Streptococcus suis serotype 2 (SS2), a zoonotic pathogen associated with meningitis in humans and pigs, disrupts the blood–brain barrier (BBB) during infection, but underlying mechanisms remain inadequately elucidated. This study demonstrated that SS2 infection induced mitophagy in human brain microvascular endothelial cells (hBMECs), with Eno (Enolase) identified as the key bacterial protein responsible for the activation of PINK1-Parkin pathway. Using Co-immunoprecipitation, mass spectrometry, and confocal microscopy, we identified that Eno interacted with the host molecular chaperone HSP90AA1 via its N-terminal domain, necessitating the integrity of the second α-helix (residues 87–98). The interaction between Eno and HSP90AA1 may promote mitochondrial proteotoxic stress and misfolded protein accumulation, thereby contributing to mitophagy induction, while the loss of mitochondrial membrane potential (MMP) alone is insufficient to initiate this process. Importantly, Eno-induced mitophagy did not result in cell death but instead led to the degradation of the tight junction protein claudin-5 (CLDN5), thus compromising BBB integrity. Both in vitro BBB models and in vivo mouse infection studies substantiated the involvement of Eno-induced mitophagy in BBB disruption. These findings reveal a novel mechanism whereby SS2 Eno exploits host HSP90AA1 to activate mitophagy, triggering the degradation of CLDN5 to breach the BBB. This study provides new insights into SS2 pathogenesis and identifies the Eno-HSP90AA1-mitophagy axis as a potential therapeutic target for preventing bacterial meningitis.
A104R of African swine fever virus (ASFV), putative histone-like protein, is known to be involved in essential processes such as viral DNA replication, repair, and transcription. Although it has been revealed that the deletion of A104R affects ASFV pathogenicity for live attenuated vaccine development, the molecular mechanism underlying those effects remains unclear. Here, we report a novel molecular mechanism of A104R that suppresses karyopherin-α (KPNA) mediated antiviral responses. A104R competitively bound to the major nuclear localization signal (NLS) region on KPNA3 and KPNA4, thereby disrupting their interaction with interferon regulatory factor 3 (IRF3). A104R also bound to the unique C terminal nonclassical NLS region of KPNA6 and competitively inhibited the interaction between KPNA6 and phosphorylated signal transducer and activator of transcription 1 (STAT1). Therefore, A104R inhibited the nuclear translocation of IRF3 and phosphorylated STAT1, thereby suppressing type I interferon (IFN) signalling and IFN-mediated antiviral responses. Consequently, ectopic expression of A104R enhanced DNA virus replication and suppressed IFN production by inhibiting the IRF3-mediated type I IFN signalling pathway and suppressed the interferon-β (IFN-β)-mediated antiviral responses. Taken together, these results highlight that A104R plays a dual role in suppressing IFN response and IFN-mediated antiviral responses. This mechanism provides new insight into the A104R induced ASFV pathogenesis, which provide a critical implication for a better understanding of the ASFV pathogenesis.
Tembusu virus (TMUV) is a neurotropic avian flavivirus that causes viral encephalitis and egg-drop syndrome in ducks, resulting in significant economic losses in duck-producing regions in Asia. Lipid droplets (LDs) are organelles derived from the endoplasmic reticulum that primarily store neutral lipids and are involved in viral replication and pathogenesis. However, little is known about the roles of LDs in TMUV replication and neuropathogenesis. Here, we report that TMUV infection increased triglyceride (TG) levels and LD accumulation in mouse neuroblastoma cells (Neuro-2a). Inhibiting TG synthesis and LD accumulation with specific drugs reduced TMUV replication, whereas oleic acid (OA) supplementation increased LD accumulation and enhanced viral replication, indicating that LDs promote TMUV replication. Furthermore, in TMUV-infected ICR mice or goslings, we assessed LD accumulation in brain tissues and its impact on viral replication and pathology. TMUV infection increased TG levels and LD accumulation in the brain. Inhibiting LD accumulation reduced viral replication and alleviated lesions, whereas enhancing LD accumulation had the opposite effects, indicating that LDs promote TMUV neuropathogenesis. We further explored the mechanism underlying TMUV-induced LD accumulation and revealed that TMUV downregulated the lipolytic enzyme adipocyte triglyceride lipase (ATGL). ATGL overexpression reduced TG production and LD accumulation, thereby inhibiting TMUV replication, whereas ATGL knockdown had the opposite effects. Moreover, TMUV activated AMP-activated protein kinase (AMPK) signalling to suppress ATGL expression, leading to increased TG levels and LD accumulation, thereby increasing viral replication. Collectively, these findings indicate that TMUV infection induced LD accumulation via AMPK-mediated ATGL suppression to promote viral replication, and that LD accumulation contributed to TMUV neuropathogenesis.
Bovine mastitis remains a major driver of antimicrobial use in dairy production, highlighting the need for physiologically relevant in vitro models for preclinical safety assessment of novel intramammary therapeutics. Here, a previously established bovine epithelial cell line co-culture mammosphere model was adapted to a single-spheroid 96-well ultra-low attachment format, improving reproducibility and longitudinal monitoring. Mammospheres maintained epithelial organization and were further characterized for secretory function through assessment of casein expression. The suitability of the model for safety assessment was subsequently evaluated using complementary fluorescence microscopy, kinetic live-cell imaging, and flow cytometry following Triton X-100-induced cytotoxicity. Using this workflow, the safety of bacteriophage-derived recombinant lysin LysMK34 and antibiotic kanamycin, alone and in combination, was evaluated, with no cytotoxic effects observed, supporting the utility of the model for future preclinical intramammary safety assessment.
H7 subtype avian influenza virus is an important zoonotic pathogen threatening the poultry industry and public health. Recombinant subunit vaccines provide a safer alternative to conventional inactivated vaccines, while yeast expression systems offer advantages of low cost, scalability, and engineering flexibility. However, their application in avian influenza vaccine development remains underexplored. In this study, we use the methylotrophic yeast Komagataella phaffii as a low-cost, industrial-scale platform to produce hemagglutinin-1 (HA1) subunits with programmable N-/O-linked glycosylation. The methanol-utilization-slow (MutS) phenotype strain secreted 1.21 g/L HA1, and compared with the standard methanol-utilization-plus (Mut+) phenotype strain, the N-linked and O-linked glycans were significantly reduced [liquid chromatography–mass spectrometry (LC–MS)/MS glycopeptide profile]. Despite hypoglycosylation, the antigen retained native conformation, binding H7-specific mAb Q74 with a KD of 1.34 × 10–7 M. SpyTag/SpyCatcher-directed conjugation to ferritin drove quantitative self-assembly into 30 nm virus-like nanoparticles. Formulated with CpG-ODN 1826, the HA1-MutS-ferritin candidate elicited Hemagglutination inhibition (HI) titers of 6.7 log2 in specific pathogen-free (SPF) leghorns at 28 days post-immunization (dpi), significantly enhancing HA1-specific IgY responses (p < 0.0001) while sustaining high-level circulating CD4+ T cells. At 28 dpi, chickens were challenged with 106 embryo infectious dose (EID)50 of H7N9 avian influenza virus; the formulation conferred 100
