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Abstract Mycobacterium tuberculosis (Mtb) employs multiple mechanisms, such as phagocytosis and autophagy, to evade innate immune clearance and establish infection. In the present study, we identified the ESX-1 secretion-associated protein EspL, which promotes Mtb survival by inhibiting phagosome maturation and autophagy initiation. EspL knockout decreased Mtb intracellular survival, while EspL overexpression increased bacterial survival by interfering with phagocytosis and autophagy. EspL interacts with ULK1 and promotes its phosphorylation at Ser757, leading to the inhibition of autophagy initiation. Additionally, overexpression of EspL reduced antigen presentation and T-cell responses both in vitro and in vivo. Our findings revealed that EspL interferes with autophagy and antigen presentation by suppressing ULK1 activation. These insights provide a novel understanding of Mtb pathogenicity.

Abstract Background It is difficult to definitively determine the degree of ischemia in the bowel in which an incarcerated groin hernia is embedded. Failure to diagnose and intervene promptly and accurately increases the rate of bowel resection and patient mortality. The aim of this study is to investigate the risk factors for incarcerated inguinal hernia complicating bowel necrosis with resection and to establish a predictive model as a reference for clinical work. Methods Patients with incarcerated groin hernia who were admitted to our hospital were retrospectively analyzed. They were divided into bowel resection and non-bowel resection groups based on whether bowel resection was performed in the surgical record and postoperative pathological results. Risk factors for the development of bowel resection in incarcerated groin hernia were analyzed by univariate analysis and multivariate logistic regression, respectively. The screened independent risk factors were used to establish a prediction model, and finally, the predictive ability and accuracy of the model were validated and the clinical benefit was analyzed. Results A total of 345 patients with incarcerated groin hernia were included, of whom 58 underwent bowel resection for bowel necrosis and 287 did not. Multifactorial logistic regression analysis identified bowel obstruction (OR, 7.285 [95% CI, 2.254–23.542], P = 0.001), peritonitis (OR, 16.786 [95% CI, 5.436–51.838], P = 0.000), duration of incarcerated groin hernia (OR, 1.009 [95% CI, 1. 001-1.018], P = 0.034), heart rate (OR, 1.109 [95% CI, 1.021–1.205], P = 0.014), and preoperative total protein (OR, 0.900 [95% CI, 0.836–0.969], P = 0.005) were independent risk factors for bowel resection in incarcerated groin hernia. The predictive value of the established prediction model was basically in agreement with the measured value with a consistency index of 0.938 (0.901–0.974) and had a good clinical benefit. Conclusion Clinical screening and management of independent risk factors for bowel resection in patients with incarcerated groin hernia should be strengthened. The predictive model developed in this study has high diagnostic efficacy for bowel resection associated with incarcerated inguinal hernia, with the aim of reducing the incidence of bowel resection and unplanned secondary surgery.

Abstract Despite the initial successes of the Bacillus Calmette-Guerin (BCG) vaccine in children, its efficacy against tuberculosis is highly variable. There is a lack of understanding about how mental conditions influence BCG vaccination. Here, we used the chronic social defeat stress (CSDS) model to explore the effects of depression on BCG vaccination efficacy. We observed higher lung and spleen bacterial loads and a lower organ index in depressed compared to BCG mice. Meanwhile, a relatively lower T cell protective efficacy was observed in both compared to control and BCG mice via a mycobacterium growth inhibition assay (MGIA). Cytokine expression of IL-12p40, IL-1β, IL-17, TNF-α and IFN-γ was reduced, whereas the expression of IL-10 and IL-5 was increased in the spleen of both compared to BCG mice. Moreover, the proportions of CD4+IFN-γ+, CD8+IFN-γ+ T lymphocytes and CD4+ effector/central memory T cells were reduced in the splenocytes of the depressed BCG mice. Depression promotes CD4+ regulatory T cells (Treg) and myeloid-derived suppressor cell (MDSC) generation in depressed mice, contributing to the reduced pro-inflammatory immune response upon BCG vaccination. This study provides insight into the decreased protective immunity by BCG vaccination attributable to depression in mice.

Activating transcription factor 6 (ATF6), an important signaling molecule in unfolded protein response (UPR), plays a role in the pathogenesis of several diseases, including diseases such as congenital retinal disease, liver fibrosis and ankylosing spondylitis. After endoplasmic reticulum stress (ERS), ATF6 is activated after separation from binding immunoglobulin protein (GRP78/BiP) in the endoplasmic reticulum (ER) and transported to the Golgi apparatus to be hydrolyzed by site 1 and site 2 proteases into ATF6 fragments, which localize to the nucleus and regulate the transcription and expression of ERS-related genes. In these diseases, ERS leads to the activation of UPR, which ultimately lead to the occurrence and development of diseases by regulating the physiological state of cells through the ATF6 signaling pathway. Here, we discuss the evidence for the pathogenic importance of ATF6 signaling in different diseases and discuss preclinical results.

ABSTRACT African swine fever virus (ASFV) causes severe acute hemorrhagic disease with high mortality in domestic pigs but minimal or no symptoms to warthogs, for which the underlying mechanism remains elusive. ASFV encodes numerous proteins to disturb host immune responses via interacting with host proteins. In this study, we deciphered the comprehensive protein-protein interaction (PPI) network between ASFV and host immune pathways by the recombination-based library vs library high-throughput yeast two-hybrid screening. This PPI network contains both ASFV-host PPI and ASFV-ASFV PPI information, providing a comprehensive ASFV-host interactome landscape. We further explored the ASFV-host PPI difference between domestic pigs and warthogs. Moreover, the inhibitory effect of ASFV proteins in the PPI with cGAS-STING pathway on IFN-I and NF-κB promoter activity was screened to investigate the ASFV-host PPI functions. Our work will help the exploration of ASFV pathogenesis and the development of anti-ASFV vaccine and ASFV-resistant transgenic pigs.IMPORTANCEAfrican swine fever (ASF), caused by African swine fever virus (ASFV), has become a major crisis for the pork industry in recent years. The mechanism for ASFV pathology and the clinical symptoms difference of ASF between domestic pigs and reservoir hosts remain to be elucidated. We deciphered the comprehensive protein-protein interaction (PPI) network between ASFV and host immune pathways. The intensive PPI network contained both ASFV-host immune pathway PPI and ASFV-ASFV PPI information, providing a comprehensive ASFV-host interaction landscape. Furthermore, the ASFV-host PPI difference between domestic pigs and warthogs was explored, which will be instructive for exploring essential candidates involved in ASFV pathology. Moreover, we screened the inhibitory effect of ASFV proteins in the PPI with cGAS-STING pathway on IFN-I and NF-κB, further providing possible functions of ASFV-host PPI network in innate immune regulation.

Abstract Mycobacterium tuberculosis (M. tuberculosis) can replicate in the macrophage by interfering with many host protein functions. While it is far from known these host proteins for controlling M. tuberculosis infection. Herein, we infected macrophages including THP-1 and Raw264.7 cells with M. tuberculosis and identified the differentially expressed genes (DEGs) in the interferon signaling pathway. Among them, 2′-5′ oligoadenylate synthetase-like (OASL) underwent the greatest upregulation in M. tuberculosis-infected macrophages. Knockdown of the expression of OASL attenuated M. tuberculosis survival in macrophages. Further, bioinformatics analysis revealed the potential interaction axis of OASL-TAB3- Rv0127, which was further validated by the yeast-two-hybrid (Y2H) assay and Co-IP. This interaction axis might regulate the M. tuberculosis survival and proliferation in macrophages. The study reveals a possible role of OASL during M. tuberculosis infection as a target to control its propagation.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Alternaria alternata (A. alternata) is a common pathogen that greatly influences apples’ quantity and quality. However, chemical treatments produce increased health risks along with decreased food and environmental safety. Advancements in plant molecular biology, such as transgenic technology, have increased apple trees’ resistance to pathogens and have therefore attracted widespread attention. WRKY transcription factors are involved in abiotic and biotic stress regulation; however, their biological role in non-model plants such as apple, is still unknown. In this investigation, MdWRKY120 was isolated from the ‘GL-3′ apple to determine its function during Alternaria alternate infection. The MdWRKY120-GFP fusion protein was located in the nucleus. MdWRKY120 in yeast cells exhibited activating transcriptional activity, meaning it is a transcription activator. MdWRKY120 overexpression transgenic plants were more sensitive to A. alternata, while RNAi transgenic plants showed increased resistance to A. alternata. This investigation demonstrates that MdWRKY120 enhances the susceptibility of apples to A. alternata.

As an intracellular pathogen, Mycobacterium tuberculosisM. tuberculosis

Abstract Background Our previous research found that YAP1 may have a role in multidrug resistance (MDR) in small cell lung cancer (SCLC). However, its underlying mechanism is unknown. Methods In this study, we investigated the expression of YAP1 using immunohistochemical staining and assessed the relationship between the expression of YAP1 and overall survival in patients with SCLC. We established H69 stable cell lines that overexpressed constitutively active YAP1 and H446 stable cell lines that dominate negative YAP1. We conducted CCK‐8, flow cytometric analysis, and in vivo chemosensitivity experiments to evaluate the function of YAP1 in drug sensitivity apoptosis in vitro and in vivo. Results The results indicated that patients with high YAP1 expression have shorter survival rates and more advanced disease stage than those with low YAP1 expression. YAP1 may induce MDR by inhibiting the apoptosis of SCLC. YAP1 induced MDR when YAP1 was hyperactivated, and drug sensitivity increased when YAP1 was inhibited in vitro and in vivo. CD74 was significantly correlated with YAP1 in SCLC samples. Inhibition of CD74 using ISO‐1 increased drug sensitivity significantly. Conclusions The expression of YAP1 is significantly correlated with overall survival and disease stage in patients with SCLC. YAP1 may play an important role in these patients. We were the first to report that YAP1 can induce MDR in SCLC in vitro and in vivo. CD74 may be involved in YAP1‐induced MDR.

The nervous necrosis virus (NNV) mainly attacks the central nervous system of fish to cause viral nervous necrosis, which is an acute and serious prevalent disease in fish. Among different genotypes of NNV, red-spotted grouper nervous necrosis virus (RGNNV) is the most widely reported, with the highest number of susceptible species. To better understand the pathogenicity of RGNNV, we first developed a reverse genetic system for recombinant RGNNV rescue using B7GG and striped snakehead (SSN-1) cells. Furthermore, we constructed attenuated RGNNV strains rRGNNV-B2-M1 and rRGNNV-B2-M2 with the loss of B2 protein expression, which grew slower and induced less Mx1 expression than that of wild-type RGNNV. Moreover, rRGNNV-B2-M1 and rRGNNV-B2-M2 were less virulent than the wild-type RGNNV. Our study provides a potential tool for further research on the viral protein function, virulence pathogenesis, and vaccine development of RGNNV, which is also a template for the rescue of other fish viruses.

Tuberculosis (TB) is a debilitating infectious disease responsible for more than one million deaths per year. The emergence of drug-resistant TB poses an urgent need for the development of new anti-TB drugs. In this study, we screened a library of over 4,000 small molecules and found that orbifloxacin and the peptide AK15 possess significant bactericidal activity against Mycobacterium tuberculosis (Mtb) in vitro. Orbifloxacin also showed an effective ability on the clearance of intracellular Mtb and protect mice from a strong inflammatory response but not AK15. Moreover, we identified 17 nucleotide mutations responsible for orbifloxacin resistance by whole-genome sequencing. A critical point mutation (D94G) of the DNA gyrase (gyrA) gene was found to be the key role of resistance to orbifloxacin. The computational docking revealed that GyrA D94G point mutation can disrupt the orbifloxacin–protein gyrase interactions mediated by magnesium ion bridge. Overall, this study indicated the potential ability of orbifloxacin as an anti-tuberculosis drug, which can be used either alone or in combination with first-line antibiotics to achieve more effective therapy on TB.

Mycobacterium tuberculosis (M.tb) secretes numerous proteins to interfere with host immune response for its long-term survival. As one of the top abundant M.tb secreted proteins, Rv3722c was found to be essential for bacilli growth. However, it remains elusive how this protein interferes with the host immune response and regulates M.tb survival. Here, we confirmed that Rv3722c interacted with host TRAF3 to promote M.tb replication in macrophages. Knock-down of TRAF3 attenuated the effect of Rv3722c on the intracellular M.tb survival. The interaction between Rv3722c and TRAF3 hampered MAPK and NF-κB pathways, resulting in a significant increase of IFN-β expression and decrease of IL-1β, IL-6, IL-12p40, and TNF-α expression. Our study revealed that Rv3722c interacted with TRAF3 and interrupted its downstream pathways to promote M.tb survival in macrophages. These findings facilitate further understanding of the mechanism of M.tb secreted proteins in regulating the host cell immune response and promoting its intracellular survival.

Background: Nuclear factor-kappaB (NF-κB) is an inducible transcription factor that plays a key role in inflammation and immune responses, as well as in the regulation of cell proliferation and survival. Previous studies by our group and others have demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection could activate NF-κB in MARC-145 cells and alveolar macrophages. The nucleocapsid (N) protein was identified as an NF-κB activator among the structural proteins encoded by PRRSV; however, it remains unclear whether the nonstructural proteins (Nsps) contribute to NF-κB activation. In this study, we identified which Nsps can activate NF-κB and investigated the potential mechanism(s) by which they act. Results: By screening the individual Nsps of PRRSV strain WUH3, Nsp2 exhibited great potential to activate NF-κB in MARC-145 and HeLa cells. Overexpression of Nsp2 induced IκBα degradation and nuclear translocation of NF-κB. Furthermore, Nsp2 also induced NF-κB-dependent inflammatory factors, including interleukin (IL)-6, IL-8, COX-2, and RANTES. Compared with the Nsp2 of the classical PRRSV strain, the Nsp2 of highly pathogenic PRRSV (HP-PRRSV) strains that possess a 30 amino acid (aa) deletion in Nsp2 displayed greater NF-κB activation. However, the 30-aa deletion was demonstrated to not be associated with NF-κB activation. Further functional domain analyses revealed that the hypervariable region (HV) of Nsp2 was essential for NF-κB activation. Conclusions: Taken together, these data indicate that PRRSV Nsp2 is a multifunctional protein participating in the modulation of host inflammatory response, which suggests an important role of Nsp2 in pathogenesis and disease outcomes. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) RNA endoribonuclease nsp11 belongs to the XendoU superfamily and plays a crucial role in arterivirus replication. Here, we report the first crystal structure of the arterivirus nsp11 protein from PRRSV, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were perfectly superimposed, especially in the "active site loop" (His129 to His144) and "supporting loop" (Val162 to Thr179) regions. Importantly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Mutations of the major dimerization site determinants (Ser74 and Phe76) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. In the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. These findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs. [ABSTRACT FROM AUTHOR]