Lianci Peng

Streptococcus suis ( S. suis ) is a prevalent zoonotic pathogen that severely threatens the swine industry and human public health. Suilysin (SLY), a major pore-forming toxin of S. suis , is indispensable for bacterial hemolytic activity and systemic pathogenicity, yet the upstream regulatory mechanism controlling SLY production remains unclear. Serine/threonine kinase (STK) has been characterized to modulate bacterial physiology and virulence by phosphorylating multiple substrate proteins. Our previous phosphoproteomics analysis indicated that the housekeeping sigma factor RpoD was a potential STK substrate. In vitro phosphorylation assays combined with mass spectrometry, co-immunoprecipitation and site-directed mutagenesis confirmed that STK directly phosphorylated RpoD at Thr-208 residue. Phenotypic tests revealed that RpoD phosphorylation had no obvious impact on bacterial growth and biofilm formation but specifically promoted the thermotolerance and hemolytic activity of S. suis . Notably, the rpoD overexpression mutant strain SC19-rpoD significantly upregulated the transcription and protein production of SLY, whereas the phosphorylation-deficient mutant SC19-rpoD T208A abolished this promotion effect, indicating that Thr-208 phosphorylation is required for sly upregulation, although the underlying molecular mechanism remains to be fully defined. Furthermore, mouse infection models demonstrated that the SC19-rpoD T208A mutant exhibited alleviated splenic pathological injury, reduced bacterial loads in multiple organs, and significantly improved host survival, indicating that the loss of RpoD phosphorylation impairs S. suis virulence. Collectively, these findings demonstrate that STK-mediated phosphorylation of RpoD at Thr-208 is required for sly upregulation and contributes to the S. suis pathogenesis.

Streptococcus suis ( S. suis ) is an important zoonotic pathogen that causes severe infections in humans and animals worldwide. Upon infection, the host innate immune system produces host defense peptides (HDPs) to resist S. suis invasion. PMAP-36, a porcine-derived HDP, has been shown to fail to kill S. suis , suggesting that this pathogen has evolved strategies to evade PMAP-36-mediated immune defense. However, the exact mechanism by which S. suis responds to PMAP-36 remains unclear. Here we showed that PMAP-36 significantly enhanced S. suis adhesion, cytotoxicity, and virulence gene expression. The two-component system (TCS) VraSR was essential for PMAP-36 to promote S. suis pathogenicity. We demonstrated that PMAP-36 directly bound to the sensor kinase VraS. Further transcriptomic analysis revealed that VraSR sensed PMAP-36 to regulate fatty acid biosynthesis, in which a key gene accD expression was significantly downregulated in both PMAP-36-treated Δ vraS and Δ vraR mutant strains. Deletion of accD attenuates S. suis in vivo pathogenicity, including reduced bacterial loads, alleviated organ damage, and decreased inflammatory response. Additionally, VraSR-mediated accD expression was critical for S. suis -induced cytotoxicity and inflammatory response, as deletion of vraS or vraR fails to reduce inflammatory cytokines production due to sustained high accD expression. This study reveals VraSR-mediated virulence regulatory mechanism in response to HDP activation, which provides new insight into the development of novel strategies to combat S. suis infection.

Lactic acid bacteria (LAB) are regarded as promising probiotics with multiple beneficial properties for humans’ and animals’ health. In this study, ten LAB isolates were screened and Lactiplantibacillus plantarum (L. plantarum) CLPX21 was selected as a candidate strain with its probiotic potential based on in vitro screening of antimicrobial activity and environmental stress tolerance, including its resistance to acidic environments and bile salts. This strain displayed adhesion capacity with an auto-aggregation rate of 47.15% at 24 h, co-aggregation rates above 67% with pathogenic bacteria, and an ability to adhere to IPEC-J2 cells (6.37%). CLPX21 showed broad-spectrum antimicrobial activity against common foodborne pathogens including Escherichia coli (E. coli), Salmonella, and Staphylococcus aureus. In addition, CLPX21 inhibited E. coli biofilm formation. Furthermore, CLPX21 significantly suppressed E. coli-induced inflammatory cytokine production in mouse peritoneal macrophages. Importantly, CLPX21 did not exhibit hemolytic activity. Genomic analysis further revealed that the CLPX21 genome encoded multiple functional genes and gene clusters, including biosynthesis of bacteriocins and secondary metabolites associated with antimicrobial and antioxidant functions, which provide a genetic basis for its beneficial phenotypic characteristics. In conclusion, L. plantarum CLPX21 displays probiotic properties with potent antimicrobial and anti-inflammatory activities, representing a promising candidate strain for applications in the food and health industries.

Porcine coronavirus is one of the prevalent enteric coronaviruses in pigs, causing watery diarrhea and even death in suckling piglets and resulting in giant losses to the pig industry. However, effective antiviral strategies against PDCoV remain limited. Notoginsenoside R1 (NG-R1), a saponin extracted from Panax notoginseng, exhibits diverse bioactivities, but its antiviral potential has not been fully characterized. Herein, we systematically investigated the anti-PDCoV effect of NG-R1 and its underlying mechanism. NG-R1 showed no cytotoxic effect on LLC-PK1 cells and exerted antiviral ability against PDCoV infection through targeting the whole life cycle of the virus. In addition, network pharmacology analysis identified calcium signaling as a potentially relevant pathway involved in the antiviral activity of NG-R1. Further data demonstrated that PDCoV infection disrupted intracellular calcium homeostasis, whereas NG-R1 treatment partially restored calcium balance and attenuated endoplasmic reticulum (ER) stress. Moreover, NG-R1 modulated the expression of SERCA2, a key regulator of ER calcium transport. Thapsigargin, an inhibitor of SERCA2, showed similar antiviral capacity to NG-R1. Collectively, our findings suggest that NG-R1 exerts antiviral activity against PDCoV, potentially through regulation of calcium homeostasis mediated by SERCA2. This study provides a theoretical basis for the development of novel antiviral agents targeting calcium signaling pathways.

Meningitis-associated extraintestinal pathogenic Escherichia coli (ExPEC) is a major cause of bacterial meningitis, yet the molecular mechanisms underlying blood-brain barrier (BBB) disruption during infection remain unclear. We employed integrated transcriptomic and proteomic analysis to investigate host responses of human cerebral microvascular endothelial cell line hCMEC/D3 to ExPEC strain RS218 infection. Multi-omics integration revealed coordinated immune activation, with upregulation of innate immune signaling pathways such as Toll-like receptor, NOD-like receptor, TNF, and IL-1 signaling, as well as antigen presentation pathways. In addition, we identified direct molecular evidence for BBB compromise, including concordant downregulation of tight junction protein ZO-1 at both transcriptomic and proteomic levels, validated by immunofluorescence showing reduced ZO-1 expression in infected cells. Several processes that may contribute to BBB breakdown were identified, such as glycosaminoglycan degradation, cytoskeletal reorganization, and suppression of TGF-β/SMAD signaling. Moreover, extensive metabolic dysregulation was evident, including downregulation of neural metabolic support functions and compromised protein homeostasis. Abundant discordance between transcriptomic and proteomic levels revealed complex post-transcriptional control mechanisms. In vitro experiments demonstrated RS218-induced cell death in brain endothelial cells, microglial cells, and peritoneal macrophages. Animal experiments confirmed systemic metabolic disruption, immune cell alteration, functional BBB disruption, and profound brain cytokine elevation. This integrated analysis advances our understanding of bacterial meningitis pathogenesis and identifies potential therapeutic targets.

Porcine antimicrobial peptides (AMPs) possess excellent antimicrobial activities and multiple biological functions, playing an essential role in the host's innate immune defense against pathogen infections. With the growing limitations of antibiotics, AMPs have been extensively studied due to their mild drug resistance and potent immunomodulatory activities. Porcine AMPs as vital components in the host defense against pathogen infections have the potential to be developed as anti-infective drugs. This review focused on the research progress in porcine AMPs regarding the structure-activity relationships and the antimicrobial and immunomodulatory activities, and discussed the potential application prospects, providing a basis for the development of anti-infective drugs in veterinary medicine.

Iron-sulfur (Fe-S) clusters are ancient prosthetic groups that are incorporated into apoproteins to generate fully functional Fe-S proteins, representing a conserved regulatory pathway in both Eukarya and Prokaryotes. In bacteria, the sulfur utilization factor (SUF) system mediates Fe-S cluster biosynthesis under stress conditions, thereby supporting essential metabolic processes to resist oxidative stress. In this study, we identify a novel substrate of serine/threonine kinase (STK), the protein with a DUF1831 domain (named as SstP1) in Streptococcus suis. GST pull-down and enzymatic activity assays demonstrated that STK-dependent phosphorylation of SstP1 at residue threonine 35 is required to sustain SUF-dependent Fe-S cluster biosynthesis under oxidative stress through direct interaction with the scaffold protein SufB. Functional analyses revealed that both deletion of sstP1 (ΔsstP1) or introduction of a phospho-ablative T35A mutant impaired bacterial growth and markedly reduced survival under oxidative stress and iron limitation conditions. In a mouse infection model, both the ΔsstP1 and T35A mutants exhibited significantly reduced bacterial burdens in the blood, lung, spleen, and brain, accompanied by attenuated virulence compared to the WT, phospho-mimetic T35E, and sstP1 complementary (CΔsstP1) mutants. Collectively, these findings reveal that STK-mediated phosphorylation of SstP1 at Thr35 is essential for oxidative stress resistance and virulence in S. suis, uncovering a novel mechanism that links phosphorylation signaling to SUF-dependent Fe-S cluster biosynthesis in bacterial pathogenesis.

Streptococcus suis ( S. suis ) induces host cell death and has the ability to invade the blood–brain barrier (BBB). However, S. suis -induced BBB disruption has not been completely elucidated. In this study, we focused on the regulatory role of lysine acetyltransferase 2A (KAT2A) on BBB, which is a key regulator of cell death. We found that S. suis strain SC19 induces cell death in human cerebral microvascular endothelial cell line D3 (hCMEC/D3) by membrane disruption and LDH release. During SC19 infection, KAT2A protein expression was markedly reduced but its mRNA expression was not affected. Further study demonstrated that SC19-induced KAT2A reduction was though ubiquitin–proteasome-mediated protein degradation pathway. KAT2A inhibition using significantly exacerbated SC19-induced cell permeability disruption by transwell infection model. Consistently, KAT2A knockdown and pharmacological inhibition significantly aggravated SC19-induced downregulation of tight junction proteins including ZO-1 and occludin, whereas KAT2A overexpression partially restored their levels. Similarly, KAT2A inhibition aggravated SC19-induced downregulation of ZO-1 in mice brain and increased mice death rate, indicating the protective role of KAT2A on BBB. Mechanistically, KAT2A was found to regulate necroptosis in hCMEC/D3. KAT2A knockdown aggravated necroptosis and upregulated phosphorylation of RIPK1, whereas RIPK1 inhibitor Nec-1 rescued SC19-induced necroptosis, indicating that KAT2A regulates RIPK1-dependent necroptosis during SC19 infection. Furthermore, quantitative proteomic analysis identified a network of putative KAT2A-mediated downstream targets and pathways that contribute to BBB integrity. Collectively, these findings reveal the underlying mechanism by which SC19 disrupts BBB through inducing KAT2A-mediated necroptosis and provide a potential therapeutic target for the treatment of bacterial meningitis.

Cathelicidin CATH-2 has been reported to exert potent anti-inflammatory activity in different species though neutralizing stimuli such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA). CATH-2 has been shown to inhibit Streptococcus suis (S. suis) -induced activation of dendritic cells and macrophages by binding to LTA. However, the exact mechanism of this prophylactically anti-inflammatory activity remains unclear. Therefore, we investigated the anti-inflammatory activity and mechanism of CATH-2 in mice peritoneal macrophages pretreated with CATH-2 followed by S. suis infection. The results showed that CATH-2 pretreatment significantly reduced S. suis -induced transcription and secretion of interleukin (IL)-1β, IL-6, and IL-12. CATH-2 also downregulated NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) expression and apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, and inhibited the maturation of IL-1β, suggesting that CATH-2 inhibits NLRP3 activation. In addition, CATH-2 significantly inhibited S. suis -induced phosphorylation of p65 and extracellular signal-regulated kinase (ERK). Further study showed that CATH-2 inhibited S. suis -induced reactive oxygen species (ROS) by upregulating the expression of ROS scavenging genes including catalase ( CAT ) and superoxide dismutase 1 ( SOD1 ). Mechanistically, transcriptome analysis revealed that CATH-2 regulated the protein kinase B (ATK)/mammalian target of rapamycin (mTOR) pathway, which was evident by the downregulation of phosphorylated (p)-ATK and p-mTOR induced by CATH-2. Notably, CATH-2 induced autophagy and autophagic flux. Inhibition of mTOR using rapamycin enhanced the CATH-2-induced autophagic efficacy, demonstrating that CATH-2 induces mTOR-dependent autophagy. However, inhibition of autophagy using 3-methyladenine (3-MA) reversed the reduction in the expression of p-p65, p-ERK, and IL-1β induced by CATH-2. Our study reveals that CATH-2 inhibits the nuclear factor kappa-B (NF-κB)/NLRP3-mediated inflammatory response through the induction of mTOR-dependent autophagy during S. suis infection, which provides new insight into the anti-inflammatory pathways of antimicrobial peptides.