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.

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.

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.

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.

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.

Prevalence and genomic-based antimicrobial resistance analysis of Avibacterium paragallinarum isolates in Guangdong Province, China.

Infectious coryza (IC) is an acute infectious respiratory disease in chickens that is caused by Avibacterium paragallinarum (A. paragallinarum). A. paragallinarum poses a significant threat to poultry health due to its virulence and multidrug resistance. This study isolated and identified 21 A. paragallinarum isolates from Guangdong between 2022 and 2023. Biochemical tests showed that 100% of A. paragallinarum isolates fermented glucose but did not ferment alginate and galactose, and only YZ18 was nicotinamide adenine dinucleotide independent. To determine the genetic relatedness between these isolates and NCBI reference strains, whole-genome-based phylogenetic analysis was employed. In addition, analysis of the 2,000 bp-length hmtp210 gene showed that the hmtp210 gene was strongly associated with A. paragallinarum serotypes. Meanwhile, a PCR assay for serotyping A. paragallinarum was developed based on the hmtp210 gene, this assay has high sensitivity and specificity. The antimicrobial susceptibility of isolates was assessed using the disk diffusion method. The antibiotic resistance genes of isolates were analyzed using the genomic method. Phenotypic resistance to ampicillin (95.2%), streptomycin (95.2%), methotrexate-sulfamethoxazole (90.5%), and tetracycline (85.7%) was most frequent among the isolates. All of the isolates exhibited resistance to multiple drugs, and furthermore, the isolates possessed a collective total of 14 genes associated with antibiotic resistance. This study will contribute to advancing our knowledge of A. paragallinarum antibiotic resistance and provide a scientific basis for the prophylaxis and treatment of IC, and the subsequent rational design of potential clinical therapeutics.

Case reports involving coinfection with Avibacterium paragallinarum and Ornithobacterium rhinotracheale in broiler chickens and Avibacterium endocarditis in broiler breeding hens in Poland.

ABSTRACTThe study describes three clinical cases of infection with Avibacterium spp.. In case no. 1, respiratory clinical signs and high mortality (0.7-4.2% daily; total 21.2%) in Ross 308 broiler chickens were shown to be caused by coinfection with sequence type 9 of O. rhinotracheale presumptive serotype A and A. paragallinarum presumptive serotype B. The identical (pulsed-field gel electrophoresis) restriction pattern (pulsotype) of seven A. paragallinarum isolates indicated that infectious coryza in broilers was caused by the same clone. In cases 2 and 3, sudden increased deaths in Ross 308 broiler breeders (especially males) with lesions in the endocardium (valvular or mural endocarditis) were shown to be caused by A. endocarditis. Among nine antibiotics tested, florfenicol was the only antibiotic to which all A. paragallinarum and O. rhinotracheale isolates were susceptible. Out of the eight antibiotics tested, 11 A. endocarditis isolates from both clinical cases of infective endocarditis were susceptible to penicillin, amoxicillin, doxycycline and florfenicol. The A. endocarditis isolates tested in both clinical cases had different PFGE patterns (pulsotypes), but identical within a case. The causes of infectious coryza and infective endocarditis in the cases presented have not been determined. In the prevention of infectious diseases in large-scale livestock farming, it is very important to follow the rules of biosecurity.

Characterization of a highly virulent Avibacterium paragallinarum isolate.

Infectious coryza (IC) is an important respiratory infectious disease in chickens. In this study, an Avibacterium paragallinarum Page serovar C strain, named ZJ-C, was isolated from a local layer flock that was routinely vaccinated with an inactivated trivalent vaccine, using reference strain Modesto as the serovar C immunogen. The pathogenicity, immunogenicity, and genetic characteristics of ZJ-C were studied. The minimum pathogenic dose of the isolate was 100 CFU, which was 1/1,000 of the dose of the serovar C reference strain Modesto. The vaccination-challenge trial in specific pathogen-free (SPF) chickens showed that the ZJ-C bacterin could provide 100% protection against challenge from both ZJ-C and Modesto strains, whereas Modesto provided 100% protection against challenge from itself, but only 70% protection against ZJ-C. Sequence analysis of the HMTp210 hypervariable region (region 2) showed that the homology of region 2 between ZJ-C and Modesto was 96.14%, whereas the homology between ZJ-C and the Kume serovar C-4 reference strain HP60 was 99.83%. Phylogenetic analysis of region 2 showed that ZJ-C was most closely related to cluster C-4, represented by HP60. The experimental data obtained in this study will help the selection of optimal vaccine strains and assist serotyping studies of Av. paragallinarum.

Vaccination with inactivated multivalent vaccines is a primary strategy to control Infectious coryza. Avibacterium paragallinarum serotyping is important for effective protection as inactivated whole-cell vaccines provide protection against only the serogroup or serovar from which the vaccine was derived. In this study, a novel serovar within the serogroup C Avibacterium paragallinarum isolate ZJ-C has been characterized first time in China. It was highly virulent and induced 100% cross-protection to Modesto bacterin vaccinated chickens, but not the other way around.

Glaesserella parasuis QseBC two-component system senses epinephrine and regulates capD expression.

IMPORTANCE: The key bacterial pathogen Glaesserella parasuis, which can cause Glässer's disease, has caused significant financial losses to the swine industry worldwide. Capsular polysaccharide (CPS) is an important virulence factor for bacteria, providing the ability to avoid recognition and killing by the host immune system. Exploring the alteration of CPS synthesis in G. parasuis in response to epinephrine stimulation can lay the groundwork for revealing the pathogenic mechanism of G. parasuis as well as providing ideas for Glässer's disease control.

Regions Important for Hemagglutination Activity and Serotypes of Avibacterium paragallinarum HMTp210 Protein.

Avibacterium paragallinarum is an important respiratory pathogen of domestic chickens. Avibacterium paragallinarum has been subtyped into three serogroups and nine serovars according to the Page and revised Kume schemes. The major hemagglutinin antigen of A. paragallinarum is HMTp210, which is a large protein of about 2000 amino acids (aa), including a 70-aa signal peptide at its N-terminal end. However, the regions important for the hemagglutination (HA) activity and serotypes of HMTp210 remain unclear. In this study we constructed a series of A. paragallinarum strains expressing HMTp210 in-frame deletion mutants and determined their HA titers to identify the regions important for the HA activity and serotypes of HMTp210. Two distinct types of HA activities were found in HMTp210. The type 1 HA activity resided in the region spanning the full-length HA (aa 71-2084), whereas the type 2 resided in the region spanning aa 1003-2084. The putative ligand binding of the type 1 HA activity was located at aa 176-360, which had a structure similar to YadA of Yersinia enterocolitica. The putative ligand binding site of the type 2 HA activity was located at aa 1003-1125, which had a structure similar to UspA1 from Moraxella catarrhalis. The type 1 HA activity appeared to be Page serogroup specific, whereas type 2 appeared to be Kume serovar specific. Finally, sequence analyses of the regions spanning aa 1-400 and aa 1100-1600 of HMTp210 could be useful for the molecular serotyping (the Page and revised Kume schemes) of A. paragallinarum isolates.

Regiones importantes para la actividad de hemaglutinación y serotipos de la proteína HMTp210 de Avibacterium paragallinarum. La bacteria Avibacterium paragallinarum es un patógeno respiratorio importante de los pollos domésticos. Avibacterium paragallinarum se subtipificó en tres serogrupos y nueve serovares de acuerdo con los esquemas revisados de Page y Kume. El principal antígeno de la hemaglutinina de A. paragallinarum es la proteína HMTp210, que es una proteína grande de unos 2000 aminoácidos (aa), que incluye un péptido señal de 70 aminoácidos en su extremo N-terminal. Sin embargo, las regiones importantes para la actividad de hemaglutinación (HA) y de los serotipos de la proteína HMTp210 siguen sin estar determinados. En este estudio, se construyó una serie de cepas de A. paragallinarum que expresaban mutantes de deleción en marco de lectura de HMTp210 y se determinaron sus títulos de hemaglutinación para identificar las regiones importantes para la actividad de hemaglutinación y de los serotipos de HMTp210. Se encontraron dos tipos distintos de actividades hemaglutinación en la proteína HMTp210. La actividad de hemaglutinación de tipo 1 residía en la región que abarcaba la longitud completa (aminoácidos 71­2084), mientras que la de tipo 2 residía en la región que abarcaba entre los aminoácidos 1003­2084. El sitio supuesto de unión al ligando de la actividad de hemaglutinación tipo 1 se ubicó entre los aminoácidos 176­360, que tenía una estructura similar a la proteína YadA de Yersinia enterocolitica. El supuesto sitio de unión del ligando de la actividad de hemaglutinación tipo 2 se ubicó entre los aminoácidos 1003­1125, que tenía una estructura similar a la proteína UspA1 de Moraxella catarrhalis. La actividad de hemaglutinación tipo 1 parecía ser específica del serogrupo Page, mientras que la hemaglutinación tipo 2 parecía ser específica del serovar Kume. Finalmente, los análisis de secuencias de las regiones que abarcan los aminácidos 1­400 y aminoácidos 1100­1600 de HMTp210 podrían ser útiles para la serotipificación molecular (por el esquema revisado de Page y Kume revisado) de aislamientos de A. paragallinarum.

Molecular characterization of the HMTp210 gene of Avibacterium paragallinarum and the proposition of a new genotyping method as alternative for classical serotyping.

Avibacterium paragallinarum (A. paragallinarum) is the aetiological agent of infectious coryza (IC) in chickens and characterized by acute respiratory distress and severe drop in egg production. Vaccination is important in the control of IC outbreaks and the efficacy of vaccination is dependent on A. paragallinarum serovars included in the vaccine. Classical serotyping of A. paragallinarum is laborious and hampered by poor availability of antigens and antisera. The haemagglutinin, important in classical serotyping, is encoded by the HMTp210 gene. HMTp210 gene analysis has been shown to have potential as alternative to classical serotyping. The aim of the present study was to further investigate the potential of sequence analyses of partial region 1 of the HMTp210 gene, the HMTp210 hypervariable region and the concatenated sequences of both fragments. For this analysis, 123 HMTp210 gene sequences (field isolates, A. paragallinarum serovar reference strains and vaccine strains) were included. Evaluation of serovar references and vaccine strains revealed a need for critical evaluation, especially within Page serovar B and C. Phylogenetic analysis of HMTp210 region 1 resulted in a separation of Page serovar A, B and C strains. Analysis of the HMTp210 HVR alone was not sufficient to discriminate all nine different Kume serovar references. The concatenated sequences of HMTp210 region 1 and HMTp210 HVR resulted in 14 clusters with a high correlation with Page serovar and with the nine currently known Kume serovars and is therefore proposed as a novel genotyping method that could be used as an alternative for classical serotyping of A. paragallinarum.

Basic Characterization of Natural Transformation in Avibacterium paragallinarum.

Avibacterium paragallinarum is the pathogen involved in infectious coryza (IC), an acute infectious upper respiratory disease in chickens. The prevalence of IC has increased in China in recent years. There is a lack of reliable and effective procedures for gene manipulation, which has limited the research on the bacterial genetics and pathogenesis of A. paragallinarum. Natural transformation has been developed as a method of gene manipulation in Pasteurellaceae by the introduction of foreign genes or DNA fragments into bacterial cells, but there has been no report on natural transformation in A. paragallinarum. In this study, we analyzed the existence of homologous genetic factors and competence proteins underlying natural transformation in A. paragallinarum and established a method for transformation in it. Through bioinformatics analysis, we identified 16 homologs of Haemophilus influenzae competence proteins in A. paragallinarum. We found that the uptake signal sequence (USS) was overrepresented in the genome of A. paragallinarum (1,537 to 1,641 copies of the core sequence ACCGCACTT). We then constructed a plasmid, pEA-KU, that carries the USS and a plasmid, pEA-K, without the USS. These plasmids can be transferred via natural transformation into naturally competent strains of A. paragallinarum. Significantly, the plasmid that carries USS showed a higher transformation efficiency. In summary, our results demonstrate that A. paragallinarum has the ability to undergo natural transformation. These findings should prove to be a valuable tool for gene manipulation in A. paragallinarum. IMPORTANCE Natural transformation is an important mechanism for bacteria to acquire exogenous DNA molecules during the process of evolution. Additionally, it can also be used as a method to introduce foreign genes into bacteria under laboratory conditions. Natural transformation does not require equipment such as an electroporation apparatus. It is easy to perform and is similar to gene transfer under natural conditions. However, there have been no reports on natural transformation in Avibacterium paragallinarum. In this study, we analyzed the presence of homologous genetic factors and competence proteins underlying natural transformation in A. paragallinarum. Our results indicate that natural competence could be induced in A. paragallinarum serovars A, B, and C. Furthermore, the method that we established to transform plasmids into naturally competent A. paragallinarum strains was stable and efficient.

Virulence assessment of four Glaesserella parasuis strains isolated in Liaoning province of China.

Glaesserella parasuis (G. parasuis) is a part of the normal upper respiratory microbiota of healthy swine. In many studies, the serovars 1, 4, 5, and 12 of G. parasuis are considered to be highly virulent and its serovars 3, 6, 7, 9, and 11 are considered to be non-virulent. Until now, researchers have found that non-virulent strains of G. parasuis cause an increasing number of diseases. However, little is known concerning why non-virulent strains cause disease with the virulence changes. In present study, four G. parasuis strains were evaluated for their cytotoxicity property, which aims to compare their virulence. The results showed that highly virulent strains XX0306 and CY1201, as well as, non-virulent strains HLD0115 and YK1603 caused a series of pathological changes, increased lactate dehydrogenase (LDH) release, and decreased cell activity. In addition, compared to the control group, both highly and non-virulent strains showed similar trends, demonstrating that the method of classifying the virulence of G. parasuis based on its serovar is worth further deliberation. Hence, we investigated the adhesion capacity and invasion rate of G. parasuis, the results indicated that XX0306 and HLD0115 had the strongest adhesion and invasion ability, which contradicts the classification of the virulence of G. parasuis based on its serovar. The apoptosis degree induced by highly virulent strains was more intensive than non-virulent strains, as measured by annexin V and propidium iodide (PI) double staining. Through testing the expression of apoptosis-related genes Bcl-2 and Bax, we found highly virulent strains induced apoptosis by inhibiting the expression of Bcl-2.

HtrA is involved in stress response and adhesion in Glaesserella parasuis serovar 5 strain Nagasaki.

Glaesserella parasuis is an important pathogen that causes fibrinous polyserositis, peritonitis and meningitis in pigs, leading to considerable economic losses to the swine industry worldwide. It is well established that the serine protease HtrA is closely associated with bacterial virulence, but the role of HtrA in G. parasuis pathogenesis remains largely unknown. To characterize the function of the htrA gene in G. parasuis, a ΔhtrA mutant was constructed. We found that the ΔhtrA mutant showed significant growth inhibition under heat shock and alkaline stress conditions, indicating HtrA is involved in stress tolerance and survival of G. parasuis. In addition, deletion of htrA gene resulted in decreased adherence to PIEC and PK-15 cells and increased phagocytic resistance to 3D4/2 macrophages, suggesting that htrA is essential for adherence of G. parasuis. Scanning electron microscopy revealed morphological surface changes of the ΔhtrA mutant, and transcription analysis confirmed that a number of adhesion-associated genes are downregulated, which corroborated the aforementioned phenomenon. Furthermore, G. parasuis HtrA induced a potent antibody response in piglets with Glässer's disease. These observations confirmed that the htrA gene is related to the survival and pathogenicity of G. parasuis.

Phylogenetic study and comparison of different TbpB obtained from Glaesserella parasuis present in Spanish clinical isolates.

Glaesserella parasuis (Gp) is the etiological agent of Glässer's disease (GD), which causes important economic losses for the pig intensive production worldwide. This organism uses a smart protein-based receptor to acquire specifically iron from the porcine transferrin. This surface receptor consists of transferrin-binding protein A (TbpA) and transferrin-binding protein B (TbpB). TbpB has been considered the most promising antigen to formulate a based-protein vaccine with broad-spectrum of protection against GD. The purpose of our study was to determine the capsular diversity of Gp clinical isolates collected in different Spanish regions between 2018 and 2021. A total of 68 Gp isolates were recovered from porcine respiratory or systemic samples. A species-specific PCR based on tbpA gene, followed by multiplex PCR for typing Gp isolates were performed. Serovars 5, 10, 2, 4 and 1 were the most prevalent and involved almost 84% of isolates. TbpB amino acid sequences from 59 of these isolates were analyzed, and a total of ten clades could be established. All of them showed a wide diversity with respect to capsular type, anatomical isolation site and geographical origin, with minor exceptions. Regardless of the serovars, the in silico analysis of TbpB sequences revealed that a vaccine based on a TbpB recombinant protein could potentially prevent Glässer's disease outbreaks in Spain.

Pili Subunit PilA Contributes to the Cytoadhesion of Glaesserella Parasuis to Host Cells and Provides Immunoprotection.

Glaesserella parasuis (G. parasuis) is commonly located in the upper respiratory tract of pigs as an opportunistic pathogen. It can cause Glässer's disease, which leads to serious economic losses in the swine industry. The occurrence of the disease is often linked with the adhesion and colonization of the pathogen. The PilA pilus subunit is important for adhesion to the host, twitching motility, and biofilm formation in many bacteria. However, no research has focused on the function of PilA in G. parasuis. To further reveal the pathogenesis of G. parasuis and to search for subunit vaccine candidates, we investigated whether PilA could adhere to cells and provide immune protection. A bioinformatic analysis showed that the protein secondary structure of the G. parasuis PilA was similar to that of Haemophilus influenzae (HI). Cell adhesion, ELISA, and far-Western blotting showed that rPilA could bind porcine-derived, porcine kidney-15 (PK-15) cells, swine tracheal epithelial cells (STECs), and the extracellular matrix components fibronectin (FN) and laminin (LN). An immunogenicity analysis showed that recombinant PilA (rPilA) reacted specifically with convalescent and hyperimmune serum. Importantly, purified rPilA elicited a strong immune response and conferred robust protection against challenges with serovar 5 G. parasuis in mice. These results suggested that the PilA protein might help G. parasuis adhere to host cells by binding to FN and LN, and its immunogenicity establishes it as a promising, novel subunit vaccine candidate against infections with G. parasuis. IMPORTANCE G. parasuis is one of the most prevalent bacterial infections in swine production and can lead to huge economic losses around the world. A full understanding of colonization and immunity with G. parasuis infections will be essential in disease control. In this study, the PilA protein, which is a common virulence factor in other bacteria that mediates adherence to the host, was assessed. The results suggested that the PilA protein of G. parasuis can mediate adhesion to host cells through FN and LN, which provides a new idea for the study of the pathogenicity of G. parasuis. Furthermore, fimbriae usually have high immunogenicity. Immunogenicity and protective capacity results showed that the use of this recombinant PilA antigen might be a promising candidate vaccine antigen with which to prevent G. parasuis infections.

Resistin secreted by porcine alveolar macrophages leads to endothelial cell dysfunction during Haemophilus parasuis infection.

Haemophilus parasuis (H. parasuis) causes exudative inflammation, implying endothelial dysfunction during pathogen infection. However, so far, the molecular mechanism of endothelial dysfunction caused by H. parasuis has not been clarified. By using the transwell-based cell co-culture system, we demonstrate that knocking out resistin in porcine alveolar macrophages (PAMs) dramatically attenuated endothelial monolayer damage caused by H. parasuis. The resistin secreted by PAMs inhibited the expression of the tight junction proteins claudin-5 and occludin rather than the adherens junction protein VE-cadherin in co-cultured porcine aortic endothelial cells (PAECs). Furthermore, we demonstrate that resistin regulated claudin-5 and occludin expression and monolayer PAEC permeability in an LKB1/AMPK/mTOR pathway-dependent manner. Additionally, we reveal that the outer membrane lipoprotein gene lppA in H. parasuis induced resistin expression in PAMs, as deleting lppA reduced resistin expression in H. parasuis-infected PAMs, causing a significant change in LKB1/AMPK/mTOR pathway activity in co-cultured PAECs, thereby restoring tight junction protein levels and endothelial monolayer permeability. Thus, we postulate that the H. parasuis lppA gene enhances resistin production in PAMs, disrupting tight junctions in PAECs and causing endothelial barrier dysfunction. These findings elucidate the pathogenic mechanism of exudative inflammation caused by H. parasuis for the first time and provide a more profound angle of acute exudative inflammation caused by bacteria.

TbpBY167A-based vaccine is safe in pregnant sows and induces high titers of maternal derived antibodies that reduce Glaesserella parasuis colonization in piglets.

Glässer's disease is one of the main diseases affecting young piglets, particularly during the nursery phase, that can significantly impact pork production. Vaccination of sows has the potential to prevent Glaesserella parasuis infection during the first weeks of life that is to a substantial degree due to the transfer of maternal derived antibodies (MDA) in colostrum. In this study we compare the antibody response to two vaccines administered to pregnant sows. A subunit vaccine containing the mutant transferrin-binding protein, TbpBY167A, and an autogenous vaccine formulated with the LM96/20 strain of G. parasuis (SV4) administered on days 65 and 86 of the gestational period were safe and induced high titers of antibodies in sows. The IgG peak was reached on day 100 of gestation, and the translocation of IgG to the mammary gland was confirmed in colostrum at the time of delivery. Piglets born from vaccinated sows maintained positive IgG titers against TbpBY167A or G. parasuis SV4 for the duration of the experiment (35 days of life). Piglets born from sows vaccinated with the TbpBY167A-based vaccine had a significantly (p = 0.001) lower load of G. parasuis in the respiratory tract compared to those born from sows vaccinated with the autogenous vaccine. Finally, we demonstrate that the LM96/20 (SV4) strain is highly virulent and a primary agent of Glässer's disease.

Immunogenicity and protection against Glaesserella parasuis serotype 13 infection after vaccination with recombinant protein LolA in mice.

Glaesserella parasuis is a pathogen causing Glässer's disease characterized by fibrinous polyserositis, polyarthritis, and meningitis. Owing to the low cross-immunogenicity of different bacterial antigens in commercial vaccines, finding and identifying effective immunoprotective antigens will facilitate the development of novel subunit vaccines. In this study, LolA, identified by bioinformatics approaches, was cloned and successfully expressed as a recombinant protein in Escherichia coli, and its immunogenicity and protection were evaluated in a mouse model. The results showed that the recombinant protein LolA can stimulate mice to produce high levels of IgG antibodies and confer 50% protection against challenge with the highly virulent G. parasuis CY1201 strain (serotype 13). By testing the cytokine levels of interleukin 4 (IL-4), IL-10, and interferon-γ (IFN-γ), it was found that the recombinant protein LolA can induce both Th1 and Th2 immune responses in mice. These results suggest that the recombinant protein LolA has the potential to serve as an alternative antigen for a novel vaccine to prevent G. parasuis infection.

TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells.

Glaesserella parasuis is a common bacterium in the porcine upper respiratory tract that causes severe Glasser's disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis. TurboID is an enzyme that mediates the biotinylation of endogenous proteins that can fuse with proteins of interest to label protein interactors and local proteomes. To reveal the host proteins that interact with outer membrane protein P2 (OmpP2) by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage iPAM cells, 0.1 and 2.58 mg/mL His-tagged TurboID-OmpP2 and TurboID recombinant proteins were expressed and purified. By mass spectrometry, we identified 948 and 758 iPAM cell proteins that interacted with His-TurboID-OmpP2 and His-TurboID, respectively. After removal of background proteins through comparison with the TurboID-treated group, 240 unique interacting proteins were identified in the TurboID-OmpP2-treated group. Ultimately, only four membrane proteins were identified, CAV1, ARF6, PPP2R1A, and AP2M1, from these 240 host proteins. Our data indicated that CAV1, ARF6, and PPP2R1A could interact with OmpP2 of G. parasuis, as confirmed by coimmunoprecipitation assay. Finally, we found that CAV1, ARF6, and PPP2R1A were involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells by using overexpression and RNA interference assays. This study provides first-hand information regarding the interaction of the iPAM cell proteomes with G. parasuis OmpP2 protein by using the TurboID proximity labeling system and identifies three novel host membrane proteins involved in the recognition and phagocytosis of G. parasuis by iPAM cells. These results provide new insight for a better understanding of Glasser's disease pathogenesis. IMPORTANCE G. parasuis can cause serious Glasser's disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis in pigs. It can cause high morbidity and mortality in swine herds and major economic losses to the global pig industry. Understanding the mechanism of interactions between alveolar macrophages and pathogenic G. parasuis is essential for developing effective vaccines and targeted drugs against G. parasuis. To reveal the host proteins interacting with OmpP2 by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage (iPAM) cells, we identified 240 unique proteins from iPAM cells that could interact with G. parasuis OmpP2. Among them, only four membrane proteins, CAV1, ARF6, PPP2R1A, and AP2M1, were identified, and further study showed that CAV1, ARF6, and PPP2R1A are involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells. This study provides new insight into proteomic interactions between hosts and pathogenic microorganisms.