proteins in the host cell cytoplasm, such as p-JNK, p-p38, and p-VPS33B, leading to suppression of host innate immunity. Here we show that, in addition, PtpA enters the nucleus of host cells and regulates the expression of host genes, some of which are known to be involved in host innate immunity or in cell proliferation and migration (such as GADD45A). PtpA can bind directly to the promoter region of GADD45A in vitro. Both phosphatase activity and DNA-binding ability of PtpA are important in suppressing host innate immune responses. Furthermore, PtpA-expressing Mycobacterium bovis BCG promotes proliferation and migration of human lung adenoma A549 cells in vitro and in a mouse xenograft model. Further research is needed to test whether mycobacteria, via PtpA, might affect cell proliferation or migration in humans. Mycobacterium tuberculosis secretes a protein, PtpA, that dephosphorylates proteins in the host cell cytoplasm, weakening immune responses. Here, the authors show that PtpA also enters the nucleus, affects the expression of several host genes, and promotes proliferation and migration of a cancer cell line.

The ubiquitin ligase TRIM27 functions as a host restriction factor antagonized by Mycobacterium tuberculosis PtpA during mycobacterial infection.

Macrophage-mediated innate immune responses play crucial roles in host defense against pathogens. Recent years have seen an explosion of host proteins that act as restriction factors blocking viral replication in infected cells. However, the essential factors restricting Mycobacterium tuberculosis (Mtb) and their regulatory roles during mycobacterial infection remain largely unknown. We previously reported that Mtb tyrosine phosphatase PtpA, a secreted effector protein required for intracellular survival of Mtb, inhibits innate immunity by co-opting the host ubiquitin system. Here, we identified a new PtpA-interacting host protein TRIM27, which is reported to possess a conserved RING domain and usually acts as an E3 ubiquitin ligase that interferes with various cellular processes. We further demonstrated that TRIM27 restricts survival of mycobacteria in macrophages by promoting innate immune responses and cell apoptosis. Interestingly, Mtb PtpA could antagonize TRIM27-promoted JNK/p38 MAPK pathway activation and cell apoptosis through competitively binding to the RING domain of TRIM27. TRIM27 probably works as a potential restriction factor for Mtb and its function is counteracted by Mtb effector proteins such as PtpA. Our study suggests a potential tuberculosis treatment via targeting of the TRIM27-PtpA interfaces.

Genomic and transcriptomic analysis of NDM-1 Klebsiella pneumoniae in spaceflight reveal mechanisms underlying environmental adaptability.

The emergence and rapid spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae strains has caused a great concern worldwide. To better understand the mechanisms underlying environmental adaptation of those highly drug-resistant K. pneumoniae strains, we took advantage of the China's Shenzhou 10 spacecraft mission to conduct comparative genomic and transcriptomic analysis of a NDM-1 K. pneumoniae strain (ATCC BAA-2146) being cultivated under different conditions. The samples were recovered from semisolid medium placed on the ground (D strain), in simulated space condition (M strain), or in Shenzhou 10 spacecraft (T strain) for analysis. Our data revealed multiple variations underlying pathogen adaptation into different environments in terms of changes in morphology, H2O2 tolerance and biofilm formation ability, genomic stability and regulation of metabolic pathways. Additionally, we found a few non-coding RNAs to be differentially regulated. The results are helpful for better understanding the adaptive mechanisms of drug-resistant bacterial pathogens.