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.

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.

Comprehensive analysis of transcriptome and metabolome analysis reveal new targets of Glaesserella parasuis glucose-specific enzyme IIBC (PtsG).

The ptsG (hpIIBCGlc) gene, belonging to the glucose-specific phosphotransferase system, encodes the bacterial glucose-specific enzyme IIBC. In this study, the effects of a deletion of the ptsG gene were investigated by metabolome and transcriptome analyses. At the transcriptional level, we identified 970 differentially expressed genes between ΔptsG and sc1401 (Padj<0.05) and 2072 co-expressed genes. Among these genes, those involved in methane metabolism, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, pyruvate metabolism, phosphotransferase system (PTS), biotin metabolism, Two-component system and Terpenoid backbone biosynthesis showed significant changes in the ΔptsG mutant strain. Metabolome analysis revealed that a total of 310 metabolites were identified, including 20 different metabolites (p < 0.05). Among them, 15 metabolites were upregulated and 5 were downregulated in ΔptsG mutant strain. Statistical analysis revealed there were 115 individual metabolites having correlation, of which 89 were positive and 26 negative. These metabolites include amino acids, phosphates, amines, esters, nucleotides, benzoic acid and adenosine, among which amino acids and phosphate metabolites dominate. However, not all of these changes were attributable to changes in mRNA levels and must also be caused by post-transcriptional regulatory processes. The knowledge gained from this lays the foundation for further study on the role of ptsG in the pathogenic process of Glaesserella parasuis (G.parasuis).

Rapid and simple detection of Glaesserella parasuis in synovial fluid by recombinase polymerase amplification and lateral flow strip.

BACKGROUND: Glaesserella parasuis (G. parasuis) is an influential pathogen of the pig, which induces high morbidity and mortality in naive pig populations in the pig industry. Accurate and rapid detection of the agent is important for disease control. In this study, a simple recombinase polymerase amplification (RPA) with a Lateral flow (LF) strip (RPA-LF-GPS) was developed to detect G. parasuis. RESULTS: The RPA-LF-GPS can specifically detect G. parasuis a limit of 100 CFU from other common related pathogens causing arthritis in the pig. The RPA-LF-GPS assay can use boiled synovial fluid samples as a template with the same sensitivity as other DNA extraction methods. In the detection of clinic positive synovial fluid sample, RPA-LF-GPS is equally sensitive (98.1%) compared with that of PCR (90.4%) (P > 0.05). The whole procedure of the RPA-LF-GPS assay could be finished in 1 hour without professional equipment. CONCLUSIONS: RPA-LF-GPS assay is a rapid and simple method for point-of-care diagnostic testing for G. parasuis infection.

Transcriptome Analysis of miRNA and mRNA in Porcine Skeletal Muscle following Glaesserella parasuis Challenge.

Glaesserella parasuis (G. parasuis) causes systemic infection in pigs, but its effects on skeletal muscle and underlying mechanisms are poorly understood. We investigated G. parasuis infection in colostrum-deprived piglets, observing decreased daily weight gain and upregulation of inflammatory factors in skeletal muscle. Muscle fiber area and diameter were significantly reduced in the treated group (n = 3) compared to the control group (n = 3), accompanied by increased expression of FOXO1, FBXO32, TRIM63, CTSL, and BNIP3. Based on mRNA and microRNA (miRNA) sequencing, we identified 1642 differentially expressed (DE) mRNAs and 19 known DE miRNAs in skeletal muscle tissues between the two groups. We predicted target genes with opposite expression patterns to the 19 miRNAs and found significant enrichment and activation of the FoxO signaling pathway. We found that the upregulated core effectors FOXO1 and FOXO4 were targeted by downregulated ssc-miR-486, ssc-miR-370, ssc-miR-615, and ssc-miR-224. Further investigation showed that their downstream upregulated genes involved in protein degradation were also targeted by the downregulated ssc-miR-370, ssc-miR-615, ssc-miR-194a-5p, and ssc-miR-194b-5p. These findings suggest that G. parasuis infection causes skeletal muscle atrophy in piglets through accelerated protein degradation mediated by the "miRNAs-FOXO1/4" axis, while further research is necessary to validate the regulatory relationships. Our results provide new insights into the understanding of systemic inflammation growth mechanisms caused by G. parasuis and the role of miRNAs in bacterial infection pathogenesis.

Salmonella enterica serovar Choleraesuis vector outperforms alum as an adjuvant, increasing a cross-protective immune response against Glaesserella parasuis.

The adjuvant and/or vector significantly affect a vaccine's efficacy. Although traditional adjuvants such as alum have contributed to vaccine development, deficiencies in the induction of cellular and mucosal immunity have limited their further promotion. Salmonella vectors have unique advantages for establishing cellular and mucosal immunity due to mucosal pathways of invasion and intracellular parasitism. In addition, Salmonella vectors can activate multiple innate immune pathways, thereby promoting adaptive immune responses. In this work, the attenuated Salmonella enterica serovar Choleraesuis (S. Choleraesuis) vector rSC0016 was used to deliver the conserved protective antigen HPS_06257 of Glaesserella parasuis (G. parasuis), generating a novel recombinant strain rSC0016(pS-HPS_06257). The rSC0016(pS-HPS_06257) can express and deliver the HPS_06257 protein to the lymphatic system of the host. In comparison to HPS_06257 adjuvanted with alum, rSC0016(pS-HPS_06257) significantly increased TLR4 and TLR5 activation in mice as well as the levels of proinflammatory cytokines. In addition, rSC0016 promoted a greater degree of maturation in bone marrow-derived dendritic cells (BMDCs) than alum. The specific humoral, mucosal, and cellular immune responses against HPS_06257 in mice immunized with rSC0016(pS-HPS_06257) were significantly higher than those of HPS_06257 adjuvanted with alum. HPS_06257 delivered by the S. Choleraesuis vector induces a Th1-biased Th1/Th2 mixed immune response, while HPS adjuvanted with alum can only induce a Th2-biased immune response. HPS_06257 adjuvanted with alum only causes opsonophagocytic activity (OPA) responses against a homologous strain (G. parasuis serotype 5, GPS5), whereas rSC0016(pS-HPS_06257) could generate cross-OPA responses against a homologous strain and a heterologous strain (G. parasuis serotype 12, GPS12). Ultimately, HPS_06257 adjuvanted with alum protected mice against lethal doses of GPS5 challenge by 60 % but failed to protect mice against lethal doses of GPS12. In contrast, mice immunized with rSC0016(pS-HPS_06257) had 100 % or 80 % survival when challenged with lethal doses of GPS5 or GPS12, respectively. Altogether, the S. Choleraesuis vector rSC0016 could potentially generate an improved innate immune response and an improved adaptive immunological response compared to the traditional alum adjuvant, offering a novel concept for the development of a universal G. parasuis vaccine.

Upregulation of TLR4-Dependent ATP Production Is Critical for Glaesserella parasuis LPS-Mediated Inflammation.

Glaesserella parasuis (G. parasuis), an important pathogenic bacterium, cause Glässer's disease, and has resulted in tremendous economic losses to the global swine industry. G. parasuis infection causes typical acute systemic inflammation. However, the molecular details of how the host modulates the acute inflammatory response induced by G. parasuis are largely unknown. In this study, we found that G. parasuis LZ and LPS both enhanced the mortality of PAM cells, and at the same time, the level of ATP was enhanced. LPS treatment significantly increased the expressions of IL-1ß, P2X7R, NLRP3, NF-κB, p-NF-κB, and GSDMD, leading to pyroptosis. Furthermore, these proteins' expression was enhanced following extracellular ATP further stimulation. When reduced the production of P2X7R, NF-κB-NLRP3-GSDMS inflammasome signaling pathway was inhibited, and the mortality of cells was reduced. MCC950 treatment repressed the formation of inflammasome and reduced mortality. Further exploration found that the knockdown of TLR4 significantly reduced ATP content and cell mortality, and inhibited the expression of p-NF-κB and NLRP3. These findings suggested upregulation of TLR4-dependent ATP production is critical for G. parasuis LPS-mediated inflammation, provided new insights into the molecular pathways underlying the inflammatory response induced by G. parasuis, and offered a fresh perspective on therapeutic strategies.

Identification of Glaesserella parasuis and Differentiation of Its 15 Serovars Using High-Resolution Melting Assays.

Glaesserella parasuis is the etiological agent of Glässer's disease, which is associated with polyserositis and arthritis and has a significant impact on the economy of the pig production industry. For the optimal surveillance of this pathogen, as well as for the investigation of G. parasuis-associated diseases, it is crucial to identify G. parasuis at the serovar level. In this work, we designed and developed new high-resolution melting (HRM) approaches, namely, the species-specific GPS-HRM1 and two serovar-specific HRM assays (GPS-HRM2 and GPS-HRM3), and evaluated the sensitivity and specificity of the assays. The HRM assays demonstrated good sensitivity, with 12.5 fg-1.25 pg of input DNA for GPS-HRM1 and 125 fg-12.5 pg for GPS-HRM2 and GPS-HRM3, as well as a specificity of 100% for the identification of all recognized 15 G. parasuis serovars. Eighteen clinical isolates obtained between 2014 and 2022 in Switzerland were tested by applying the developed HRM assays, which revealed a heterogeneous distribution of serovars 2, 7, 4, 13, 1, and 14. The combination with virulence marker vtaA (virulence-associated trimeric autotransporters) allows for the prediction of potentially virulent strains. The assays are simple to execute and enable a reliable low-cost approach, thereby refining currently available diagnostic tools.

Genomic insight into the integrative conjugative elements from ICEHpa1 family.

Integrative conjugative elements (ICEs) are important carriers for disseminating resistance genes. We have previously reported a novel element ICEHpa1 carrying seven antibiotic resistance genes, which could be self-transmissible relying on the novel T4SS. To identify novel ICEHpa1 variants from 211 strains and novel T4SS encoded in ICEHpa1, and to explore the relationships in these ICEs, four complete sequences of ICEs were identified by WGS analysis and antimicrobial susceptibility testing was determined by broth microdilution. In addition, a comparative analysis of these ICEs was conducted with bioinformatic tools, and the transfer abilities of these ICEs were confirmed by conjugation. Four ICEHpa1 variants ICEGpa1818, ICEGpa1808, ICEGpa1807, and ICEGpa1815 with different resistance gene profiles were characterized, and their hosts showed different resistance spectrums. All ICEs shared the same backbone and were inserted into the tRNALeu site, and all resistance regions were inserted into the same target site between the accessory and integration regions. This study analyzed complete sequences of ICEs from the ICEHpa1 family and identified novel T4SS and insertion element ISGpa2. Diverse resistance genes extensively exist in these ICEs, serving as a reservoir for resistance genes and facilitating their dissemination.