Streptococcus suis serotype 2 (SS2) is a prominent pathogen that impacts swine and presents a zoonotic threat to humans; it is a cause of bacterial meningitis, a severe condition linked to neurological impairment and elevated mortality rates. For SS2 to access the central nervous system, it must traverse the blood–brain barrier (BBB); however, the precise mechanisms underlying this process remain incompletely elucidated. In this study, we demonstrate that the RTX family exoprotein A (RfeA), which is secreted by SS2, can be internalized by human brain microvascular endothelial cells (hBMECs) via a caveolae/lipid raft-dependent pathway. RfeA subsequently induces pyroptosis through the NLRP3/Caspase-1 pathway, a process attributed to the increase in mitochondrial reactive oxygen species (mtROS). The interaction between the N-terminus of RfeA and voltage-dependent anion channel 1 (VDAC1) leads to mtROS production, which can be suppressed by a VDAC1 oligomerization inhibitor. RfeA-induced pyroptosis results in disruption of the BBB in both the hBMEC monolayer model and the mouse infection model, thereby promoting bacterial infection of the brain. These findings elucidate a novel mechanism by which SS2 induces pyroptosis to breach the BBB, suggesting a potential target for the prevention and treatment of SS2 infection.

Staphylococcus aureus is a leading cause for morbidity and mortality associated with skin and burn wound infections. Therapeutic options for methicillin-resistant S. aureus (MRSA) have dwindled and therefore alternative treatments are urgently needed. In this study, the immuno-stimulating and anti-MRSA effects of cyclic di-guanosine monophosphate (c-di-GMP), a uniquely bacterial second messenger and immuno-modulator, were investigated in HaCaT human epidermal keratinocytes and a murine skin wound infection model. Stimulation of HaCaT cells with 125 μM c-di-GMP for 12 h prior to MRSA challenge resulted in a 20-fold reduction in bacterial colonization compared with untreated control cells, which was not the result of a direct c-di-GMP toxic effect, since bacterial viability was not affected by this dose in the absence of HaCaT cells. C-di-GMP-stimulated or MRSA-challenged HaCaT cells displayed enhanced secretion of the antimicrobial peptides human β-defensin 1 (hBD-1), hBD-2, hBD-3 and LL-37, but for hBD1 and LL-37 the responses were additive in a c-di-GMP-dose-dependent manner. Secretion of the chemokines CXCL1 and CXCL8 was also elevated after stimulation of HaCaT cells with lower c-di-GMP doses and peaked at a dose of 5 μM. Finally, pre-treatment of mice with a 200 nmol dose of c-di-GMP 24 h before a challenge with MRSA in skin wound infection model resulted in a major reduction (up to 1,100-fold by day 2) in bacterial CFU counts recovered from challenged skin tissue sections compared PBS-treated control animals. Tissue sections displayed inflammatory cell infiltration and enhanced neutrophil influx in the c-di-GMP pre-treated animals, which might account for the reduced ability of MRSA to colonize c-di-GMP pre-treated mice. These results demonstrate that c-di-GMP is a potent immuno-modulator that can stimulate anti-MRSA immune responses in vivo and might therefore be a suitable alternative prophylactic or therapeutic agent for MRSA skin or burn wound infections.

Distinct toxinotypes of Clostridium perfringens cause different diseases in animals and humans. Rapid and accurate typing methods remain essential for early diagnosis, effective intervention, and reduced mortality. In this study, we designed specific primer pairs and crRNA sequences targeting the α, β, ε, and ι toxin genes of C. perfringens and constructed a rapid, sensitive, accurate and instrument-free method for typing of C. perfringens . This typing method of C. perfringens based on the multiplex recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a system, termed Cp-MRC12a, can be completed in 1 h. The Cp-MRC12a assay shows high sensitivity with a detection limit of 10 copies/μL for the type A strain and 100 copies/μL for the type B-E strains and high specificity without cross-reactivity to non-target bacteria, and demonstrates a reliable performance in detecting clinical and spiked samples. Collectively, Cp-MRC12a provides a robust and practical approach for the typing of C. perfringens strains, offering substantial advances for early disease diagnosis and pathogen identification.

Vibrio vulnificus ( V. vulnificus ) is a highly virulent zoonotic pathogen causing severe septicemia and tissue necrosis in humans, characterized by a cytokine storm. While bacterial outer membrane vesicles (OMVs) are known to mediate host-pathogen interactions, their specific contribution to V. vulnificus -induced immunopathology remains obscure. In this study, we isolated and characterized V. vulnificus -derived OMVs and investigated their impact on murine macrophages (J774A.1). We demonstrate that these OMVs are actively internalized by macrophages in a time-dependent manner, triggering a robust pro-inflammatory response. Transcriptomic analysis and subsequent validation revealed that internalized OMVs activated the TNF signaling pathway, leading to the phosphorylation of NF-κB and MAPK (p38/JNK) cascades. Crucially, OMV exposure induced severe mitochondrial dysfunction, evidenced by ultrastructural disruption and a significant reduction in mitochondrial membrane potential. Notably, pharmacological blockade of the TNF receptor with the antagonist R-7050 effectively attenuated this mitochondrial damage. These findings establish a novel pathogenic mechanism wherein V. vulnificus OMVs exploit the host TNF signaling axis to drive mitochondrial injury, providing new insights into the cellular mechanisms of V. vulnificus lethality and suggesting the TNF-mitochondria axis as a potential therapeutic target.