Pathogens have evolved diverse strategies to counteract host immunity. Ubiquitylation of lipopolysaccharide (LPS) on cytosol-invading bacteria by the E3 ligase RNF213 creates 'eat me' signals for antibacterial autophagy, but whether and how cytosol-adapted bacteria avoid LPS ubiquitylation remains poorly understood. Here, we show that the enterobacterium Shigella flexneri actively antagonizes LPS ubiquitylation through IpaH1.4, a secreted effector protein with ubiquitin E3 ligase activity. IpaH1.4 binds to RNF213, ubiquitylates it and targets it for proteasomal degradation, thus counteracting host-protective LPS ubiquitylation. To understand how IpaH1.4 recognizes RNF213, we determined the cryogenic electron microscopy structure of the IpaH1.4-RNF213 complex. The specificity of the interaction is achieved through the leucine-rich repeat of IpaH1.4, which binds the RING domain of RNF213 by hijacking the conserved RING interface required for binding to ubiquitin-charged E2 enzymes. IpaH1.4 also targets other E3 ligases involved in inflammation and immunity through binding to the E2-interacting face of their RING domains, including the E3 ligase LUBAC that is required for the synthesis of M1-linked ubiquitin chains on cytosol-invading bacteria downstream of RNF213. We conclude that IpaH1.4 has evolved to antagonize multiple antibacterial and proinflammatory host E3 ligases.

Regulated cell death and xenophagy constitute fundamental cellular mechanisms against invading microorganisms. Staphylococcus aureus, a notorious pathogen, can invade and persist within host cells for extended periods. Here, we describe a novel mechanism by which S. aureus subverts these host defenses through the manipulation of the CASP8 (caspase 8) signaling pathway. Upon invasion, S. aureus triggers the assembly of a RIPK3 (receptor interacting serine/threonine kinase 3) complex to induce CASP8 autoprocessing. However, the bacterium inhibits CUL3 (cullin 3)-dependent K63-linked ubiquitination, leading to an atypical activation of CASP8. This non-canonical activation does not initiate the CASP8-CASP3 cascade but instead suppresses RIPK3-dependent necroptosis, a regulated cell death pathway typically activated when apoptosis fails. The resulting non-apoptotic, cleaved CASP8 redirects its enzymatic activity toward cleaving SQSTM1/p62, a selective macroautophagy/autophagy receptor, thus enabling S. aureus to evade antimicrobial xenophagy. The results of this study suggest that S. aureus reprograms the CASP8 signaling pathway from inducing cell death to preserving cell survival and inhibiting xenophagy, a critical strategy that supports its stealthy replication and persistence within host cells.Abbreviations: CASP3: caspase 3; CASP8: caspase 8; CFU: colony-forming units; CUL3: cullin 3; DUB: deubiquitinating enzyme; MAP1LC3B-II/LC3B-II: microtubule associated protein 1 light chain 3 beta-II; MOI: multiplicity of infection; RIPK1: receptor interacting protein kinase 1; RIPK3: receptor interacting protein kinase 3; S. aureus: Staphylococcus aureus.

Many intracellular bacteria secrete deubiquitinase (DUB) effectors into eukaryotic host cells to keep the bacterial surface or the enclosing vesicle membrane free of ubiquitin marks. This study describes a family of DUBs from several bacterial genera, including Simkania, Parachlamydia, Burkholderia, and Pigmentiphaga, which is structurally related to eukaryotic Josephin-type DUBs but contains members that catalyze a unique destructive substrate deubiquitination. These ubiquitin C-terminal clippases (UCCs) cleave ubiquitin before the C-terminal diGly motif, thereby truncating the modifier and leaving a remnant on the substrate. By comparing the crystal structures of substrate-bound clippases and a closely related conventional DUB, we identified the factors causing this shift and found them to be conserved in other clippases, including one highly specific for M1-linked ubiquitin chains. This enzyme class has great potential to serve as tools for studying the ubiquitin system, particularly aspects involving branched chains.

Host-directed therapies (HDTs) have been investigated as a potential solution to combat intracellular and drug-resistant bacteria. HDTs stem from extensive research on the intricate interactions between the host and intracellular bacteria, leading to a treatment approach that relies on immunoregulation. To improve the bioavailability and safety of HDTs, researchers have utilized diverse drug delivery systems (DDS) to encapsulate and transport therapeutic agents to target cells. In this review, we first introduce the three mechanisms of bactericidal action and intracellular bacterial evasion: autophagy, reactive oxygen species (ROS), and inflammatory cytokines, with a particular focus on autophagy. Special attention is given to the detailed mechanism of xenophagy in clearing intracellular bacteria, a crucial selective autophagy process that specifically targets and degrades intracellular pathogens. Following this, we present the application of DDS to modulate these regulatory methods for intracellular bacteria elimination. By integrating insights from immunology and nanomedicine, this review highlights the emerging role of DDS in advancing HDTs for intracellular bacterial infections and paving the way for innovative therapeutic interventions.

Xenophagy plays a crucial role in restraining the growth of intracellular bacteria in macrophages. However, the machinery governing autophagosome‒lysosome fusion during bacterial infection remains incompletely understood. Here, we utilize leprosy, an ideal model for exploring the interactions between host defense mechanisms and bacterial infection. We highlight the glycoprotein nonmetastatic melanoma protein B (GPNMB), which is highly expressed in macrophages from lepromatous leprosy (L-Lep) patients and interferes with xenophagy during bacterial infection. Upon infection, GPNMB interacts with autophagosomal-localized STX17, leading to a reduced N-glycosylation level at N296 of GPNMB. This modification promotes the degradation of SNAP29, thus preventing the assembly of the STX17-SNAP29-VAMP8 SNARE complex. Consequently, the fusion of autophagosomes with lysosomes is disrupted, resulting in inhibited cellular autophagic flux. In addition to Mycobacterium leprae, GPNMB deficiency impairs the proliferation of various intracellular bacteria in human macrophages, suggesting a universal role of GPNMB in intracellular bacterial infection. Furthermore, compared with their counterparts, Gpnmbfl/fl Lyz2-Cre mice presented decreased Mycobacterium marinum amplification. Overall, our study reveals a previously unrecognized role of GPNMB in host antibacterial defense and provides insights into its regulatory mechanism in SNARE complex assembly.

Repair of infectious bone defects remains a serious problem in clinical practice owing to the high risk of infection and excessive reactive oxygen species (ROS) during the early stage, and the residual bacteria and delayed Osseo integrated interface in the later stage, which jointly creates a complex and dynamic microenvironment and leads to bone non-union. The melatonin carbon dots (MCDs) possess antibacterial and osteogenesis abilities, greatly simplifying the composition of a multifunctional material. Therefore, a multifunctional hydrogel containing MCDs (GH-MCD) is developed to meet the multi-stage and complex repair needs of infectious bone injury in this study. The GH-MCD can intelligently release MCDs responding to the acidic microenvironment to scavenge intracellular ROS and exhibit good antibacterial activity by inducing the production of ROS in bacteria and inhibiting the expression of secA2. Moreover, it has high osteogenesis and long-lasting antimicrobial activity during bone repair. RNA-seq results reveal that the hydrogels promote the repair of infected bone healing by enhancing cellular resistance to bacteria, balancing osteogenesis and osteoclastogenesis, and regulating the immune microenvironment. In conclusion, the GH-MCD can promote the repair of infectious bone defects through the programmed transformation of the microenvironment, providing a novel strategy for infectious bone defects.

The hair coat is an adaptive evolutionary trait unique to mammals, aiding them in adapting to complex environmental challenges. Although some of the factors involved in regulating hair follicle development have been characterized, further in-depth research is still needed. Retinoic acid receptor-related orphan receptor alpha (RORA), as a member of the nuclear receptor family, is highly involved in the regulation of cellular states. Previous studies have shown that autophagy plays a significant role in hair follicle development. This study uses rat hair follicle stem cells (HFSCs) as a model to analyze the impact of RORA on the autophagy levels of HFSCs. Upon activation of RORA, autophagy indicators such as the LC3-II/LC3-I ratio and MDC staining significantly increased, suggesting an elevated level of autophagy in HFSCs. Following treatment with chloroquine, the LC3-II/LC3-I ratio, as well as the expression levels of BECN1 protein and SQSTM1 protein, were markedly elevated in the cells, indicating that the autophagic flux was unobstructed and ruling out the possibility that RORA activation impeded autophagy. Additionally, the level of the Sqstm1 gene increased markedly after RORA activation promoted autophagy in the cells. We found that RORA regulates the transcription level of Sqstm1 by binding to its promoter region. We believe that RORA activation significantly promotes the level of autophagy, particularly selective autophagy, in HFSCs, suggesting that RORA has the potential to become a new target for research on hair follicle development. This research provides a theoretical foundation for studies on hair follicle development and also offers new insights for the treatment of diseases such as alopecia.

Mycobacteria have several mechanisms for evasion of protective responses mounted by the host. In this study, we unravel yet another mechanism that is mediated by Toll-Like Receptors TLR2, TLR4, and TLR7 in epithelial cells. We show that mycobacterial infection of epithelial cells increases the expression of TLR2, TLR4, and TLR7. Stimulation of either TLR along with mycobacterial infection results in an inhibition of oxidative burst resulting in increased survival of mycobacteria inside epithelial cells. TLR stimulation along with mycobacterial infection also inhibits activation of epithelial cells for T cell responses by differentially regulating the activation of ERK-MAPK and p38-MAPK along with inhibition of co-stimulatory molecule CD86 expression. Furthermore, stimulation of either TLR inhibits the induction of apoptosis and autophagy. Knockdown of either TLR by specific siRNAs reverses the inhibition by ROS and apoptosis by mycobacteria and results in reduced intracellular survival of mycobacteria in a MyD88-dependent manner. These results point towards a negative role for TLR2, TLR4, and TLR7 in regulating protective responses to M. bovis BCG infection in epithelial cells.

Interleukin-27 (IL-27) is a cytokine that is reported to be highly expressed in the peripheral blood of patients with pulmonary tuberculosis (PTB). IL-27-mediated signaling pathways, which exhibit anti- Mycobacterium tuberculosis (Mtb) properties, have also been demonstrated in macrophages infected with Mtb. However, the exact mechanism remains unclear. This study aimed to clarify the potential molecular mechanisms through which IL-27 enhances macrophage resistance to Mtb infection. Both normal and PTB patients provided bronchoalveolar lavage fluid (BALF). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals and stimulated with 50 ng/mL macrophage-colony stimulating factor (M-CSF) to obtain monocyte-derived macrophages (MDMs). Using 100 ng/mL phorbol 12-myristate 13-acetate (PMA), THP-1 cells were induced to differentiate into THP-1-derived macrophage-like cells (TDMs). Both MDMs and TDMs were subsequently infected with the Mtb strain H37Rv and treated with 50 ng/mL IL-27 prior to infection. The damage and inflammation of macrophages were examined using flow cytometry, enzyme-linked immunosorbent assay (ELISA), and Western blotting. Patients with PTB had elevated levels of IL-27 in their BALF. Preconditioning with IL-27 was shown to reduce H37Rv-induced MDMs and TDMs apoptosis while also decreasing the levels of Cleaved Caspase-3, Bax and the proinflammatory cytokines TNF-α, IL-1β, and IL-6, promoting the expression of Bcl-2 and the anti-inflammatory factors IL-10 and IL-4. Silencing of the IL-27 receptor IL-27Ra increased macrophage damage and inflammation triggered by H37Rv. Mechanistically, IL-27 activates autophagy by inhibiting TLR4/NF-κB signaling and activating the PI3K/AKT signaling pathway, thereby inhibiting H37Rv-induced macrophage apoptosis and the inflammatory response. Our study suggests that IL-27 alleviates H37Rv-induced macrophage injury and the inflammatory response by activating autophagy and that IL-27 may be a new target for the treatment of PTB.

Gastric cancer remains a significant global health challenge, with Helicobacter pylori (H. pylori) recognized as a major etiological agent, affecting an estimated 50% of the world's population. There has been a rapidly expanding knowledge of the molecular and pathogenetic mechanisms of H. pylori over the decades. This review summarizes the latest research advances to elucidate the molecular mechanisms underlying the H. pylori infection in gastric carcinogenesis. Our investigation of the molecular mechanisms reveals a complex network involving STAT3, NF-κB, Hippo, and Wnt/β-catenin pathways, which are dysregulated in gastric cancer caused by H. pylori. Furthermore, we highlight the role of H. pylori in inducing oxidative stress, DNA damage, chronic inflammation, and cell apoptosis-key cellular events that pave the way for carcinogenesis. Emerging evidence also suggests the effect of H. pylori on the tumor microenvironment and its possible implications for cancer immunotherapy. This review synthesizes the current knowledge and identifies gaps that warrant further investigation. Despite the progress in our previous knowledge of the development in H. pylori-induced gastric cancer, a comprehensive investigation of H. pylori's role in gastric cancer is crucial for the advancement of prevention and treatment strategies. By elucidating these mechanisms, we aim to provide a more in-depth insights for the study and prevention of H. pylori-related gastric cancer.

Exposure to Group A Streptococcus leads to a broad spectrum of disease and sequelae, as the bacterium employs a wide range of virulence factors to facilitate colonization of the host, propagation and onward transmission, disrupting both innate and adaptive immune responses. The protease SpyCEP has a crucial role in contributing to bacterial immune evasion by impairing neutrophil recruitment and killing of bacteria through the cleavage of interleukin-8 (IL-8). Given this critical function, SpyCEP represents a key vaccine antigen and quantifying functional anti-SpyCEP antibodies represents not only an important marker of vaccine efficacy, but also a tool to dissect the natural immune response. Here, we report the development and characterization of an IL-8 cleavage inhibition assay to measure the function of anti-SpyCEP antibodies in human sera. The assay was demonstrated to be sensitive, highly specific, linear and reproducible, and suitable for evaluating the function of anti-SpyCEP antibodies induced in humans in vaccine clinical trials and in observational studies of natural immunity.

Intracellular bacterial infections are a serious threat to human health due to their ability to escape immunity and develop drug resistance. Recent attention has been devoted to identifying and ablating intracellular bacteria with fluorescence probes. Aggregation-induced emission luminogens (AIEgens) photosensitizers as fluorescence probes possess excellent photostability and rapid response, which have emerged as powerful fluorescent tools for intracellular bacterial detection and antibacterial therapy. This review is intended to highlight the current advances in AIEgens on intracellular bacteria imaging and elimination, which covers topics from intracellular AIE mechanism, intracellular bacteria imaging of AIEgens to the elimination of intracellular bacteria with AIEgens. AIEgens utilized different interactions to detect intracellular bacteria, emitting bright light due to restricted intramolecular movement to visualize intracellular bacteria. Photosensitive AIEgens generate reactive oxygen species (ROS) in the aggregate state to elimate intracellular bacteria. Moreover, the prospects and application of AIEgens in intracellular bacteria imaging and elimination are also discussed, which provides insights for the development of AIE-based diagnostic and therapeutic materials and technologies.

The public's health and the financial sustainability of international societies remain threatened by bacterial zoonoses, with limited reliable diagnostic and therapeutic options available for bacterial diseases. Bacterial infections influence mammalian miRNA expression in host-pathogen interactions. In order to counteract bacterial infections, miRNAs participate in gene-specific expression and play important regulatory roles that rely on translational inhibition and target gene degradation by binding to the 3' non-coding region of target genes. Intriguingly, according to current studies, that exogenous miRNAs derived from plants could potentially serve as effective medicinal components sourced from traditional Chinese medicine plants. These exogenous miRNAs exhibit stable functionality in mammals and mimic the regulatory roles of endogenous miRNAs, illuminating the molecular processes behind the therapeutic effects of plants. This review details the immune defense mechanisms of inflammation, apoptosis, autophagy and cell cycle disturbance caused by some typical bacterial infections, summarizes the role of some mammalian miRNAs in regulating these mechanisms, and introduces the cGAS-STING signaling pathway in detail. Evidence suggests that this newly discovered immune defense mechanism in mammalian cells can also be affected by miRNAs. Meanwhile, some examples of transboundary regulation of mammalian mRNA and even bacterial diseases by exogenous miRNAs from plants are also summarized. This viewpoint provides fresh understanding of microbial tactics and host mechanisms in the management of bacterial illnesses.

Many pathogens have evolved sophisticated strategies to evade autophagy, a crucial cellular defense mechanism that typically targets and degrades invading microorganisms. By subverting or inhibiting autophagy, these pathogens can create a more favorable environment for their replication and survival within the host. For instance, some bacteria secrete factors that block autophagosome formation, while others might escape from autophagosomes before degradation. These evasion tactics are critical for the pathogens' ability to establish and maintain infections. Understanding the mechanisms by which pathogens avoid autophagy is crucial for developing new therapeutic strategies, as enhancing autophagy could bolster the host's immune response and aid in the elimination of pathogenic bacteria. Francisella tularensis can manipulate host cell pathways to prevent its detection and destruction by autophagy, thereby enhancing its virulence. Given the potential for F. tularensis to be used as a bioterrorism agent due to its high infectivity and ability to cause severe disease, research into how this pathogen evades autophagy is of critical importance. By unraveling these mechanisms, new therapeutic approaches could be developed to enhance autophagic responses and strengthen host defense against this and other similarly evasive pathogens.

Bacteria-mediated antitumor therapy has gained widespread attention for its innate tumor-targeting capability and excellent immune activation properties. Nevertheless, the clinical approval of bacterial therapies remains elusive primarily due to the formidable challenge of balancing safety with enhancing in vivo efficacy. In this study, leveraging the probiotic Escherichia coli Nissle1917 (EcN) emerges as a promising approach for colon cancer therapy, offering a high level of safety attributed to its lack of virulence factors and its tumor-targeting potential owing to its obligate anaerobic nature. Specifically, we delineate the erythrocyte (RBC) membrane-camouflaged EcN, termed as Trojan horse EcN@RBC, which triggers apoptosis in tumor cells by mitigating mitochondrial membrane potential (MMP) and subsequently activating the PINK1/Parkin pathway associated with mitophagy. Concurrently, the decline in MMP induced by mitophagy disrupts the mitochondrial permeability transition pore (MPTP), leading to the release of Cytochrome C and subsequent apoptosis induction. Moreover, synergistic effects were observed through the combination of the autophagy activator rapamycin, bolstering the antitumor efficacy in vivo. These findings offer novel insights into probiotic-mediated antitumor mechanisms and underscore the therapeutic potential of EcN@RBC for colon cancer patients.

LC3-associated phagocytosis (LAP) is critical in host defense against invading pathogens, but the molecular mechanism for LAP activation is still unclear. Here, we find programmed cell death 6 (PDCD6) as a negative regulator of LAP. PDCD6 deficiency in mice and macrophages induces enhanced bactericidal activity and LAP formation. In parallel, lactate dehydrogenase A (LDHA) activity and lactate production is induced in macrophages challenged with bacteria, Zymosan or Pam3CSK4, while genetic ablation or pharmacological inhibition of LDHA reduces lactate levels and impairs bactericidal activity in vivo and in vitro. Mechanistically, PDCD6 interacts with LDHA to downregulate lactate metabolism, leading to reduced RUBCN lactylation at lysine33 (K33). By contrast, PDCD6-deficiency increases RUBCN lactylation, thereby promotes RUBCN interaction with VPS34, LAP formation, and protective responses. Our results thus suggest a PDCD6-LDHA-lactate-RUBCN axis of innate immunity regulation that may both contribute to protection from infectious diseases and serve as targets for therapeutic development.

The thioredoxin (Trx) system plays a vital role in protecting against oxidative stress and ensures correct disulfide bonding to maintain protein function. Our previous research demonstrated that TrxA of Streptococcus suis Serotype 2 (SS2), a clinical strain from the lung of a diseased pig, contributes to virulence but is not involved in antioxidative stress. In this study, we identified another gene in the Trx family, TrxC, which encodes a protein of 104 amino acids with a CGDC active motif and 22.4 % amino acid sequence homology with TrxA. Unlike the TrxA, TrxC mutant strains were more susceptible to oxidative stresses induced by hydrogen peroxide and paraquat. In vitro experiments, the survival rate of the TrxC deletion mutant in RAW264.7 macrophages was only one-eighth of that of TrxA mutant strains. Transcriptome analysis revealed that autophagy-related genes were significantly upregulated in the TrxC mutant compared to those in the wild-type or TrxA mutant strains. Co-localization of LC3 puncta with TrxC was confirmed using laser confocal microscopy, and autophagy-related indicators were quantified using western blotting. Autophagy deficiency induced by ATG5 knockout significantly increased SS2 survival rate, especially in TrxC mutant strains. For the upstream signal regulation pathways, we found ΔTrxC strains regulate autophagy by activation of PI3K/Akt/mTOR signaling in RAW264.7 macrophages. In the Akt1-overexpressing cell line, ΔTrxC infection significantly decreased the autophagic response and promoted ΔTrxC mutant strain survival, while inhibition of Akt with MK2206 resulted in reduced ΔTrxC mutant strain survival and enhance the autophagic response. Furthermore, loss of TrxC increased the activity of MSR1, thereby inducing cellular autophagy and phagocytosis. Our data demonstrate that TrxC of SS2 contributes to virulence by inducing antioxidative stress and inhibits autophagy via the PI3K-Akt-mTOR pathway in macrophages, with MSR1 acting as a key factor in controlling infection.

Identifying soft selective sweeps using genomic data is a challenging yet crucial task in population genetics. In this study, we present HaploSweep, a novel method for detecting and categorizing soft and hard selective sweeps based on haplotype structure. Through simulations spanning a broad range of selection intensities, softness levels, and demographic histories, we demonstrate that HaploSweep outperforms iHS, nSL, and H12 in detecting soft sweeps. HaploSweep achieves high classification accuracy-0.9247 for CHB, 0.9484 for CEU, and 0.9829 YRI-when applied to simulations in line with the human Out-of-Africa demographic model. We also observe that the classification accuracy remains consistently robust across different demographic models. Additionally, we introduce a refined method to accurately distinguish soft shoulders adjacent to hard sweeps from soft sweeps. Application of HaploSweep to genomic data of CHB, CEU, and YRI populations from the 1000 genomes project has led to the discovery of several new genes that bear strong evidence of population-specific soft sweeps (HRNR, AMBRA1, CBFA2T2, DYNC2H1, and RANBP2 etc.), with prevalent associations to immune functions and metabolic processes. The validated performance of HaploSweep, demonstrated through both simulated and real data, underscores its potential as a valuable tool for detecting and comprehending the role of soft sweeps in adaptive evolution.

Western diets are the underlying cause of metabolic and liver diseases. Recent trend to limit the consumption of protein-rich animal products has become more prominent. This dietary change entails decreased protein consumption; however, it is still unknown how this affects innate immunity. Here, we studied the influence of a low protein diet (LPD) on the liver response to bacterial infection in mice. We found that LPD protects from Salmonella enterica serovar Typhimurium (S. Typhimurium)-induced liver damage. Bulk and single-cell RNA sequencing of murine liver cells showed reduced inflammation and upregulation of autophagy-related genes in myeloid cells in mice fed with LPD after S. Typhimurium infection. Mechanistically, we found reduced activation of the mammalian target of rapamycin (mTOR) pathway, whilst increased phagocytosis and activation of autophagy in LPD-programmed macrophages. We confirmed these observations in phagocytosis and mTOR activation in metabolically programmed human peripheral blood monocyte-derived macrophages. Together, our results support the causal role of dietary components on the fitness of the immune system.

Urinary tract infections are primarily caused by uropathogenic Escherichia coli (UPEC). UPEC infects bladder epithelial cells (BECs) via fusiform vesicles and escapes into the cytosol by disrupting fusiform vesicle membrane using outer membrane phospholipase PldA, and establishes biofilm-like intracellular bacterial communities (IBCs) for protection from host immune clearance. Cytosolic UPEC is captured by autophagy to form autophagosomes, then transported to lysosomes, triggering the spontaneous exocytosis of lysosomes. The mechanism by which UPEC evades autophagy to recognize and form IBCs remains unclear. Here, we demonstrate that by inhibiting autophagic flux, UPEC PldA reduces the lysosome exocytosis of BECs. By reducing intracellular phosphatidylinositol 3-phosphate levels, UPEC PldA increases the accumulation of NDP52 granules and decreases the targeting of NDP52 to autophagy, hence stalling preautophagosome structures. Thus, our results uncover a critical role for PldA to inhibit autophagic flux, favoring UPEC escapes from lysosome exocytosis, thereby contributing to acute urinary tract infection.

Tuberculosis (TB), predominantly caused by Mycobacterium tuberculosis (MTB) infection, remains a prominent global health challenge. Macrophages are the frontline defense against MTB, relying on autophagy for intracellular bacterial clearance. However, MTB can combat and evade autophagy, and it influences macrophage polarization, facilitating immune evasion and promoting infection. We previously found that heparin-binding hemagglutinin (HBHA) inhibits autophagy in A549 cells; however, its role in macrophage autophagy and polarization remains unclear.

Bacterial cultures, cell cultures, Western blotting, immunofluorescence, macrophage infection assays, siRNA knockdown, and enzyme-linked immunosorbent assay were used to investigate HBHA's impact on macrophages and its relevance in Mycobacterium infection.

HBHA inhibited macrophage autophagy. Expression of recombinant HBHA in Mycobacterium smegmatis (rMS-HBHA) inhibited autophagy, promoting bacterial survival within macrophages. Conversely, HBHA knockout in the Mycobacterium bovis bacillus Calmette-Guérin (BCG) mutant (BCG-ΔHBHA) activated autophagy and reduced bacterial survival. Mechanistic investigations revealed that HBHA may inhibit macrophage autophagy through the Toll-like receptor 4-dependent PI3K-AKT-mTOR signaling pathway. Furthermore, HBHA induced macrophage M2 polarization.

Mycobacterium may exploit HBHA to suppress the antimicrobial immune response in macrophages, facilitating intracellular survival and immune evasion through autophagy inhibition and M2 polarization induction. Our findings may help identify novel therapeutic targets and develop more effective treatments against MTB infection.

The genetic signatures associated with the susceptibility to nontuberculous mycobacterial pulmonary disease (NTM-PD) are still unknown. In this study, we performed RNA sequencing to explore gene expression profiles and represent characteristic factor in NTM-PD.

Peripheral blood samples were collected from patients with NTM-PD and healthy individuals (controls). Differentially expressed genes (DEGs) were identified by RNA sequencing and subjected to functional enrichment and immune cell deconvolution analyses.

We enrolled 48 participants, including 26 patients with NTM-PD (median age, 58.0 years; 84.6% female), and 22 healthy controls (median age, 58.5 years; 90.9% female). We identified 21 upregulated and 44 downregulated DEGs in the NTM-PD group compared to those in the control group. NTM infection did not have a significant impact on gene expression in the NTM-PD group compared to the control group, and there were no differences in the proportion of immune cells. However, through gene ontology (GO), gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) analysis, we discovered that PARK2 is a key factor associated with NTM-PD. The PARK2 gene, which is linked to the ubiquitination pathway, was downregulated in the NTM-PD group (fold change, - 1.314, P = 0.047). The expression levels of PARK2 remained unaltered after favorable treatment outcomes, suggesting that the gene is associated with host susceptibility rather than with the outcomes of infection or inflammation. The area under the receiver operating characteristic curve for the PARK2 gene diagnosing NTM-PD was 0.813 (95% confidence interval, 0.694-0.932).

We identified the genetic signatures associated with NTM-PD in a cohort of Korean patients. The PARK2 gene presents as a potential susceptibility factor in NTM-PD .

Autophagy is a unique catabolic process that degrades irrelevant or damaged components in eukaryotic cells to maintain homeostasis and eliminate infections from pathogenesis. Pathogenic bacteria have developed many autophagy manipulation techniques that affect host immune responses and intracellular bacterial pathogens have evolved to avoid xenophagy. However, reducing its effectiveness as an innate immune response has not yet been elucidated. Bacterial pathogens cause autophagy in infected cells as a cell-autonomous defense mechanism to eliminate the pathogen. However, harmful bacteria have learned to control autophagy and defeat host defenses. Intracellular bacteria can stimulate and control autophagy, while others inhibit it to prevent xenophagy and lysosomal breakdown. This review evaluates the putative functions for xenophagy in regulating bacterial infection, emphasizing that successful pathogens have evolved strategies to disrupt or exploit this defense, reducing its efficiency in innate immunity. Instead, animal models show that autophagy-associated proteins influence bacterial pathogenicity outside of xenophagy. We also examine the consequences of the complex interaction between autophagy and bacterial pathogens in light of current efforts to modify autophagy and develop host-directed therapeutics to fight bacterial infections. Therefore, effective pathogens have evolved to subvert or exploit xenophagy, although autophagy-associated proteins can influence bacterial pathogenicity outside of xenophagy. Finally, this review implies how the complex interaction between autophagy and bacterial pathogens affects host-directed therapy for bacterial pathogenesis.

Infection with Glaesserella parasuis, the primary pathogen behind Glässer's disease, is often associated with diverse clinical symptoms, including serofibrinous polyserositis, arthritis, and meningitis. Autophagy plays a dual role in bacterial infections, exerting either antagonistic or synergistic effects depending on the nature of the pathogen. Our previous studies have demonstrated that autophagy serves as a defense mechanism, combating inflammation and invasion caused by infection of highly virulent G. parasuis. However, the precise mechanisms remain to be elucidated. Pathogens exhibit distinct interactions with inflammasomes and autophagy processes. Herein, we explored the effect of autophagy on inflammasomes during G. parasuis infection. We found that G. parasuis infection triggers NLRP3-dependent pro-CASP-1-IL-18/IL-1β processing and maturation pathway, resulting in increased release of IL-1β and IL-18. Inhibition of autophagy enhances NLRP3 inflammasome activity, whereas stimulation of autophagy restricts it during G. parasuis infection. Furthermore, assembled NLRP3 inflammasomes undergo ubiquitination and recruit the autophagic adaptor, p62, facilitating their sequestration into autophagosomes during G. parasuis infection. These results suggest that the induction of autophagy mitigates inflammation by eliminating overactive NLRP3 inflammasomes during G. parasuis infection. Our research uncovers a mechanism whereby G. parasuis infection initiates inflammatory responses by promoting the assembly of the NLRP3 inflammasomes and activating NLRP3-CASP-1, both of which processes are downregulated by autophagy. This suggests that pharmacological manipulation of autophagy could be a promising approach to modulate G. parasuis-induced inflammatory responses.

Crohn disease (CD) is an inflammatory bowel disease whose pathogenesis involves inappropriate immune responses toward gut microbiota on genetically predisposed backgrounds. Notably, CD is associated with single-nucleotide polymorphisms affecting several genes involved in macroautophagy/autophagy, the catabolic process that ensures the degradation and recycling of cytosolic components and microorganisms. In a clinical translation perspective, monitoring the autophagic activity of CD patients will require some knowledge on the intrinsic functional status of autophagy. Here, we focused on monocyte-derived dendritic cells (DCs) to characterize the intrinsic quantitative features of the autophagy flux. Starting with DCs from healthy donors, we documented a reprogramming of the steady state flux during the transition from the immature to mature status: both the autophagosome pool size and the flux were diminished at the mature stage while the autophagosome turnover remained stable. At the cohort level, DCs from CD patients were comparable to control in term of autophagy flux reprogramming capacity. However, the homozygous presence of ATG16L1 rs2241880 A>G (T300A) and ULK1 rs12303764 (G/T) polymorphisms abolished the capacity of CD patient DCs to reprogram their autophagy flux during maturation. This effect was not seen in the case of CD patients heterozygous for these polymorphisms, revealing a gene dose dependency effect. In contrast, the NOD2 rs2066844 c.2104C>T (R702W) polymorphism did not alter the flux reprogramming capacity of DCs. The data, opening new clinical translation perspectives, indicate that polymorphisms affecting autophagy-related genes can differentially influence the capacity of DCs to reprogram their steady state autophagy flux when exposed to proinflammatory challenges.Abbreviation: BAFA1: bafilomycin A1, CD: Crohn disease; DC: dendritic cells; HD: healthy donor; iDCs: immature DCs; IL: interleukin; J: autophagosome flux; LPS: lipopolysaccharide; MHC: major histocompatibility complex; nA: autophagosome pool size; SNPs: single-nucleotide polymorphisms; PCA: principal component analysis; TLR: toll like receptor; τ: transition time; TNF: tumor necrosis factor.

Galectins, a family of glycan-binding proteins have been shown to bind a wide range of glycans. In the cytoplasm, these glycans can be endogenous (or "self"), originating from damaged endocytic vesicles, or exogenous (or "non-self"), found on the surface of invading microbial pathogens. Galectins can detect these unusual cytosolic exposures to glycans and serve as critical regulators in orchestrating immune responses in innate and adaptive immunity. This review provides an overview of how galectins modulate host cellular responses, such as autophagy, xenophagy, and inflammasome-dependent cell death program, to infection.

Listeria monocytogenes, a prominent foodborne pathogen responsible for zoonotic infections, owes a significant portion of its virulence to the presence of the phospholipase PlcB. In this study, we performed an in-depth examination of the intricate relationship between L. monocytogenes PlcB and host cell mitochondria, unveiling a novel participant in bacterial survival: the mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA). Our investigation uncovered previously unexplored levels of interaction and colocalization between PCCA and PlcB within host cells, with particular emphasis on the amino acids 504-508 of PCCA, which play a pivotal role in this partnership. To assess the effect of PCCA expression on L. monocytogenes proliferation, PCCA expression levels were manipulated by siRNA-si-PCCA or pCMV-N-HA-PCCA plasmid transfection. Our findings demonstrated a clear inverse correlation between PCCA expression levels and the proliferation of L. monocytogenes. Furthermore, the effect of L. monocytogenes infection on PCCA expression was investigated by assessing PCCA mRNA and protein expression in HeLa cells infected with L. monocytogenes. These results indicate that L. monocytogenes infection did not significantly alter PCCA expression. These findings led us to propose that PCCA represents a novel participant in L. monocytogenes survival, and its abundance has a detrimental impact on bacterial proliferation. This suggests that L. monocytogenes may employ PlcB-PCCA interactions to maintain stable PCCA expression, representing a unique pro-survival strategy distinct from that of other intracellular bacterial pathogens.

Mitochondria represent attractive targets for pathogenic bacteria seeking to modulate host cellular processes to promote their survival and replication. Our current study has uncovered mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA) as a novel host cell protein that interacts with L. monocytogenes PlcB. The results demonstrate that PCCA plays a negative regulatory role in L. monocytogenes infection, as heightened PCCA levels are associated with reduced bacterial survival and persistence. However, L. monocytogenes may exploit the PlcB-PCCA interaction to maintain stable PCCA expression and establish a favorable intracellular milieu for bacterial infection. Our findings shed new light on the intricate interplay between bacterial pathogens and host cell mitochondria, while also highlighting the potential of mitochondrial metabolic enzymes as antimicrobial agents.

Conserved molecular signatures in multidrug-resistant Salmonella typhi can serve as novel therapeutic targets for mitigation of infection. In this regard, we present the S. typhi cell division activator protein (StCAP) as a conserved target across S. typhi variants. From in silico and fluorimetric assessments, we found that StCAP is a DNA-binding protein. Replacement of the identified DNA-interacting residue Arg34 of StCAP with Ala34 showed a dramatic (15-fold) increase in Kd value compared to the wild type (Kd 546 nm) as well as a decrease in thermal stability (10 °C shift). Out of the two screened molecules against the DNA-binding pocket of StCAP, eltrombopag, and nilotinib, the former displayed better binding. Eltrombopag inhibited the stand-alone S. typhi culture with an IC50 of 38 μM. The effect was much more pronounced on THP-1-derived macrophages (T1Mac) infected with S. typhi where colony formation was severely hindered with IC50 reduced further to 10 μM. Apoptotic protease activating factor1 (Apaf1), a key molecule for intrinsic apoptosis, was identified as an StCAP-interacting partner by pull-down assay against T1Mac. Further, StCAP-transfected T1Mac showed a significant increase in LC3 II (autophagy marker) expression and downregulation of caspase 3 protein. From these experiments, we conclude that StCAP provides a crucial survival advantage to S. typhi during infection, thereby making it a potent alternative therapeutic target.

Ammonia in aquatic environments is toxic to fish, directly impacting their growth performance and development. Activation of autophagy can facilitate intracellular component renewal and enhance an organism's adaptability to adverse environments. Therefore, this study investigates the impact of autophagy on the yellow catfish under acute ammonia stress. In this study, the yellow catfish intraperitoneally injected with 0.9 % sodium chloride were placed with 0 (CON group) and 125 (HA group) mg/L T-AN (Total ammonia nitrogen) dechlorinated water. The yellow catfish intraperitoneally injected with 30 mg/kg fish CQ (Chloroquine, HA + CQ group) and 1.5 mg/kg fish RAPA (rapamycin, HA + RAPA group) were placed in dechlorinated water containing 125 mg/L T-AN. The results showed that activation of autophagy by injecting with RAPA can alleviate oxidative stress (catalase, superoxide dismutase, total antioxidant capacity significantly increased, H2O2 content significantly decreased), and inflammatory response (pro-inflammatory factors TNF-α, MyD88, IL 1-β gene expression decreased significantly), apoptosis (baxa, Bcl2, Tgf-β, Smad2, Caspase3, Caspase 9 gene expression decreased significantly) induced by ammonia stress. In addition, activation of autophagy in yellow catfish can enhance ammonia detoxification by promoting the urea cycle and synthesis of glutamine (the mRNA level of CPS Ⅰ, ARG, OTC, ASS, ASL, and GS increased in the HA + RAPA group). The data above demonstrates that activating autophagy can alleviate oxidative stress, inflammatory responses, and cell apoptosis induced by ammonia stress. Therefore, enhancing autophagy is proposed as a potential strategy to mitigate the detrimental impacts of ammonia stress on yellow catfish.

Emamectin benzoate (EMB) is extensively used as a crop protection agent. Overuse of EMB poses a serious threat to the quality of water and non-target organisms in the environment. Resveratrol (RES) is a natural phytoalexin with the function of anti-oxidation and anti-inflammation. Nonetheless, it is unclear whether EMB affects the expression of cytokines and induces autophagy, apoptosis, and necroptosis of hepatocytes (L8824 cell) in grass carp (Ctenopharyngodon idella), and whether RES has an attenuate function in this process. Therefore, we established the L8824 cells model of EMB exposure and treated it with RES. The results showed that compared with the control (CON) group, EMB exposure significantly increased the nitric oxide (NO) content, inducible nitric oxide synthase (iNOS) activity, and the expression of iNOS and phosphorylated nuclear factor kappa B (p-NF-κB) (P < 0.05). In addition, compared with the CON group, the results of flow cytometry and dansylcadaverine (MDC) staining showed a significant increase in apoptosis and autophagy in the EMB-exposed group (P < 0.05) with the activation of the B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X (Bax)/cysteine-aspartic acid protease 3 (Caspase-3)/cysteine-aspartic acid protease 9 (Caspase-9) pathway and microtubule-associated protein light chain 3 (LC3)/sequestosome 1 (p62)/Beclin1 pathway. EMB exposure significantly increased the mRNA and protein expression of receptor-interacting protein 1 (RIPK1)/receptor-interacting protein 3 (RIPK3)/mixed the lineage kinase domain-like (MLKL) pathway (P < 0.05). Moreover, EMB exposure significantly increased the expression of genes related to immunity (immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin D (IgD), and antimicrobial peptide-related genes expression including β-defensin and hepcidin) (P < 0.05). The addition of RES significantly diminished autophagy, apoptosis, necroptosis, and immunity-related gene expression by inhibiting iNOS activity, NO content, and the protein expression of iNOS and p-NF-κB. In conclusion, RES attenuated autophagy, apoptosis, and necroptosis in EMB-exposed L8824 cells via suppression of the NO system/NF-κB signaling pathway.

The facultative intracellular bacterium Listeria (L.) monocytogenes may cause severe diseases in humans and animals. The control of listeriosis/L. monocytogenes requires the concerted action of cells of the innate and adaptive immune systems. In this regard, cell-intrinsic immunity of infected cells, activated by the immune responses, is crucial for the control and elimination intracellular L. monocytogenes. Both the immune response against L. monocytogenes and cell intrinsic pathogen control are critically regulated by post-translational modifications exerted by the host ubiquitin system and ubiquitin-like modifiers (Ubls). In this review, we discuss our current understanding of the role of the ubiquitin system and Ubls in listeriosis, as well as future directions of research.

Tuberculosis (TB) remains a leading cause of death among infectious diseases, particularly in poor countries. Viral infections, multidrug-resistant and ex-tensively drug-resistant TB strains, as well as the coexistence of chronic illnesses such as diabetes mellitus (DM) greatly aggravate TB morbidity and mortality. DM [particularly type 2 DM (T2DM)] and TB have converged making their control even more challenging. Two contemporary global epidemics, TB-DM behaves like a syndemic, a synergistic confluence of two highly prevalent diseases. T2DM is a risk factor for developing more severe forms of multi-drug resistant-TB and TB recurrence after preventive treatment. Since a bidirectional relationship exists between TB and DM, it is necessary to concurrently treat both, and promote recommendations for the joint management of both diseases. There are also some drug-drug interactions resulting in adverse treatment outcomes in TB-DM patients including treatment failure, and reinfection. In addition, autophagy may play a role in these comorbidities. Therefore, the TB-DM comorbidities present several health challenges, requiring a focus on multidisciplinary collaboration and integrated strategies, to effectively deal with this double burden. To effectively manage the comorbidity, further screening in affected countries, more suitable drugs, and better treatment strategies are required.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

The consumption of dietary fiber is beneficial for gut health, but the role of bound polyphenols in dietary fiber has lacked systematic study. The aim of this study is to evaluate the ameliorative effect of mung bean coat dietary fiber (MDF) on DSS-induced ulcerative colitis in mice in the presence and absence of bound polyphenols. Compared to polyphenol-removed MDF (PR-MDF), MDF and formulated-MDF (F-MDF,backfilling polyphenols by the amount of extracted from MDF into PR-MDF) alleviated symptoms such as weight loss and colonic injury in mice with colitis, effectively reduced excessive inflammatory responses, and the bound polyphenols restored the integrity of the intestinal barrier by promoting the expression of tight junction proteins. Additionally, bound polyphenols restored the expression of autophagy-related proteins (mTOR, beclin-1, Atg5 and Atg7) and inhibited the excessive expression of apoptotic-related proteins (Bax, caspase-9, and caspase-3). Furthermore, bound polyphenols could ameliorate the dysregulation of the intestinal microbiota by increasing the abundance of beneficial bacteria and inhibiting the abundance of harmful bacteria. Thus, it can be concluded that the presence of bound polyphenols in MDF plays a key role in the alleviation of DSS-induced ulcerative colitis.

Aspergillus fumigatus (A. fumigatus) is one of the common pathogens of fungal keratitis. Fungal growth and invasion cause excessive inflammation and corneal damage, leading to severe vision loss. Neutrophils are the primary infiltrating cells critical for fungal clearance. Cathelicidin [LL-37 in humans and cathelicidin-related antimicrobial peptide (CRAMP) in mice], a natural antimicrobial peptide, can directly inhibit the growth of many pathogens and regulate immune responses. However, the role of cathelicidin and its effect on neutrophils in A. fumigatus keratitis remain unclear. By establishing A. fumigatus keratitis mouse models, we found that cathelicidin was increased in A. fumigatus keratitis. It could reduce fungal loads, lower clinical scores, and improve corneal transparency. Restriction of CRAMP on fungal proliferation was largely counteracted in CD18-/- mice, in which neutrophils cannot migrate into infected sites. When WT neutrophils were transferred into CD18-/- mice, corneal fungal loads were distinctly reduced, indicating that neutrophils are vital for CRAMP-mediated resistance. Furthermore, cathelicidin promoted neutrophils to phagocytose and degrade conidia both in vitro and in vivo. CXC chemokine receptor 2 (CXCR2) was reported to be a functional receptor of LL-37 on neutrophils. CXCR2 antagonist SB225002 or phospholipase C (PLC) inhibitor U73122 weakened LL-37-induced phagocytosis. Meanwhile, LL-37 induced PLC γ phosphorylation, which was attenuated by SB225002. SB225002 or the autophagy inhibitors Bafilomycin-A1 and 3-Methyladenine weakened LL-37-induced degradation of conidia. Transmission electron microscopy (TEM) observed that LL-37 increased autophagosomes in Aspergillus-infected neutrophils. Consistently, LL-37 elevated autophagy-associated protein expressions (Beclin-1 and LC3-II), but this effect was weakened by SB225002. Collectively, cathelicidin reduces fungal loads and improves the prognosis of A. fumigatus keratitis. Both in vitro and in vivo, cathelicidin promotes neutrophils to phagocytose and degrade conidia. LL-37/CXCR2 activates PLC γ to amplify neutrophils' phagocytosis and induces autophagy to eliminate intracellular conidia.

Targeted protein degradation (TPD) has been established as a viable alternative to attenuate the function of a specific protein of interest in both biological and clinical contexts. The unique TPD mode-of-action has allowed previously undruggable proteins to become feasible targets, expanding the landscape of "druggable" properties and "privileged" target proteins. As TPD continues to evolve, a range of innovative strategies, which do not depend on recruiting E3 ubiquitin ligases as in proteolysis-targeting chimeras (PROTACs), have emerged. Here, we present an overview of direct lysosome- and proteasome-engaging modalities and discuss their perspectives, advantages, and limitations. We outline the chemical composition, biochemical activity, and pharmaceutical characteristics of each degrader. These alternative TPD approaches not only complement the first generation of PROTACs for intracellular protein degradation but also offer unique strategies for targeting pathologic proteins located on the cell membrane and in the extracellular space.

Intratumoral microbiota has become research hotspots, and emerges as a non-negligent new component of tumor microenvironments (TME), due to its powerful influence on tumor initiation, metastasis, immunosurveillance and prognosis despite in low-biomass. The accumulations of microbes, and their related components and metabolites within tumor tissues, endow TME with additional pluralistic features which are distinct from the conventional one. Therefore, it's definitely necessary to comprehensively delineate the sophisticated landscapes of tumor microbe microenvironment, as well as their functions and related underlying mechanisms. Herein, in this review, we focused on the fields of tumor microbe microenvironment, including the heterogeneity of intratumor microbiota in different types of tumors, the controversial roles of intratumoral microbiota, the basic features of tumor microbe microenvironment (i.e., pathogen-associated molecular patterns (PAMPs), typical microbial metabolites, autophagy, inflammation, multi-faceted immunomodulation and chemoresistance), as well as the multidisciplinary approach-based intervention of tumor microbiome for cancer therapy by applying wild-type or engineered live microbes, microbiota metabolites, antibiotics, synthetic biology and rationally designed biomaterials. We hope our work will provide valuable insight to deeply understand the interplay of cancer-immune-microbial, and facilitate the development of microbes-based tumor-specific treatments.