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.

Pyroptosis modulation by bacterial effector proteins.

Pyroptosis is a proinflammatory form of programmed cell death featured with membrane pore formation that causes cellular swelling and allows the release of intracellular inflammatory mediators. This cell death process is elicited by the activation of the pore-forming proteins named gasdermins, and is intricately orchestrated by diverse regulatory factors in mammalian hosts to exert a prompt immune response against infections. However, growing evidence suggests that bacterial pathogens have evolved to regulate host pyroptosis for evading immune clearance and establishing progressive infection. In this review, we highlight current understandings of the functional role and regulatory network of pyroptosis in host antibacterial immunity. Thereafter, we further discuss the latest advances elucidating the mechanisms by which bacterial pathogens modulate pyroptosis through adopting their effector proteins to drive infections. A better understanding of regulatory mechanisms underlying pyroptosis at the interface of host-bacterial interactions will shed new light on the pathogenesis of infectious diseases and contribute to the development of promising therapeutic strategies against bacterial pathogens.

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.

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.

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 bacterial phospholipid phosphatase inhibits host pyroptosis by hijacking ubiquitin.

The inflammasome-mediated cleavage of gasdermin D (GSDMD) causes pyroptosis and inflammatory cytokine release to control pathogen infection, but how pathogens evade this immune response remains largely unexplored. Here we identify the known protein phosphatase PtpB from Mycobacterium tuberculosis as a phospholipid phosphatase inhibiting the host inflammasome-pyroptosis pathway. Mechanistically, PtpB dephosphorylated phosphatidylinositol-4-monophosphate and phosphatidylinositol-(4,5)-bisphosphate in host cell membrane, thus disrupting the membrane localization of the cleaved GSDMD to inhibit cytokine release and pyroptosis of macrophages. Notably, this phosphatase activity requires PtpB binding to ubiquitin. Disrupting phospholipid phosphatase activity or the ubiquitin-interacting motif of PtpB enhanced host GSDMD-dependent immune responses and reduced intracellular pathogen survival. Thus, pathogens inhibit pyroptosis and counteract host immunity by altering host membrane composition.

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.

The deubiquitinase OTUD1 inhibits colonic inflammation by suppressing RIPK1-mediated NF-κB signaling.

The E3 ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination processes are closely associated with the occurrence and development of colonic inflammation. Ovarian tumor deubiquitinase 1 (OTUD1) is involved in immunoregulatory functions linked to infectious diseases. However, the effect of OTUD1 on intestinal immune responses during colonic inflammatory disorders such as inflammatory bowel disease (IBD) remains unclear. Here, we show that loss of OTUD1 in mice contributes to the pathogenesis of dextran sulfate sodium (DSS)-induced colitis via excessive release of proinflammatory cytokines. In addition, bone marrow transplantation experiments revealed that OTUD1 in hematopoietic cells plays a dominant role in protection against colitis. Mechanistically, OTUD1 physically interacts with receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and selectively cleaves K63-linked polyubiquitin chains from RIPK1 to inhibit the recruitment of NF-κB essential modulator (NEMO). Moreover, the expression of OTUD1 in mucosa samples from ulcerative colitis (UC) patients was lower than that in mucosa samples from healthy controls. Furthermore, we demonstrate that the UC-associated OTUD1 G430V mutation abolishes the ability of OTUD1 to inhibit RIPK1-mediated NF-κB activation and intestinal inflammation. Taken together, our study unveils a previously unexplored role of OTUD1 in moderating intestinal inflammation by inhibiting RIPK1-mediated NF-κB activation, suggesting that the OTUD1-RIPK1 axis could be a potential target for the treatment of IBD.

Ubiquitination-Dependent Regulation of Small GTPases in Membrane Trafficking: From Cell Biology to Human Diseases.

Membrane trafficking is critical for cellular homeostasis, which is mainly carried out by small GTPases, a class of proteins functioning in vesicle budding, transport, tethering and fusion processes. The accurate and organized membrane trafficking relies on the proper regulation of small GTPases, which involves the conversion between GTP- and GDP-bound small GTPases mediated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Emerging evidence indicates that post-translational modifications (PTMs) of small GTPases, especially ubiquitination, play an important role in the spatio-temporal regulation of small GTPases, and the dysregulation of small GTPase ubiquitination can result in multiple human diseases. In this review, we introduce small GTPases-mediated membrane trafficking pathways and the biological processes of ubiquitination-dependent regulation of small GTPases, including the regulation of small GTPase stability, activity and localization. We then discuss the dysregulation of small GTPase ubiquitination and the associated human membrane trafficking-related diseases, focusing on the neurological diseases and infections. An in-depth understanding of the molecular mechanisms by which ubiquitination regulates small GTPases can provide novel insights into the membrane trafficking process, which knowledge is valuable for the development of more effective and specific therapeutics for membrane trafficking-related human diseases.

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.

High- and low-virulent bovine Pasteurella multocida induced differential NLRP3 inflammasome activation and subsequent IL-1ß secretion.

Pasteurella multocida is a gram-negative bacterial pathogen, which causes a large number of diseases in mammals, birds and human. Although the bacterium has been known for decades, the pathogenesis and the mechanisms of P. multocida induced host immunity are poorly understood. Recently, we have reported that nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome plays an important role in caspase-1 activation and IL-1ß secretion in macrophages infected with P. multocida. In this study, the inflammasome activation and IL-1ß secretion were further demonstrated by using high- and low-virulent bovine P. multocida isolates. The results showed that, comparing with macrophages infected with the high-virulent PmCQ2 isolates, the low-virulent PmCQ6 induced higher levels of NLRP3 transcription, caspase-1 activation and mature IL-1ß secretion. Furthermore, the capsule of the high-virulent PmCQ2 was much thicker than that of low-virulent PmCQ6, which indicating that capsular thickness might influence the bacteria colonization and NLRP3 inflammasome activation. The results suggested that differences in maturation of IL-1ß in macrophages upon high- and low- virulent P. multocida infection are critically dependent on the differential activation of NLRP3 inflammasome. This study provided more understanding for the host immune responses induced by P. multocida and further extended the knowledge of P. multocida virulence from the view of host innate immunity.

Pneumolysin-Dependent Calpain Activation and Interleukin-1α Secretion in Macrophages Infected with Streptococcus pneumoniae.

Pneumolysin (PLY), a major virulence factor of Streptococcus pneumoniae, is a pore-forming cytolysin that modulates host innate responses contributing to host defense against and pathogenesis of pneumococcal infections. Interleukin-1α (IL-1α) has been shown to be involved in tissue damage in a pneumococcal pneumonia model; however, the mechanism by which this cytokine is produced during S. pneumoniae infection remains unclear. In this study, we examined the role of PLY in IL-1α production. Although the strains induced similar levels of pro-IL-1α expression, wild-type S. pneumoniae D39, but not a deletion mutant of the ply gene (Δply), induced the secretion of mature IL-1α from host macrophages, suggesting that PLY is critical for the maturation and secretion of IL-1α during S. pneumoniae infection. Further experiments with calcium chelators and calpain inhibitors indicated that extracellular calcium ions and calpains (calcium-dependent proteases) facilitated the maturation and secretion of IL-1α from D39-infected macrophages. Moreover, we found that PLY plays a critical role in calcium influx and calpain activation, as elevated intracellular calcium levels and the degradation of the calpain substrate α-fodrin were detected in macrophages infected with D39 but not the Δply strain. These results suggested that PLY induces the influx of calcium in S. pneumoniae-infected macrophages, followed by calpain activation and subsequent IL-1α maturation and secretion.