Transcriptome analysis reveals a new virulence-associated trimeric autotransporter responsible for Glaesserella parasuis autoagglutination.

Capsular polysaccharide is an important virulence factor of Glaesserella parasuis. An acapsular mutant displays multiple phenotype variations, while the underlying mechanism for these variations is unknown. In this study, we created an acapsular mutant by deleting the wza gene in the capsule locus. We then used transcriptome analysis to compare the gene expression profiles of the wza deletion mutant with those of the parental strain to understand the possible reasons for the phenotypic differences. The mutant Δwza, which has a deleted wza gene, secreted less polysaccharide and lost its capsule structure. The Δwza exhibited increased autoagglutination, biofilm formation and adherence to eukaryotic cells, while the complementary strain C-Δwza partially restored the phenotype. Transcriptome analysis revealed several differentially expressed genes (DEGs) in Δwza, including up-regulated outer membrane proteins and proteins involved in peptidoglycan biosynthesis, suggesting that wza deletion affects the cell wall homeostasis of G. parasuis. Transcriptome analysis revealed the contribution of non-coding RNAs in the regulation of DEGs. Moreover, a new virulence-associated trimeric autotransporter, VtaA31 is upregulated in Δwza. It is responsible for enhanced autoagglutination but not for enhanced biofilm formation and adherence to eukaryotic cells in Δwza. In conclusion, these data indicate that wza affects the expression of multiple genes, especially those related to cell wall synthesis. Furthermore, they provide evidence that vtaA31 is involved in the autoagglutination of G. parasuis.

Research progress into the principles and methods underlying capsular typing of Glaesserella parasuis.

Glaesserella parasuis (GPS) is an important bacterial pathogen of swine. Serotype identification has presented a bottleneck in GPS research since it was first identified as the pathogen causing Glässer's disease in pigs in 1910. This paper presents a systematic review of the history of the development and application of gel immunodiffusion (GID), indirect hemagglutination assay (IHA), and polymerase chain reaction (PCR) typing methods for GPS, and the discovery of their shared antigenic basis. It provides a systematic theoretical overview of the immunology and principles underlying the three typing methods and offers new ideas for research into the prevention and control of Glässer's disease. In 1992, GPS was first classified into serotypes 1-15 using GID based on GPS heat-stable antigens, but about 25% of the strains were found to be non-typeable, and the composition of their antigens for serotyping was unclear. In 2003, the IHA method was established based on saline-extracted antigens of GPS, whose sensitivity and typing rate were higher than for GID, although about 15% of strains were still found to be non-typeable. The results of IHA and GID typing are roughly consistent, since they share the same GPS surface polysaccharide serotyping antigens, although whether these are capsular polysaccharides, lipopolysaccharides, or other polysaccharides, remains to be determined. In 2013, the Capsular polysaccharide (CPS) synthetic gene clusters from GPS serotypes 1-15 were successfully analyzed, confirming that CPS is essential for the formation of antigens for serotyping. In 2015, primers were designed based on the specific target genes of GPS capsules to establish a PCR typing method (H-PCR) for GPS, which, however, could not identify serotypes 5 and 12. In 2017, a new PCR typing method (J-PCR) was established based on the specific target genes of GPS capsules, which could identify serotypes 5 and 12. A combination of the two PCR typing methods enables the typing of almost all GPS strains, and the consistency with GID and IHA was verified using molecular biological methods. The antigenic basis of the three typing methods was shown to involve the GPS capsule. PCR typing methods are characterized by simple operation, fast speed, and low cost, and can successfully solve many problems in GID and IHA serotyping, and so have become widely adopted.

A marker-free genetic manipulation method for Glaesserella parasuis strains developed by alternately culturing transformants at 37°C and 30°C.

BACKGROUND: Glaesserella parasuis (G. parasuis) is the causative agent of Glässer's disease, which causes significant economic losses in the swine industry. However, research on the pathogenesis of G. parasuis has been hampered by the lack of a simple and efficient marker-free knockout system. RESULTS: In this study, a marker-free knockout system was developed for G. parasuis using a temperature-sensitive vector. By alternating the incubation of transformants at 30°C and 37°C, we optimized the screening process for this system. The system was successfully applied to knockout the KanR cassette from JS0135ΔnanH::KanR, achieving a knockout efficiency of 90% in the final round of screening. To confirm that temperature variation was a key factor, we proceeded with knocking out the nanH and apd genes in the CF7066 strain. The knockout efficiency reached up to 100%, with the shortest screening time being only four days. The knockout of the nanH gene resulted in a significant reduction in the growth vitality of the strains, while the knockout of the apd gene led to an approximate 56% improvement in the adhesion rate. Additionally, we observed that the expression of recombinant genes in transformants was higher at 30℃ than at 37℃, with the recC gene being upregulated approximately 7-fold. In contrast, there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in the wild-type strains. This discrepancy was likely due to an elevated copy number of target plasmids at 30℃, which may have resulted in the enhanced expression of recombinant genes. CONCLUSIONS: In conclusion, this newly developed gene knockout system for G. parasuis presents a valuable tool for advancing research on this organism.

Glaesserella parasuis serotype 4 exploits fibronectin via RlpA for tracheal colonization following porcine circovirus type 2 infection.

Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality, but the role played by PCV2 and bacterial and host factors contributing to this process have not been defined. Bacterial attachment is assumed to occur via specific receptor-ligand interactions between adhesins on the bacterial cell and host proteins adsorbed to the implant surface. Mass spectrometry (MS) analysis of PCV2-infected swine tracheal epithelial cells (STEC) revealed that the expression of Extracellular matrix protein (ECM) Fibronectin (Fn) increased significantly on the infected cells surface. Importantly, efficient G. parasuis serotype 4 (GPS4) adherence to STECs was imparted by interactions with Fn. Furthermore, abrogation of adherence was gained by genetic knockout of Fn, Fn and Integrin ß1 antibody blocking. Fn is frequently exploited as a receptor for bacterial pathogens. To explore the GPS4 adhesin that interacts with Fn, recombinant Fn N-terminal type I and type II domains were incubated with GPS4, and the interacting proteins were pulled down for MS analysis. Here, we show that rare lipoprotein A (RlpA) directly interacts with host Fibronectin mediating GPS4 adhesion. Finally, we found that PCV2-induced Fibronectin expression and adherence of GPS4 were prevented significantly by TGF-ß signaling pathway inhibitor SB431542. Our data suggest the RlpA-Fn interaction to be a potentially promising novel therapeutic target to combat PCV2 and GPS4 coinfection.

Genomic insight into the diversity of Glaesserella parasuis isolates from 19 countries.

Glaesserella parasuis is a commensal bacterial organism found in the upper respiratory tract of healthy pigs and the etiological agent of Glässer's disease, which causes severe economic losses in the swine industry. This study aimed to better understand the epidemiological characteristics of this opportunistic pathogen. We investigated the prevalence and distribution of sequence types (STs), serovars, antimicrobial resistance genes (ARGs), and potential virulence factors (VFs) in 764 G. parasuis isolates collected from diseased and healthy pigs from 19 countries, including China. Multilocus sequence typing showed a high degree of variation with 334 STs, of which 93 were not previously recognized. Phylogenetic analysis revealed two major clades distinguished by isolation year, source, country, and serovar. The dominant serovars of G. parasuis were serovars 4 (19.50%), 7 (15.97%), 5/12 (13.87%), and 13 (12.30%). Serovar 7 gradually became one of the dominant serovars in G. parasuis with more VFs and fewer ARGs. Serovars 4 and 5/12 were the most frequent serovars in diseased pigs, whereas serovars 2, 8, and 11 were predominant in healthy pigs. Serovars 7 and 13 possessed more VFs than the other serovars. This study provides novel insights into the global prevalence and epidemiology of G. parasuis and valuable clues for further investigation into the pathogenicity of G. parasuis, which will facilitate the development of effective vaccines.IMPORTANCEGlaesserella parasuis is a clinically important gram-negative opportunistic pathogen, which causes serious financial losses in swine industry on a global scale. No vaccine is known that provides cross-protection against all 15 serovars; furthermore, the correlation between serovar and virulence is largely unknown. This study provides a large number of sequenced strains in 19 countries and compares the genomic diversity of G. parasuis between diseased and healthy pigs. We found a slight change in the dominant serovar of G. parasuis in the world, with serovar 7 gradually emerging as one of the predominant serovars. The observed higher average number of VFs in this particular serovar strain challenges the previously held notion that serovar 7 is non-virulent, indicating a more complex virulence landscape than previously understood. Our analysis indicating that six ARGs [tet(B), sul2, aph(3')-Ia, aph (6)-Id, blaROB-1, and aph(3'')-Ib] are likely to be transmitted horizontally in their entirety. By analyzing VFs, we provided an improved understanding of the virulence of G. parasuis, and these key findings suggest that vaccine development will be challenging.

Transcriptome Analysis Reveals Novel Inflammatory Signalings to Glaesserella parasuis Infection.

Glaesserella parasuis (GPS) can cause severe systemic inflammation in pigs, resulting in huge economic losses to the pig industry. At present, no effective method is available for the prevention and control of GPS infection. Molecular breeding for disease resistance is imminent, but disease-resistance genes have not been identified. To study the mechanism of systemic acute inflammation caused by GPS, we established three in vitro infection models (3D4/21 cells, PK15 cells, and PAVEC cells) according to its infection path. There was no significant difference in apoptosis among the three kinds of cells after 12 h of continuous GPS stimulation, while inflammatory factors were significantly upregulated. Subsequent transcriptome analysis revealed 1969, 1207, and 3564 differentially expressed genes (DEGs) in 3D4/21 cells, PK15 cells, and PAVEC cells, respectively, after GPS infection. Many of the DEGs were predicted to be associated with inflammatory responses (C3, CD44, etc.); cell proliferation, growth and apoptosis; gene expression; and protein phosphorylation. Key signaling pathways, including S100 family signaling, bacteria and virus recognition, and pathogen-induced cytokine storm signaling, were enriched based on Ingenuity Pathway Analysis (IPA). Furthermore, a total of three putative transmembrane receptors and two putative G-protein-coupled receptors, namely F3, ICAM1, PLAUR, ACKR3, and GPRC5A, were identified by IPA among the three types of cells. ACKR3 and GPRC5A play pivotal roles in bacterial adhesion, invasion, host immune response and inflammatory response through the S100 family signaling pathway. Our findings provide new insights into the pathological mechanisms underlying systemic inflammation caused by GPS infection in pigs, and they lay a foundation for further research on disease-resistance breeding to GPS.

HbpA from Glaesserella parasuis induces an inflammatory response in 3D4/21 cells by activating the MAPK and NF-κB signalling pathways and protects mice against G. parasuis when used as an immunogen.

Glaesserella parasuis is usually a benign swine commensal in the upper respiratory tract, but virulent strains can cause systemic infection characterized by pneumonia, meningitis, and fibrinous polyserositis. The intensive pulmonary inflammatory response following G. parasuis infection is the main cause of lung injury and death in pigs. Vaccination has failed to control the disease due to the lack of extended cross-protection. Accumulating evidence indicates that the heme-binding protein A (HbpA) is a potential virulence determinant and a promising antigen candidate for the development of a broader range of vaccines. However, it is not yet known whether HbpA contributes to G. parasuis virulence or has any potential immune protective effects against G. parasuis. Here, we show that HbpA can induce the transcription and secretion of proinflammatory cytokines (IL-6, TNF-α, and MCP-1) in porcine alveolar macrophages (PAM, 3D4/31). The HbpA protein is recognized by Toll-like receptors 2 and 4 on 3D4/21 macrophages, resulting in the activation of MAP kinase and NF-κB signalling cascades and the transcription and secretion of proinflammatory cytokines. HbpA contributes to virulence and bacterial pulmonary colonization in C57BL/6 mice and plays a role in adhesion to host cells and evasion of the bactericidal effect of pulmonary macrophages. In addition, mice immunized with HbpA were partially protected against challenge by G. parasuis SC1401. The results suggest that HbpA plays an important role in the pathogenesis of disease caused by G. parasuis and lay a foundation for the development of a subunit or chimeric anti-G. parasuis vaccine.

Baicalin attenuates PD-1/PD-L1 axis-induced immunosuppression in piglets challenged with Glaesserella parasuis by inhibiting the PI3K/Akt/mTOR and RAS/MEK/ERK signalling pathways.

Infection of piglets with Glaesserella parasuis (G. parasuis) induces host immunosuppression. However, the mechanism underlying the immunosuppression of piglets remains unclear. Activation of the PD-1/PD-L1 axis has been shown to trigger host immunosuppression. Baicalin possesses anti-inflammatory and immunomodulatory functions. However, whether baicalin inhibits PD-1/PD-L1 activation and thus alleviates host immunosuppression has not been investigated. In this study, the effect of baicalin on the attenuation of piglet immunosuppression induced by G. parasuis was evaluated. Seventy piglets were randomly divided into the control group, infection group, levamisole group, BMS-1 group, 25 mg/kg baicalin group, 50 mg/kg baicalin group and 100 mg/kg baicalin group. Following pretreatment with levamisole, BMS-1 or baicalin, the piglets were challenged with 1 × 108 CFU of G. parasuis. Our results showed that baicalin, levamisole and BMS-1 modified routine blood indicators and biochemical parameters; downregulated IL-1ß, IL-10, IL-18, TNF-α and IFN-γ mRNA expression; and upregulated IL-2 and IL-8 mRNA expression in blood. Baicalin, levamisole and BMS-1 increased the proportions of CD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells and CD3-CD21+ B cells in the splenocyte population, increased the proportions of CD3+ T cells, CD3+CD4+ T cells and CD3+CD8+ T cells in the blood, and inhibited PD-1/PD-L1 and TIM-3 activation. Baicalin, levamisole and BMS-1 reduced p-PI3K, p-Akt, and p-mTOR expression, the p-MEK1/2/MEK1/2 and p-ERK1/2/ERK1/2 ratios and increased RAS expression. Baicalin, levamisole and BMS-1 provided substantial protection against G. parasuis challenge and relieved tissue histopathological damage. Our findings might provide new strategies for controlling G. parasuis infection and other immunosuppressive diseases.

The role of cytolethal distending toxin in Glaesserella parasuis JS0135 strain infection: Cytotoxicity, phagocytic resistance and pathogenicity.

Glaesserella parasuis is an important porcine pathogen that commonly colonizes the upper respiratory tract of pigs and is prone to causing Glässer's disease under complex conditions. As yet, the disease has led to serious economic losses to the swine industry worldwide. Studies so far have found that several virulence factors are associated with the pathogenicity of G. parasuis, but the pathogenic mechanism is still not fully understood. Cytolethal distending toxin (CDT), a potential virulence factor in G. parasuis, is involved in cytotoxicity, serum resistance, adherence to and invasion of host cells in vitro. Here, to further investigate the pathogenic role of CDT during G. parasuis infection in vitro and in vivo, a double cdt1 and cdt2 deletion mutant (Δcdt1Δcdt2) without selectable marker was first generated in G. parasuis JS0135 strain by continuous natural transformations and replica plating. Morphological observation and lactate dehydrogenase assay showed that the Δcdt1Δcdt2 mutant was defective in cytotoxicity. Additionally, the Δcdt1Δcdt2 mutant was more susceptible to phagocytosis caused by 3D4/2 macrophages compared to the wild-type JS0135 strain. Moreover, by focusing on clinical signs, necropsy, bacterial recovery and pathological observation, we found that the deletion of cdt1 and cdt2 genes led to a significant attenuation of virulence in G. parasuis. Taken together, these findings suggest that as an important virulence factor, CDT can significantly affect the pathogenicity of G. parasuis.

Baicalin and probenecid protect against Glaesserella parasuis challenge in a piglet model.

Glaesserella parasuis (G. parasuis) induces vascular damage and systemic inflammation. However, the mechanism by which it causes vascular damage is currently unclear. Baicalin has important anti-inflammatory, antibacterial and immunomodulatory functions. In this study, we explored the ability of baicalin and probenecid to protect against G. parasuis challenge in a piglet model. Sixty piglets were randomly divided into a control group; an infection group; a probenecid group; and 25 mg/kg, 50 mg/kg and 100 mg/kg baicalin groups. The probenecid group and the 25 mg/kg, 50 mg/kg and 100 mg/kg baicalin groups were injected intramuscularly with 20 mg/kg body weight (BW) probenecid and 25 mg/kg BW, 50 mg/kg BW and 100 mg/kg BW baicalin, respectively. All piglets except those from the control group were injected intraperitoneally with 1 × 108 CFU of G. parasuis. The control group was injected intraperitoneally with TSB. The results showed baicalin and probenecid protected piglets against G. parasuis challenge, improved body weight and decreased temperature changes in piglets. Baicalin and probenecid attenuated IL-1ß, IL-10, IL-18, TNF-α and IFN-γ mRNA levels in the blood for 48 h, inhibited the production of the nucleosides ATP, ADP, AMP and UMP from 24 to 72 h, reduced Panx-1/P2Y6/P2X7 expression, weakened NF-kB, AP-1, NLRP3/Caspase-1 and ROCK/MLCK/MLC signalling activation, and upregulated VE-cadherin expression in the blood vessels of piglets challenged with G. parasuis. Baicalin and probenecid alleviated pathological tissue damage in piglets induced by G. parasuis. Our results might provide a promising strategy to control and treat G. parasuis infection in the clinical setting.

Expression profiles of miRNAs in the lung tissue of piglets infected with Glaesserella parasuis and the roles of ssc-miR-135 and ssc-miR-155-3p in the regulation of inflammation.

MicroRNAs (miRNAs) have been shown to play an important regulatory role in the process of pathogenic infection. However, the miRNAs that regulate the pathogenic process of G. parasuis and their functions are still unknown. Here, high-throughput sequencing was used to quantify the expression of miRNA in piglet lung tissue after G. parasuis XX0306 strain infection. A total of 25 differentially expressed microRNAs (DEmiRNAs) were identified. GO and KEGG pathway enrichment analysis showed that many of the functions of genes that may be regulated by DEmiRNA are related to inflammatory response and immune regulation. Further studies found that ssc-miR-135 may promote the expression of inflammatory factors through NF-κB signaling pathway. Whereas, ssc-miR-155-3p inhibited the inflammatory response induced by G. parasuis, and its regulatory mechanism remains to be further investigated. This study provides a valuable reference for revealing the regulatory effects of miRNAs on the pathogenesis of G. parasuis. DATA AVAILABILITY: The datasets generated during the current study are not publicly available due to this study is currently in the ongoing research stage, and some of the data cannot be made public sooner yet, but are available from the corresponding author on reasonable request.

Activation of the NLRP3-CASP-1 inflammasome is restrained by controlling autophagy during Glaesserella parasuis infection.

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-1ß processing and maturation pathway, resulting in increased release of IL-1ß 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.

Histidine Kinase QseC in Glaesserella parasuis Enhances the Secretion of Proinflammatory Cytokines by Macrophages via the p38 and NF-κB Signaling Pathways.

The qseC gene is a two-component system that encodes a histidine protein kinase and is highly conserved among different Glaesserella parasuis strains. In this study, we used qRT-PCR and enzyme-linked immunosorbent assay to confirm that Toll-like receptor 4 (TLR4) plays a role in the expression of proinflammatory cytokines interleukin (IL)-1ß and IL-6 by stimulating RAW 264.7 macrophages with QseC. Furthermore, we revealed that blocking the p38 and NF-κB pathways that regulate signaling can significantly reduce the production of proinflammatory cytokines induced by QseC. In summary, our data suggest that QseC is a novel proinflammatory mediator that induces TLR4-dependent proinflammatory activity in RAW 264.7 macrophages through the p38 and NF-κB pathways.

Quercetin Protects Blood-Brain Barrier Integrity via the PI3K/Akt/Erk Signaling Pathway in a Mouse Model of Meningitis Induced by Glaesserella parasuis.

Glaesserella parasuis (G. parasuis) causes serious inflammation and meningitis in piglets. Quercetin has anti-inflammatory and anti-bacterial activities; however, whether quercetin can alleviate brain inflammation and provide protective effects during G. parasuis infection has not been studied. Here, we established a mouse model of G. parasuis infection in vivo and in vitro to investigate transcriptome changes in the mouse cerebrum and determine the protective effects of quercetin on brain inflammation and blood-brain barrier (BBB) integrity during G. parasuis infection. The results showed that G. parasuis induced brain inflammation, destroyed BBB integrity, and suppressed PI3K/Akt/Erk signaling-pathway activation in mice. Quercetin decreased the expression of inflammatory cytokines (Il-18, Il-6, Il-8, and Tnf-α) and BBB-permeability marker genes (Mmp9, Vegf, Ang-2, and Et-1), increased the expression of angiogenetic genes (Sema4D and PlexinB1), reduced G. parasuis-induced tight junction disruption, and reactivated G. parasuis-induced suppression of the PI3K/Akt/Erk signaling pathway in vitro. Thus, we concluded that quercetin may protect BBB integrity via the PI3K/Akt/Erk signaling pathway during G. parasuis infection. This was the first attempt to explore the protective effects of quercetin on brain inflammation and BBB integrity in a G. parasuis-infected mouse model. Our findings indicated that quercetin is a promising natural agent for the prevention and treatment of G. parasuis infection.

AMPK signaling pathway regulated the expression of the ApoA1 gene via the transcription factor Egr1 during G. parasuis stimulation.

Glaesserella parasuis (G. parasuis) is the causative agent of porcine Glässer's disease, resulting in high mortality rates in pigs due to excessive inflammation-induced tissue damage. Previous studies investigating the protective effects of G. parasuis vaccination indicated a possible role of ApoA1 in reflecting disease progression following G. parasuis infection. However, the mechanisms of ApoA1 expression and its role in these infections are not well understood. In this investigation, newborn porcine tracheal (NPTr) epithelial cells infected with G. parasuis were used to elucidate the molecular mechanism and role of ApoA1. The study revealed that the AMPK pathway activation inhibited ApoA1 expression in NPTr cells infected with G. parasuis for the first time. Furthermore, Egr1 was identified as a core transcription factor regulating ApoA1 expression using a CRISPR/Cas9-based system. Importantly, it was discovered that APOA1 protein significantly reduced apoptosis, pyroptosis, necroptosis, and inflammatory factors induced by G. parasuis in vivo. These findings not only enhance our understanding of ApoA1 in response to bacterial infections but also highlight its potential in mitigating tissue damage caused by G. parasuis infection.

Glaesserella parasuis serotype 5 induces pyroptosis via the RIG-I/MAVS/NLRP3 pathway in swine tracheal epithelial cells.

Glaesserella parasuis (G. parasuis) is a common Gram-negative commensal bacterium in the upper respiratory tract of swine that can cause Glässer's disease under stress conditions. Pyroptosis is an important immune defence mechanism of the body that plays a crucial role in clearing pathogen infections and endogenous danger signals. This study aimed to investigate the mechanism of G. parasuis serotype 5 SQ (GPS5-SQ)-induced pyroptosis in swine tracheal epithelial cells (STECs). The results of the present study demonstrated that GPS5-SQ infection induces pyroptosis in STECs by enhancing the protein level of the N-terminal domain of gasdermin D (GSDMD-N) and activating the NOD-like receptor protein 3 (NLRP3) inflammasome. Furthermore, the levels of pyroptosis-related proteins, including GSDMD-N and cleaved caspase-1 were considerably decreased in STECs after the knockdown of retinoic acid inducible gene-I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). These results indicated that GPS5-SQ might trigger pyroptosis through the activation of the RIG-I/MAVS/NLRP3 signaling pathway. More importantly, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) repressed the activation of the RIG-I/MAVS/NLRP3 signaling and rescued the decrease in Occludin and zonula occludens-1 (ZO-1) after GPS5-SQ infection. Overall, our findings show that GPS5-SQ can activate RIG-I/MAVS/NLRP3 signaling and destroy the integrity of the epithelial barrier by inducing ROS generation in STECs, shedding new light on G. parasuis pathogenesis.

Evaluation of immunoregulation and immunoprotective efficacy of Glaesserella parasuis histidine kinase QseC.

QseC is a membrane sensor kinase that enables bacteria to perceive autoinducers -3, adrenaline, and norepinephrine to initiate downstream gene transcription. In this study, we found that the QseC protein of Glaesserella parasuis can serve as an effective antigen to activate the host's immune response. Therefore, we investigated the immunogenicity and host protective effect of this protein. ELISA and indirect immunofluorescence results showed that QseC protein can induce high titer levels of humoral immunity in mice and regularly generate specific serum antibodies. We used MTS reagents to detect lymphocyte proliferation levels and found that QseC protein can cause splenic lymphocyte proliferation with memory and specificity. Further immunological analysis of the spleen cell supernatant revealed significant upregulation of levels of IL-1ß, IL-4 and IFN-γ in the QseC + adjuvant group. In the mouse challenge experiment, it was found that QseC + adjuvant can provide effective protection. The results of this study demonstrate that QseC protein provides effective protection in a mouse model and has the potential to serve as a candidate antigen for a novel subunit vaccine for further research.

PD-1/PD-L1 axis induced host immunosuppression via PI3K/Akt/mTOR signalling pathway in piglets infected by Glaesserella Parasuis.

Glaesserella parasuis, an important respiratory bacterial pathogen, causes Glässer's disease in piglets, with potential immunosuppression. We established a piglet infection model and explored the immunosuppression mechanism to improve our understanding of the host immune response to G. parasuis. Twenty piglets were randomly divided into two groups (n = 10). The infection group was intraperitoneally challenged with 2 × 108 CFU of G. parasuis in 2 mL TSB. The control group was intraperitoneally injected with equivalent TSB. After 72 h, the piglets were sacrificed, and spleen tissue was collected. PD-1/PD-L1 expression was determined. The splenocytes were isolated to detect CD3+ T, CD3+CD4+ T, CD3+CD8+ T and CD3-CD21+cell differentiation. Via data-independent acquisition (DIA), we compared the proteomics of healthy and infected spleen tissues. Glaesserella parasuis modified CD3+ T, CD3+CD4+ T, CD3+CD8+ T and CD3-CD21+ cell differentiation and PD-1/PD-L1 expression in the spleen. The infection group had 596 proteins with significant differences in expression, of which 301 were significantly upregulated and 295 downregulated. Differentially expressed proteins (DEPs) were mainly related to immune responses. This is the first study on PD-1/PD-L1 expression in the spleen associated with immunosuppression in a piglet model to explore the protein changes related to immune responses via DIA.

EspP2 Regulates the Adhesion of Glaesserella parasuis via Rap1 Signaling Pathway.

Different levels of EspP2 expression are seen in strains of Glaesserella parasuis with high and low pathogenicity. As a potential virulence factor for G. parasuis, the pathogenic mechanism of EspP2 in infection of host cells is not clear. To begin to elucidate the effect of EspP2 on virulence, we used G. parasuis SC1401 in its wild-type form and SC1401, which was made EspP2-deficient. We demonstrated that EspP2 causes up-regulation of claudin-1 and occludin expression, thereby promoting the adhesion of G. parasuis to host cells; EspP2-deficiency resulted in significantly reduced adhesion of G. parasuis to cells. Transcriptome sequencing analysis of EspP2-treated PK15 cells revealed that the Rap1 signaling pathway is stimulated by EspP2. Blocking this pathway diminished occludin expression and adhesion. These results indicated that EspP2 regulates the adhesion of Glaesserella parasuis via Rap1 signaling pathway.

Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for naked-eye detection of Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions. RESULTS: In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min. SIGNIFICANCE: We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis.