The mosquito gut microbiota plays a pivotal role in regulating arbovirus transmission, yet the specific antiviral metabolites produced by native symbiotic bacteria and their underlying mechanisms remain poorly understood. In this study, we isolated a natural gut symbiotic bacterium, Serratia marcescens strain WZ1, from field-caught Aedes albopictus in Wenzhou, China, and demonstrated its potent ability to inhibit dengue virus (DENV) infection. Through integrated metabolomic analysis, we identified the red pigment prodigiosin (PG) as a functional antiviral metabolite secreted by this strain. PG treatment suppressed DENV infection in mosquito cells in a dose- and time-dependent manner and significantly reduced DENV2 RNA levels in adult Ae. albopictus midguts. Mechanistic investigations revealed that PG preferentially localizes to the endoplasmic reticulum (ER), where it induces ER stress and upregulates the chaperone protein GRP78. This process subsequently activates a complete autophagic flux, as evidenced by enhanced conversion of Atg8-I to Atg8-II, increased autophagosome formation, and elevated lysosomal activity. Crucially, we further demonstrated that PG facilitates the convergence of autophagosomes and lysosomes, culminating in the colocalization of DENV with lysosomal compartments and subsequent viral clearance. Both genetic knockdown of the autophagy gene Atg8 and pharmacological inhibition of ER stress substantially attenuated PG-mediated viral suppression, confirming the functional link between PG-induced ER stress, autophagy activation, and viral clearance. Our findings elucidate a novel mechanism by which a native mosquito gut symbiont metabolite restricts arboviral infection through activation of the host ER stress-autophagy pathway, providing a mechanistic basis and candidate leads for future transmission-blocking studies.
The mycobacterial ESX-1 (Type VII) secretion system is essential for virulence and induces the NLRP3 inflammasome activation. However, the specific bacterial effectors involved in this process, beyond the well-characterized EsxA (ESAT-6), remain largely unidentified. Through systematic screening of ESX-1 effectors, we identified EspF as a potent and evolutionarily conserved activator of the NLRP3 inflammasome across pathogenic mycobacterial species, including Mycobacterium tuberculosis, M. bovis, and M. marinum. Our results demonstrate that EspF significantly augments the mature IL-1β and IL-18 release, ASC speck formation, caspase-1 activation, and gasdermin D (GSDMD)-mediated pyroptosis in THP-1 cells. Notably, infection with M. smegmatis or M. bovis BCG strain overexpressing EspF significantly enhanced NLRP3 inflammasome activation and pyroptosis, which were completely abolished in NLRP3-deficient cells. Mechanistically, EspF directly interacts with the NACHT and LRR domains of NLRP3. Furthermore, unbiased proteomic screening identified the host E3 ubiquitin ligase TRIM25 as an indispensable binding partner. In THP-1 cells, we confirmed the endogenous interaction and colocalization of the EspF-TRIM25-NLRP3 complex, demonstrating that EspF functions as a molecular scaffold that bridges TRIM25 to NLRP3. This proximity interaction, further validated in situ during mycobacterial infection, facilitates TRIM25-mediated K63-linked ubiquitination of NLRP3. In vivo, mice infected with M. bovis BCG strains overexpressing EspF exhibited exacerbated lung lesions, increased inflammatory cell infiltration, and higher bacterial burdens. Collectively, these findings reveal EspF-TRIM25-NLRP3 axis is a novel mechanism of mycobacterial pathogenesis that drives hyperinflammation to facilitate bacterial survival and dissemination.
The widespread dissemination of New Delhi metallo-β-lactamase-1 (NDM-1) has severely compromised the clinical efficacy of carbapenem antibiotics, highlighting the need for strategies to restore meropenem activity. SKQ1 was identified through surface plasmon resonance (SPR)-based screening. Broth microdilution checkerboard assays demonstrated strong synergy between SKQ1 and meropenem (FICI = 0.25-0.5). Enzyme kinetic analyses revealed that SKQ1 acts as a noncompetitive inhibitor of NDM-1 (IC50 = 34.99 ± 3.13 μg/mL). Molecular docking, molecular dynamics simulations, microscale thermophoresis (MST) and thermal stability assays collectively supported the direct binding of SKQ1 to NDM-1, resulting in the inhibition of its hydrolytic activity. Further analyses showed that SKQ1 affected bacterial envelope physiology, including membrane integrity, membrane potential, ATP distribution and oxidative stress, suggesting additional envelope-associated effects beyond NDM-1 inhibition. Both in vitro and in vivo models confirmed the efficacy of the SKQ1-meropenem combination. SKQ1 enhanced the antibiofilm activity and therapeutic efficacy of meropenem while attenuating lipopolysaccharide (LPS)-induced inflammatory responses. Together, these findings support SKQ1 as a repurposed meropenem potentiator that acts through NDM-1 inhibition and envelope-associated effects, offering a potential strategy for combating NDM-mediated carbapenem resistance.
Bacteria pose a serious threat to hosts through adaptive mutations that confer stress resistance and promote persistent colonization. Here, we describe an adaptive evolution event involving eight highly similar ST11-KL64 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains, isolated from a non-infected inpatient who acquired two distinct CRKP strains, CRKP-F1 and CRKP-S2 during the first hospitalization, recovered, and was discharged after receiving antimicrobial therapy but subsequently experienced two additional recurrent febrile episodes and re-admission. The strain CRKP-S2 showed significantly enhanced resistance to oxidative stress, survival within macrophages, and internalization ability, and carried an additional ~72 kb fragment containing oxidative stress response factors (including NAD(P)-dependent oxidoreductases), and a ~ 19kb plasmid fragment harboring catA2, sul2, umuC/D genes, compared to the initial strain CRKP-F1. All four strains, CRKP-B3, CRKP-U4, CRKP-F5 and CRKP-S6, from the second hospitalization exhibited higher genetic similarity to CRKP-S2 than each other, and each of these strains has its own unique mutations compared to CRKP-S2. The third-hospitalization strain CRKP-U7 displayed the highest average nucleotide identity (ANI) with CRKP-S2, and possessed unique mutations in cecR, rlmA1, and selB, distinct from second-hospitalization strains. However, the last strain, CRKP-B8, carries a new gene mutation based on CRKP-U7 and exhibits greater host adaptability than all other isolates. While these findings are suggestive, whether the ~72-kb and ~19-kb fragments and mutations in CRKP-S2 drove enhanced colonization, and whether subsequent mutations contributed to subclones linked to recurrent febrile, or merely coincided, remains unclear. The possibility of mixed colonization by co-circulating subclones cannot be excluded, and functional validation is needed.
The rapid expansion of cervid farming raises concerns about antimicrobial resistance (AMR) dissemination, yet its impact on the Cervidae gut microbiome remains poorly characterized. We integrated 89 newly sequenced fecal metagenomes with 599 publicly available datasets, comprising 285 metagenomes from farmed cervids and 370 from wild cervids, to construct a catalog of 15,494 non-redundant metagenome-assembled genomes (MAGs) representing 2,401 species. Our analysis demonstrates that farming profoundly reshapes the gut microbiome's functional composition. Specifically, farmed cervids exhibited significantly higher relative abundance, diversity, and heterogeneity of antimicrobial resistance genes (ARGs) compared to wild counterparts. We observed a robust synergistic relationship between ARGs, virulence factor genes, and mobile genetic element (MGE)-associated genes, identifying 70 ARG-MGE combinations as evidence of potential horizontal gene transfer. Plasmid profiling further suggested that a subset of ARGs may be associated with conjugative plasmids, with plasmid-associated ARGs being significantly more abundant in farmed than in wild cervids. Virome analyses indicated that bacteriophages, particularly Siphoviridae, may serve as mobile reservoirs for ARGs. Notably, Cervidae shared 268 ARG types with humans, including 23 high-risk genes associated with resistance to clinically important antibiotics (e.g. tetX1, vanRD, and bla-CTX-M-178), with Escherichia coli as a key cross-host carrier. These findings highlight that human-impacted cervid gut microbiomes are significant environmental reservoirs of clinically relevant AMR, underscoring the necessity for enhanced antibiotic stewardship and resistance surveillance in managed wildlife within a One Health framework.
Feline calicivirus (FCV) and feline herpesvirus 1 (FHV-1) are major pathogens threatening feline health, and prophylactic immunity remains the primary preventive approach. To develop a safe and effective multivalent vaccine against the two viruses, two recombinant rabies virus (RABV) vaccines expressing the FCV VP1 and FHV-1 gB/gD genes were constructed. The exogenous genes showed stable inheritance following serial viral passages, and the recombinant RABVs exhibited growth kinetics comparable to the parental strain, with a delayed peak titer. After verifying the viral pathogenicity, the recombinant RABV vaccines elicited specific IgG antibodies against VP1, gB, and gD, as well as neutralizing antibodies against FCV, FHV-1, and RABV in mice. Immunized mice were also protected against lethal RABV challenge. In cats, the vaccines induced neutralizing antibodies against the three pathogens, and immunized cats showed milder clinical signs and less severe histopathological lesions following FHV-1 and FCV challenge. Collectively, these findings indicate that the two recombinant vaccines induce robust humoral immune responses against FCV, FHV-1, and RABV in both mice and cats and confer protection against viral challenges.
Allergic diseases are a class of important immune imbalance diseases that lack effective cures. Helminth-derived serine protease inhibitors (serpins) exert immunoregulatory effects similar to those of helminth, are controllable, have few side effects, and have great application potential in correcting unbalanced immune responses. However, the immune regulatory effects and mechanisms mediated by the enzyme inhibitory activity of helminth-derived serpins have not been investigated or clarified in previous studies. We obtained mutant serpin proteins with significantly reduced enzyme inhibitory activity by predicting and mutating the single key amino acid. The therapeutic effect of immunoregulation mediated by enzyme inhibitory activity of serpin on allergic inflammation was investigated based on an acute allergy model constructed in OVA-specific T-cell receptor transgenic C57BL/6 mice. Unlike the Trichinella spiralis-derived serpin (Ts-serpin), the mutant proteins showed significantly reduced inhibitory activity against the chymotrypsin and elastase. Meanwhile, the immunosuppressive ability of the mutant proteins was weakened and mutant protein intervention groups showed more severe allergic inflammation in the lungs. Additionally, enzyme inhibitory activity regulates the differentiation of macrophages and Treg cells to establish immune tolerance, which is the key to rapidly improving lung injury at the challenge and treatment stages of allergy. These findings suggest that the rapid regulatory properties of enzymatic reactions have great potential in the prevention and treatment of allergic inflammation.
Aspergillus fumigatus is a major opportunistic fungal pathogen, and increasing azole resistance poses a challenge for aspergillosis treatment. Squalene is an upstream precursor of ergosterol biosynthesis and may also be utilized by SHC-like triterpene cyclases, suggesting a potential link between squalene-associated metabolism, membrane adaptation, and azole response. However, the roles of SHC-like triterpene cyclase genes in A. fumigatus remain unclear. Here, we characterized three candidates, shc1, shc2, and shc3, using comparative bioinformatic analysis, gene deletion, phenotypic assays, azole susceptibility testing, transcriptomics, and host-interaction models. Sequence, genomic-context, phylogenetic, and structural analyses suggested divergence among the three candidates. Individual shc deletion caused limited effects on vegetative growth, whereas loss of shc1 mildly reduced susceptibility to voriconazole and posaconazole, as reflected by twofold MIC increases and lower inhibition rates. Transcriptomic analysis revealed distinct remodeling patterns, with Δshc3 showing the broadest transcriptional changes despite no detectable MIC shift. Targeted metabolite profiling and PI uptake analysis further supported an association between shc deletion, sterol/hopane-type triterpenoid balance, and membrane-associated properties. shc deletion also altered epithelial cell interaction phenotypes, while Δshc1 showed reduced lethality in Galleria mellonella. In clinical isolates, elevated shc transcription was associated with azole-resistant backgrounds. These findings suggest functional diversification among SHC-like triterpene cyclase genes and indicate that shc1 may contribute to azole-associated adaptation and virulence-related traits in A. fumigatus.
The link between diet and overall human health is well established, with certain diets known to promote better health and be associated with a reduced risk of developing chronic diseases. Previous research has also demonstrated the direct effects of diet on microbiome composition, diversity, and fitness, which can, in turn, alter colonization resistance and the immune response against enteric pathogens. However, much less is known regarding the direct effect of diet on the virulence of pathogenic bacteria. To examine the effect of diet on bacterial virulence while maintaining constant macronutrient composition, we used peptones from different sources (plant- and animal-based) to simulate various dietary protein sources. The peptones were examined for their effect on the virulence of enterohemorrhagic Escherichia coli (EHEC). We found that bacteria grown with peptone derived from casein - the main protein component of milk - exhibit a significant reduction in their type III secretion system (T3SS) activity. This effect manifested as a significant reduction in bacterial adherence to host cells and limited translocation activity of the T3SS effector Tir. We additionally found evidence suggesting that milk-derived peptone causes a shift in bacterial behavior from hyper-virulent, adherent bacteria to a more motile state. Finally, we observed that the inhibitory effects of milk-derived peptone extend beyond EHEC to other T3SS-possessing pathogens. While the specific inhibitory components in casein remain to be identified, our findings provide a foundation for developing casein-based, non-antibiotic therapies against bacterial diarrheagenic pathogens.
Chronic hepatitis B remains difficult to cure because the viral covalently closed circular DNA (cccDNA) minichromosome can persist and sustain viral transcription, creating a need for scalable, reporter readouts that facilitate early discovery of cccDNA-modulating agents. Here, we developed two complementary hepatocyte HiBiT reporter models: a replication-competent HBV reporter in HepaRG cells (HepaRG-Hibit16), in which a secreted split-NanoLuc HiBiT signal is linked to cccDNA-associated expression, and a Cre/Lox-based recombinant cccDNA (rcccDNA) reporter in HepG2 cells (HepG2-Rccc1a) that rapidly generates rcccDNA with a matched HiBiT readout. Screening of 1,403 FDA-approved compounds across both models identified 13 concordant, non-cytotoxic hits. Palovarotene, a retinoic acid receptor-γ agonist, was selected as an exemplar concordant hit and reduced HBV antigens, HBV DNA, and cccDNA and inhibited HBV infection in multiple hepatocyte-based in vitro systems without overt cytotoxicity at the tested concentrations. Together, this dual-reporter strategy supports efficient cross-model triage of candidate cccDNA modulators for subsequent orthogonal validation.
Getah virus (GETV) is an emerging arthropod-borne zoonotic alphavirus that poses a growing threat to animal and public health, yet its virulence determinants remain poorly understood. Here, we report the first identification of a natural GETV variant isolated from the brain of diseased piglets. This variant harbors a 9-nucleotide deletion in the E1 glycoprotein stem region, resulting in the deletion of glutamine 397 (Q397) and valine 398 (V398), along with an asparagine‑to‑isoleucine substitution at position 396 (N396I) and the loss of phenylalanine 399 (F399). It also carries a threonine-to-methionine substitution at position 49 (T49M) in nonstructural protein 2 (NSP2). The NQVF motif (residues 396-399) in E1 protein is highly conserved among GETV strains and related alphaviruses, suggesting a shared functional role. Using reverse genetics, we demonstrated that the E1 deletion-but not the NSP2 mutation-drastically attenuates viral replication in vitro and completely abrogates lethality in neonatal mice by restricting systemic dissemination. Stepwise mutagenesis further revealed that the four‑residue motif functions synergistically as a virulence switch: single deletions partially reduce pathogenicity, while the quadruple deletion confers full attenuation, limiting tissue tropism and histopathology. Although both wild‑type and mutant viruses crossed the placental barrier in pregnant mice, the mutant exhibited reduced vertical transmission and maternal tissue replication. Our findings identify a highly conserved E1 stem motif as a critical regulator of GETV virulence, offering a strategic target for developing attenuated vaccines against GETV and other alphaviruses.
Gene therapy has emerged as a promising strategy for cancer treatment, yet challenges in efficient gene delivery remain a major barrier. Herpes simplex virus type 1 (HSV-1), as an oncolytic virus, has garnered attention for its potential in cancer therapy due to its replicative capacity, large genomic payload, and relatively low toxicity. Notably, syncytium-forming HSV-1 (SF-HSV-1) not only exhibits enhanced and sustained antitumor efficacy but also triggers profound immune responses. However, the exact molecular mechanisms orchestrating HSV-1-induced syncytium formation, its resulting cytotoxicity, and its precise role in immune modulation remain incompletely understood. This review aims to provide an in-depth exploration of the mechanisms underlying HSV-1 syncytium formation and its therapeutic implications in cancer gene therapy.
Human cytomegalovirus (HCMV) profoundly reprograms host transcription and RNA metabolism, yet its impact on transcription start site (TSS) regulation of host genes remains poorly understood. Here, we employed NanoCap Analysis of Gene Expression sequencing (NanoCAGE-seq) to investigate HCMV-driven changes in alternative TSS usage across the host transcriptome. We identified widespread TSS switching, with ribosomal protein genes (RPGs) emerging as a highly enriched category. Alternative TSS usage produced isoforms with distinct 5’untranslated regions (UTRs), thereby altering cis-regulatory elements that shape translational efficiency. Integrative transcriptomic and proteomic analyses revealed a paradoxical accumulation of RPG proteins despite transcriptional downregulation during infection. Using 5’ Rapid Amplification of cDNA Ends (5’RACE), we characterized four RPGs of RPL4, RPS11, RPS23, and RPS24 that generated 5’UTR variants through alternative TSS usage. Notably, isoforms containing a 5’terminal oligopyrimidine (5’TOP) motif were significantly enriched, correlating with mTOR activation induced by HCMV. Functional assays with bicistronic reporter constructs in HEK293 cells and infection models in human embryonic lung fibroblasts demonstrated that the RPL4 5’TOP isoform exhibited enhanced mTORC1-driven translation compared with non-5’TOP counterparts. Importantly, RPL4 upregulation facilitated viral protein synthesis and boosted production of infectious virions. Together, our findings reveal that dynamic TSS switching of RPGs provides a simple, yet effective, mechanism for fine-tuning mTORC1-responsive translation. By co-opting host transcriptional and translational programs, HCMV enhances ribosome function to optimize the cellular environment for productive viral replication.
Chinese cordyceps consists of fruiting body and sclerotia (larvae part) formed through the parasitism of the insect Hepialidae by the fungus Ophiocordyceps sinensis, but artificial cultivation is hindered by fruiting body abortion, severely impacting yield and quality. The interactions between the fungus, its host insects, and soil are critical for its development. Multi-omics analyses were conducted to compare normally and abnormally developing samples, examining microbial communities and metabolites in fungus-colonized host larvae and the mycosphere soil. Abnormal fruiting body development was associated with profound shifts in microbial ecology: fungal diversity increased significantly in both endophytic larvae and mycosphere soil, whereas bacterial diversity decreased within host larvae. The microbial composition in and around abnormal samples was markedly altered, characterized by a high enrichment of Penicillium fungi and a depletion of Bacillus bacteria. Cross-kingdom microbial network analysis showed fewer connections and lower stability in abnormally developing samples. Untargeted metabolomic profiling revealed significant accumulation of the antibiotics N1-hydroxy-roquefortine C and glandicoline A, both roquefortine C derivatives characteristic of Penicillium metabolism, along with enriched pathways involved in antibiotic biosynthesis. Exploratory Partial Least Squares Structural Equation Modeling (PLS-SEM) analysis supported an associative pathway in which Penicillium-mediated toxicity is correlated with microbial dysbiosis, which in turn is associated with fruiting body abortion. Conversely, Bacillus may play a critical role in suppressing Penicillium overgrowth and maintaining microbial homeostasis, representing a promising target for biocontrol strategies. This study is the first to reveal potential links between Penicillium‑mediated toxicity, microbial imbalance, and developmental disorders of Chinese cordyceps.
The synergistic interaction between Staphylococcus aureus and Candida albicans exacerbates polymicrobial infection severity and mortality compared to monomicrobial infections. C. albicans metabolism is known to enhance S. aureus virulence, whereas the role of S. aureus metabolic reprogramming in this cross-species synergy remains poorly defined. In this study, we demonstrate that C. albicans remodels S. aureus glycolytic pathways, thereby increasing its virulence in glucose-rich environments. C. albicans triggers metabolic reprogramming in S. aureus, characterized by a reduction of lactate and accumulation of upstream glycolytic intermediates, including 3-phosphoglycerate, 2-phospho-D-glycerate, and phosphoenolpyruvate. The metabolic reprogramming phenotype is glycolysis-dependent, which is evidenced by 2-deoxy-D-glucose-mediated inhibition of virulence enhancement. Addition of exogenous lactate lowers extracellular pH and reduces the hemolytic activity of S. aureus-C. albicans co-cultures. Inhibition of lactate dehydrogenase by oxamate or extracellular alkalinization with sodium hydroxide significantly increases hemolysis in S. aureus mono-cultures. S. aureus-C. albicans co-infections promote inflammatory cell infiltration, but do not affect the quantity of S. aureus, indicating that increased toxin levels, rather than bacterial quantity, drive the enhanced virulence. Collectively, these findings indicate that C. albicans enhances S. aureus virulence through metabolic reprogramming. Targeting lactate metabolism or glycolytic intermediates may disrupt this cooperative interaction, offering a novel therapeutic strategy against these polymicrobial infections.
Scedosporium is the second most prevalent fungal colonizer of the airways of cystic fibrosis (CF) patients after Aspergillus fumigatus. Chronic bacterial and fungal colonization drives persistent inflammation via damage-associated molecular patterns, contributing to lung injury. Owing to their distinct structural and biological properties, S. apiospermum and A. fumigatus may trigger different immune responses and exhibit different susceptibilities to macrophage mediated killing. We compared the transcriptomic and inflammatory profiles of human macrophages challenged with S. apiospermum (Sap) or A. fumigatus (Afu) for 4 h and 12 h, and assessed fungal survival. Sap activated multiple pro-inflammatory pathways, overlapping with those induced by Afu. At 4 h, 15 inflammation-related pathways were differentially regulated in Sap-infected macrophages (FDR 4 × 10-2 to 7 × 10-13), 12 of which were also upregulated in Afu-infected cells. The most highly dysregulated pathways involved TNF, IL-17, NF-κB signaling, and cytokine-cytokine receptor interactions. Sap induced a stronger, earlier inflammatory response, with three times more genes upregulated at 4 h than for Afu (671 vs. 200), and significantly higher levels of IL-6, IL-1β, TNF-α, IL-23, IL-10 (p <0.001), and IFN-α2 (p <0.05) secretion. The proportions of macrophages infected at 4 h were similar for the two fungi, but Sap conidia were more efficiently killed after 6 h (46.6% vs. 34.5%, p <0.05). Thus, S. apiospermum elicits a faster and stronger inflammatory macrophage response than A. fumigatus, potentially enhancing fungal clearance but also exacerbating airway inflammation in CF.
Porcine epidemic diarrhea virus (PEDV) causes substantial economic losses in the swine industry globally. Host factors regulating the intracellular replication of PEDV, particularly early RNA synthesis and structural protein production, are not well understood, limiting antiviral strategies. We report that glycoprotein non‑metastatic melanoma protein B (GPNMB) is a key host factor promoting PEDV infection. Initially identified as a PEDV S1-binding partner, GPNMB was confirmed to enhance infection via loss-and gain-of-function experiments in Vero and IPEC-J2 cells. Genetic knockout of GPNMB inhibited PEDV replication without affecting viral attachment or internalization. We show that GPNMB is necessary for the accumulation of double-membrane vesicles (DMVs) and promotes early viral RNA synthesis. Notably, GPNMB directly interacts with the PEDV spike (S) and nucleocapsid (N) proteins, increases their abundance, and facilitates their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, implicating it in structural protein maturation. Our work reveals a pivotal role for GPNMB in PEDV replication and nominates it as a target for host-directed antiviral intervention.
Fusobacterium necrophorum is a major opportunistic pathogen of ruminants that causes foot rot and liver abscesses and contributes to substantial economic losses. Current vaccines provide limited protection. Here, we prepared and evaluated outer membrane vesicles (OMVs)-based vaccine candidate derived from F. necrophorum. OMVs were purified by density-gradient centrifugation and were characterized for morphology, composition, and immunological activity. The purified OMVs showed intact vesicle structure and a uniform particle size distribution and contained virulence-associated antigens and pathogen-associated molecular patterns. OMVs were rapidly taken up by dendritic cells and promoted dendritic cell maturation, as indicated by activation of PI3K/AKT signaling and increased expression of MHC I, MHC II, CD80, and CD86. In vivo imaging showed persistent fluorescence at the injection site and signal accumulation in draining lymph nodes and the spleen. In mice, OMVs immunization induced high titers of antigen-specific IgG and elicited a Th1-biased cellular immune response. In a lethal challenge, OMVs immunization achieved 100% survival, significantly reduced liver bacterial loads, and alleviated inflammatory and necrotic liver lesions. In sheep, OMVs immunization also induced progressively increased antigen-specific IgG and a predominantly Th1 type cellular immune response, supporting strong immunogenicity in a natural host. Together, these results support F. necrophorum OMVs as a promising vaccine candidate containing multiple native antigens for the prevention and control of F. necrophorum - associated diseases in ruminants.
Exopolysaccharides (EPS) are carbohydrate polymers produced by various bacteria, including species of the genus Lactobacillus. Until now, the link between the functional properties of these bacteria and their EPS remains poorly understood. Lactobacilli are used in a wide range of applications due to their protective effects against inflammation and their role in maintaining intestinal homeostasis. However, the functional properties of their EPS remain weakly characterized. This study investigated the EPS produced by Lactobacillus gasseri (Lg-EPS), a human-derived probiotic, focusing on their immunomodulatory activity and inhibitory effects on the growth and virulence of Candida albicans, an opportunistic fungal pathogen associated with dysbiosis and immune dysfunction. The results show that Lg-EPS are composed of 59.1% glucose, 26.0% galactose, 14.6% rhamnose, and 0.3% N-acetyl-glucosamine. Functionally, Lg-EPS reduced the expression of pro-inflammatory cytokines (IL-6, IL-8, CCL-2) and IL-6 protein levels, while increasing IL-10 expression and secretion. These effects were associated with downregulation of TLRs, MyD88 and NF-κB, alongside upregulation of the aryl hydrocarbon receptor (AhR), an intracellular transcription factor involved in immune modulation. Additionally, Lg-EPS increase induction of AhR activity in HT29-Lucia™ AhR cells. Furthermore, Lg-EPS inhibited C. albicans biofilm formation and fungal growth. These findings suggest that Lg-EPS contribute to immune regulation and antifungal defense via AhR activation. This receptor, known for sensing microbial metabolites, mediates anti-inflammatory effects by suppressing NF-κB signaling, enhancing IL-10 production, and reinforcing epithelial barrier function. This is the first report to demonstrate the AhR-dependent anti-inflammatory effect of bacterial EPS.
The type IV secretion system (T4SS) acts as the central virulence determinant of Brucella, facilitating intracellular survival via the secretion of effector proteins. In this study, we combined bioinformatic prediction with translocation assays to identify seven novel VirB-dependent effectors. Functional characterization revealed distinct roles for these proteins in both bacterial physiology and host-pathogen interactions. We identified BT4E19 and BT4E43 as critical determinants of cell envelope integrity: BT4E19 is required for core oligosaccharide maintenance and nitrosative stress tolerance, whereas BT4E43 is essential for O-antigen biosynthesis and oxidative stress resistance. Furthermore, BT4E43 is required for the efficient avoidance of lysosomal trafficking during intracellular infection. Notably, BT4E4 displays dual functions under tested conditions, being essential for oxidative stress resistance while simultaneously functioning to inhibit Caspase-5-mediated pyroptosis. In vivo assays further demonstrated that both BT4E19 and BT4E43 are indispensable for establishing chronic infection in mice. Collectively, these findings expand the Brucella effector repertoire and uncover the dual functions of specific effectors in maintaining bacterial structural integrity and orchestrating immune evasion, highlighting them as potential targets for anti-virulence therapies.
