LRRC8A promotes Glaesserella parasuis cytolethal distending toxin-induced p53-dependent apoptosis in NPTr cells.

Glaesserella parasuis is an early colonizer of the swine upper respiratory tract and can break through the respiratory barrier for further invasion. However, the mechanisms underlying G. parasuis increases epithelial barrier permeability remain unclear. This study demonstrates that G. parasuis cytolethal distending toxin (CDT) induces p53-dependent apoptosis in new-born piglet tracheal (NPTr) cells. Moreover, we report for the first time that leucine-rich repeat-containing protein 8A (LRRC8A), an essential subunit of the volume-regulated anion channel (VRAC), involves in apoptosis of NPTr cells mediated by G. parasuis CDT. Pharmacological inhibition of VRAC with either PPQ-102 or NS3728 largely attenuated CDT-induced apoptosis in NPTr cells. Additionally, experiments with cells knocked down for LRRC8A using small interfering ribonucleic acid (siRNA) or knocked out LRRC8A using CRISPR/Cas9 technology showed a significant reduction in CDT-induced apoptosis. Conversely, re-expression of Sus scrofa LRRC8A in LRRC8A-/- NPTr cells efficiently complemented the CDT-induced apoptosis. In summary, these findings suggest that LRRC8A is pivotal for G. parasuis CDT-induced apoptosis, providing novel insights into the mechanism of apoptosis caused by CDT.

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.

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.

Development and evaluation of an indirect enzyme-linked immunosorbent assay based on a recombinant SifA protein to detect Salmonella infection in poultry.

Salmonella is an important zoonotic pathogen that not only endangers food safety and human health, but also causes considerable economic losses to the poultry industry. Therefore, it is essential to establish a rapid, sensitive, and specific diagnostic method for the early detection of Salmonella infection in poultry. In this study, we developed a novel enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Salmonella antibodies using a recombinant SifA protein. Amino acid sequence comparison revealed that SifA is a relatively conserved secretory protein across Salmonella serotypes. Therefore, we hypothesized that SifA can serve as a detection antigen for diagnostic testing. The SifA protein was expressed in Escherichia coli and used as a coating antigen to establish an SifA-ELISA. Control sera from specific-pathogen-free (SPF) chickens infected with Salmonella or several other non-Salmonella pathogens were then tested using the SifA-ELISA. Specificity testing demonstrated that the SifA-ELISA could detect antibodies against 3 different serotypes of Salmonella, whereas antibodies against other non-Salmonella pathogens could not be detected. Compared to the SifA-ELISA, the Salmonella plate agglutination test (PAT) failed to detect antibodies in serum samples from chickens infected with Salmonella Typhimurium. This result suggests that our SifA-ELISA may be better than PAT at detecting Salmonella infection. Comparing clinical sera, we observed a similar rate of Salmonella positivity between SifA-ELISA and PAT (92.6%). In addition, anti-SifA antibodies were continuously detected during Salmonella infection of SPF chickens, demonstrating that SifA-ELISA could consistently detect high levels of antibodies for at least 8 wk. Furthermore, the intra-assay and interassay coefficients of variation (CV) of the SifA-ELISA were below 10%, which is considered acceptable. In summary, the SifA-ELISA established here is a promising and reliable method for detection of anti-Salmonella antibodies in poultry and may contribute to the early diagnosis of Salmonella infection.

Generation of markerless and multiple-gene knockout in Glaesserella parasuis based on natural transformation and Flp recombinase.

Glaesserella parasuis is an important bacterial pathogen that affects the swine industry worldwide. Research on the pathogenic mechanism and genetically engineered vaccine remains undeveloped because an effective markerless and multiple-gene knockout system is unavailable for G. parasuis yet. To establish a markerless knockout, deleted allelic genes with kanamycin resistance (KanR) cassettes were introduced into the genome of G. parasuis by using natural transformation with suicide plasmids. Then, the KanR cassette was excised with a thermosensitive plasmid pGF conferring a constitutive Flp expression. To realize the markerless and multiple-gene knockout, plasmid pGAF was constructed by placing the Flp gene under the control of an arabinose-inducible promoter. Firstly, pGAF was introduced into G. parasuis by electroporation, and the marked mutants were produced following natural transformation. Finally, the KanR cassette was excised from the genome by the inducible expression of Flp upon arabinose action. Based on the natural transformation and the inducible expression of Flp, the markerless single-gene knockout mutants of ΔhsdR, ΔneuA2, ΔespP2, Δapd, and ΔnanH were constructed. In addition, a five-gene knockout mutant of ΔhsdRΔneuA2ΔespP2ΔapdΔnanH was generated by successive natural transformation with five suicide plasmids. Taken together, a markerless and multiple-gene deletion system was established for G. parasuis in the present study for the first time. This system is simple, efficient, and easy to manipulate for G. parasuis; thus, our technique will substantially aid the understanding of the etiology, pathogenesis, and genetic engineering of G. parasuis and other bacteria that can be naturally transformed in laboratory conditions. KEY POINTS: • Flp recombinase excised the KanR gene flanked by FRT sites in Glaesserella parasuis. • The regulatory expression of Flp enabled a multiple-gene knockout forG. parasuis. • The technique will promote the understanding of Glässer's disease pathogens.

Campylobacter jejuni Cytolethal Distending Toxin Induces GSDME-Dependent Pyroptosis in Colonic Epithelial Cells.

Background: Cytolethal distending toxin (CDT) is a critical virulence factor of Campylobacter jejuni, and it induces cell death and regulates inflammation response in human epithelial cells. Pyroptosis is an inflammatory form of programmed cell death (PCD), but whether it is involved in CDT-mediated cytotoxicity remains elusive. Aims: This study explores the role and mechanism of pyroptosis in CDT-mediated cytotoxicity. Methods: HCT116 and FHC cell lines were treated with CDT. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability. Western blotting was used to measure the expression of related proteins in the pathway, and cell morphology observation, annexin V/propidium iodide (PI) staining and lactate dehydrogenase (LDH) release assay were performed to evaluate the occurrence of pyroptosis. Result: Our results show that C. jejuni CDT effectively induces pyroptosis in a dose- and time- dependent manner in human colonic epithelial cells owing to its DNase activity. Specific pyroptotic features including large bubbles emerging from plasma membrane and LDH release were observed upon CDT treatment. Moreover, CDT-induced pyroptosis involves the caspase-9/caspase-3 axis, which is followed by gasdermin E (GSDME) cleavage rather than gasdermin D (GSDMD). N-acetyl cysteine (NAC), a reactive oxygen species (ROS) inhibitor, attenuates the activation of caspase-9/3, the cleavage of GSDME and pyroptotic characteristic, therefore demonstrating ROS initiates pyroptotic signaling. Conclusions: We first clarify a molecular mechanism that CDT induces pyroptosis via ROS/caspase-9/caspase-3/GSDME signaling. These findings provide a new insight on understanding of CDT-induced pathogenesis at the molecular level.

Glaesserella parasuis autotransporters EspP1 and EspP2 are novel IgA-specific proteases.

Background: Glaesserella parasuis causes Glässer's disease, which is associated with severe polyarthritis, fibrinous polyserositis and meningitis, and leads to significant economic losses to the swine industry worldwide. IgA is one of the most important humoral immune factors present on mucosal surfaces, and it plays a crucial role in neutralizing and removing pathogens. G. parasuis is able to colonize the mucosal membrane of respiratory tract without being eliminated. Nevertheless, the immune evasion mechanism of G. parasuis in thwarting IgA remains unclear. Aims: The object of this study is to characterize the IgA degradation activity of Mac-1-containing autotransporter EspP1 and EspP2 from G. parasuis. Methods: The swine IgA was purified and incubated with EspP1 and EspP2 respectively. Western blotting was used to detect the cleavage of swine IgA. Generation of EspP1 and EspP2 mutant protein were used to explore the putative active sites of EspPs. LC-MS/MS based N/C-terminal sequencing was performed to measure the cleavage sites in swine IgA. Result: Our results show that G. parasuis EspP1 and EspP2 cleave swine IgA in a dose- and time- dependent manner. G. parasuis lose the IgA protease activity after simultaneously delete espP1 and espP2 indicating that EspP1 and EspP2 are the only two IgA proteases in G. parasuis. The IgA protease activity of EspP1 and EspP2 is affected by the putative active sites which contain Cys47, His172 and Asp194/195. Swine IgA is cleaved within Cα1 and Cα3 domains upon incubation with EspPs. Moreover, EspPs can degrade neither IgG nor IgM while G. parasuis possess the ability to degrade IgM unexpectedly. It suggests that G. parasuis can secrete other proteases to cleave IgM which have never been reported. Conclusion: We report for the first time that both EspP1 and EspP2 are novel IgA-specific proteases and cleave swine IgA within the Cα1 and Cα3 domains. These findings provide a theoretical basis for the EspPs-induced immune evasion.

A previously uncharacterized two-component signaling system in uropathogenic Escherichia coli coordinates protection against host-derived oxidative stress with activation of hemolysin-mediated host cell pyroptosis.

Uropathogenic Escherichia coli (UPEC) deploy an array of virulence factors to successfully establish urinary tract infections. Hemolysin is a pore-forming toxin, and its expression correlates with the severity of UPEC infection. Two-component signaling systems (TCSs) are a major mechanism by which bacteria sense environmental cues and respond by initiating adaptive responses. Here, we began this study by characterizing a novel TCS (C3564/C3565, herein renamed orhK/orhR for oxidative resistance and hemolysis kinase/regulator) that is encoded on a UPEC pathogenicity island, using bioinformatic and biochemical approaches. A prevalence analysis indicates that orhK/orhR is highly associated with the UPEC pathotype, and it rarely occurs in other E. coli pathotypes tested. We then demonstrated that OrhK/OrhR directly activates the expression of a putative methionine sulfoxide reductase system (C3566/C3567) and hemolysin (HlyA) in response to host-derived hydrogen peroxide (H2O2) exposure. OrhK/OrhR increases UPEC resistance to H2O2 in vitro and survival in macrophages in cell culture via C3566/C3567. Additionally, OrhK/OrhR mediates hemolysin-induced renal epithelial cell and macrophage death via a pyroptosis pathway. Reducing intracellular H2O2 production by a chemical inhibitor impaired OrhK/OrhR-mediated activation of c3566-c3567 and hlyA. We also uncovered that UPEC links the two key virulence traits by cotranscribing the c3566-c3567 and hlyCABD operons. Taken together, our data suggest a paradigm in which a signal transduction system coordinates both bacterial pathogen defensive and offensive traits in the presence of host-derived signals; and this exquisite mechanism likely contributes to hemolysin-induced severe pathological outcomes.

Bis-molybdopterin guanine dinucleotide modulates hemolysin expression under anaerobiosis and contributes to fitness in vivo in uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs). Successful urinary tract colonization requires appropriate expression of virulence factors in response to host environmental cues, such as limited oxygen and iron availability. Hemolysin is a pore-forming toxin, and its expression correlates with the severity of UPEC infection. Previously, we showed that hemolysin expression is enhanced under anaerobic conditions; however, the genetic basis and regulatory mechanisms involved remain undefined. Here, a transposon-based forward screen identified bis-molybdopterin guanine dinucleotide cofactor (bis-MGD) biosynthesis as an important factor for a full transcription of hemolysin under anaerobiosis but not under aerobiosis. bis-MGD positively influences hemolysin transcription via c3566-c3568, an operon immediately upstream of and cotranscribed with hlyCABD. Furthermore, suppressor mutation analysis identified the nitrogen regulator NtrC as a direct repressor of c3566-c3568-hlyCABD expression, and intact bis-MGD biosynthesis downregulated ntrC expression, thus at least partially explaining the positive role of bis-MGD in modulating hemolysin expression. Finally, bis-MGD is involved in hemolysin-mediated uroepithelial cell death and contributes to the competitive fitness of UPEC in a murine model of UTI. Collectively, our data establish that bis-MGD biosynthesis plays a crucial role in UPEC fitness in vivo, thus providing a potential target for combatting UTIs.

Sialidase of Glaesserella parasuis Augments Inflammatory Response via Desialylation and Abrogation of Negative Regulation of Siglec-5.

Siglecs are sialic acid-binding immunoglobulin-like lectins that play an important role in tissue homeostasis, immune response, and pathogen infection. Bacterial sialidases act on natural ligands of Siglecs, interfering with the Siglec-mediated immune response. Glaesserella parasuis is a porcine bacterial pathogen that secretes sialidase. However, little is known about the sialidase of G. parasuis and its impact on immune regulation. Here, we used wild-type G. parasuis, a sialidase-deficient mutant, and complementary strains to investigate the role of sialidase in porcine alveolar macrophage infection. Sialidase induced the release of proinflammatory cytokines, such as interleukin-1α (IL-1α), IL-6, and tumor necrosis factor alpha, from porcine alveolar macrophages. Moreover, sialidase desialylated the surface of porcine alveolar macrophages and altered the expression of Siglecs (the expression of Siglec-5 was reduced). Furthermore, sialidase led to a reduction in endogenous SH2 domain-containing protein tyrosine phosphatase (SHP-2) recruitment to Siglec-5 and simultaneously activated the inflammatory response via the mitogen-activated protein kinase and nuclear factor kappa light chain enhancer of activated B cell signaling pathways. This desialylation occurred before the release of proinflammatory cytokines, suggesting that the sialidase-induced inflammatory response was followed by reduced recruitment of SHP-2 to Siglec-5. Thus, this study is the first to demonstrate the role of sialidase in the inflammatory response of G. parasuis. This role resulted from the abrogation of negative regulation of Siglec-5 on proinflammatory cytokine release. This study helps to understand the molecular mechanism underlying the inflammatory response induced by sialidase secreted by G. parasuis and the acute inflammation caused by G. parasuis.

Ct value-based real time PCR serotyping of Glaesserella parasuis.

Glaesserella parasuis is the causative agent of Glässer's disease in swine. Serotyping plays an essential role in prevalence investigations and in the development of vaccination strategies for the prevention of this disease. Molecular serotyping based on variation within the capsule loci of the 15 serovars is more accurate and efficient than traditional serological serotyping. To reduce the running time and facilitate ease of data interpretation, we developed a simple and rapid cycle threshold (Ct) value-based real time PCR (qPCR) method for the identification and serotyping of G. parasuis. The qPCR method distinguished between all 15 serovar reference strains of G. parasuis with efficiency values ranging between 85.5 % and 110.4 % and, R2 values > 0.98. The qPCR serotyping was evaluated using 83 clinical isolates with 43 of the isolates having been previously assigned to a serovar by the gel immuno-diffusion (GID) assay and 40 non-typeable isolates. The qPCR results of 41/43 (95.3 %) isolates were concordant with the GID assay except two isolates of serovar 12 were assigned to serovar 5. In addition, the qPCR serotyping assigned a serovar to each of the 40 non-typeable isolates. Of the 83 isolates tested to assign a serovar, a concordance rate of 98.8 % (82/83) was determined between the qPCR and the previously reported multiplex PCR of Howell et al. (2015) (including those that were either serovars 5 or 12). Despite the inability to differentiate between serovars 5 and 12, the Ct value-based qPCR serotyping represents an attractive alternative to current molecular serotyping method for G. parasuis and could be used for both epidemiological monitoring and the guidance of vaccination programs.

Identification of Haemophilus parasuis genes uniquely expressed during infection using in vivo-induced antigen technology.

Haemophilus parasuis is the etiological agent of Glässer's disease which is characterized by fibrinous polyserositis, arthritis and meningitis. The pathogenesis of this bacterium remains largely unknown. Genes expressed in vivo may play an important role in the pathogenicity of H. parasuis. The development of in vivo-induced antigen technology (IVIAT) has provided a valuable tool for the identification of in vivo-induced genes during bacterial infection. In this study, IVIAT was applied to identify in vivo-induced antigens of H. parasuis. Pooled swine H. parasuis-positive sera, adsorbed against in vitro-grown cultures of H. parasuis SH0165 and Escherichia coli BL21 (DE3), were used to screen the inducible expression library of genomic proteins from whole genome sequenced H. parsuis SH0165. Finally, 24 unique genes expressed in vivo were successfully identified after secondary and tertiary screening with IVIAT. These genes were implicated in cell surface proteins, metabolism, stress response, regulation, transportation and other processes. Quantitative real-time PCR showed that the mRNA levels of 24 genes were all upregulated in vivo relative to in vitro, with 13 genes were detected significantly upregulated in H. parasuis infected pigs. Several potential virulence-associated genes were found to be uniquely expressed in vivo, including espP, lnt, hutZ, mreC, vtaA, pilB, tex, sunT and aidA. The results indicated that the proteins identified using IVIAT may play important roles in the pathogenesis of H. parasuis infection in vivo.

Development and application of an antibody detection ELISA for Haemophilus parasuis based on a monomeric autotransporter passenger domain.

BACKGROUND: Haemophilus parasuis is a commensal pathogen in the swine upper respiratory tract and causes Glässer's disease. Surveillance, screening for infection, and vaccination response of H. parasuis is hindered by the lack of a rapid antibody detection method. RESULTS: In the present study, a monomeric autotransporter was identified as a novel antigen for developing an indirect ELISA. The autotransporter passenger domain (Apd) was expressed, purified, and demonstrated to be specific in ELISA and western blotting. Mouse antiserum of recombinant Apd (rApd) recognized native Apd in the 15 serotype reference strains and five non-typeable isolate stains, but showed no reaction with seven other bacterial pathogens. The rApd ELISA was optimized and validated using 67 serum samples with known background, including 27 positive sera from experimentally infected and vaccinated pigs along with 40 negative sera that had been screened with H. parasuis whole cell ELISA from clinically healthy herds. The rApd ELISA provided positive and negative percent agreements of 96.4 and 94.9%, respectively, and an AUC value of 0.961, indicating that the assay produced accurate results. CONCLUSION: Apd was a universal antigen component among 15 serotype and non-typeable strains of H. parasuis and was also specific to this pathogen. The rApd ELISA could detect antibodies elicited by H. parasuis infection and vaccination, thereby exhibiting the potential to be applied for Glässer's disease diagnosis, H. parasuis vaccination evaluation, and large-scale serological surveillance.

Porcine Alveolar Macrophages' Nitric Oxide Synthase-Mediated Generation of Nitric Oxide Exerts Important Defensive Effects against Glaesserella parasuis Infection.

Glaesserella parasuis is a habitual bacterium of pigs' upper respiratory tracts. Its infection initiates with the invasion and colonization of the lower respiratory tracts of pigs, and develops as the bacteria survive host pulmonary defenses and clearance by alveolar macrophages. Alveolar macrophage-derived nitric oxide (NO) is recognized as an important mediator that exerts antimicrobial activity as well as immunomodulatory effects. In this study, we investigated the effects and the signaling pathway of NO generation in porcine alveolar macrophages 3D4/21 during G. parasuis infection. We demonstrated a time and dose-dependent generation of NO in 3D4/21 cells by G. parasuis, and showed that NO production required bacterial viability and nitric oxide synthase 2 upregulation, which was largely contributed by G. parasuis-induced nuclear factor-κB signaling's activation. Moreover, the porcine alveolar macrophage-derived NO exhibited prominent bacteriostatic effects against G. parasuis and positive host immunomodulation effects by inducing the production of cytokines and chemokines during infection. G. parasuis in turn, selectively upregulated several nitrate reductase genes to better survive this NO stress, revealing a battle of wits during the bacteria-host interactions. To our knowledge, this is the first direct demonstration of NO production and its anti-infection effects in alveolar macrophages with G. parasuis infection.

Uropathogenic Escherichia coli preferentially utilize metabolites in urine for nucleotide biosynthesis through salvage pathways.

Growth in urinary tract depends on the ability of uropathogenic E. coli to adjust metabolism in response to available nutrients, especially to synthesize metabolites that are present in urinary tract with limited concentrations. In this study, a genome-wide assay was applied and identified five nucleotide biosynthetic genes purA, guaAB and carAB that are required for optimal growth of UPEC in human urine and colonization in vivo. Subsequent functional analyses revealed that either interruption of de novo nucleotide biosynthesis or blocking of salvage pathways alone could not decrease UPEC's growth, while only simultaneous interruption of both two pathways significantly reduced UPEC's growth in urine. Evidences showed that uracil, xanthine, and hypoxanthine in human urine could support nucleotide biosynthesis through salvage pathways when the de novo pathways were interrupted. Moreover, the expression of genes involved in salvage pathways of nucleotide biosynthesis were significantly upregulated when UPEC are cultured in human urine and artificial urine medium with uracil, xanthine or hypoxanthine. Finally, animal tests showed that further deletion of genes involved in salvage nucleotide biosynthesis from mutants with defects in de novo pathways significantly reduced UPEC's colonization in host bladders and kidneys. These results indicated that UPEC preferentially utilize abundant metabolites in urine for nucleotide biosynthesis through salvage pathways, which is not like in serum, where the limiting amounts of substrates for salvage biosynthesis force invading pathogens to rely on de novo nucleotide biosynthesis. Taken together, our study implied the importance of salvage pathways of nucleotides biosynthesis for UPEC's fitness during urinary tract infection.

Haemophilus parasuis α-2,3-sialyltransferase-mediated lipooligosaccharide sialylation contributes to bacterial pathogenicity.

Bacterial lipooligosaccharide (LOS) is an important virulence-associated factor, and its sialylation largely confers its ability to mediate cell adhesion, invasion, inflammation, and immune evasion. Here, we investigated the function of the Haemophilus parasuis α-2,3-sialyltransferase gene, lsgB, which determines the terminal sialylation of LOS, by generating a lsgB deletion mutant as well as a complementation strain. Our data indicate a direct effect of lsgB on LOS sialylation and reveal important roles of lsgB in promoting the pathogenicity of H. parasuis, including adhesion to and invasion of porcine cells in vitro, bacterial load and survival in vivo, as well as a contribution to serum resistance. These observations highlight the function of lsgB in mediating LOS sialylation and more importantly its role in H. parasuis infection. These findings provide a more profound understanding of the pathogenic mechanism of this disease-causing bacterium.

Improvement in the efficiency of natural transformation of Haemophilus parasuis by shuttle-plasmid methylation.

Some Haemophilus parasuis strains display resistance to transformation with Escherichia.coli-derived plasmids. This property limits the application of genetic approaches previously developed for H. parasuis. The present study showed that natural transformation with the shuttle plasmid pS2UK led to allelic exchange in H. parasuis strains SH0165 and CF7066. Furthermore, natural transformation with pS2UK yielded allelic exchange mutants in 10 of 17 H. parasuis strains, similar to results using the suicide plasmid pK2UK. Subsequently, 17 H. parasuis strains were transformed with pS2UK by electroporation and 13 obtained the transformants harboring the complete plasmid molecules. As a result, natural transformation of homologous blank strains with the H. parasui-derived plasmids significantly improved the transformation efficiency targeted at obtaining allelic exchange mutants. In addition, shuttle plasmids pS1UG and pSHUK that carried the different homologous arm sequences also displayed the increased transformation efficiency after they were replicated in homologous H. parasuis cells. The approach described here not only improved the efficiency of natural transformation of H. parasuis, but also enlarged the range of transformable H. parasuis strains, thereby enabling application of H. parasuis-specific genetic manipulation techniques in a wider range of isolates.

Haemophilus parasuis CpxRA two-component system confers bacterial tolerance to environmental stresses and macrolide resistance.

Haemophilus parasuis is an opportunistic pathogen localized in the upper respiratory tracts of pigs, its infection begins from bacterial survival under complex conditions, like hyperosmosis, oxidative stress, phagocytosis, and sometimes antibiotics as well. The two-component signal transduction (TCST) system serves as a common stimulus-response mechanism that allows microbes to sense and respond to diverse environmental conditions via a series of phosphorylation reactions. In this study, we investigated the role of TCST system CpxRA in H. parasuis in response to different environmental stimuli by constructing the ΔcpxA and ΔcpxR single deletion mutants as well as the ΔcpxRA double deletion mutant from H. parasuis serotype 4 isolate JS0135. We demonstrated that H. parasuis TCST system CpxRA confers bacterial tolerance to stresses and bactericidal antibiotics. The CpxR was found to play essential roles in mediating oxidative stress, osmotic stresses and alkaline pH stress tolerance, as well as macrolide resistance (i.e. erythromycin), but the CpxA deletion did not decrease bacterial resistance to abovementioned stresses. Moreover, we found via RT-qPCR approach that HAPS_RS00160 and HAPS_RS09425, both encoding multidrug efflux pumps, were significantly decreased in erythromycin challenged ΔcpxR and ΔcpxRA mutants compared with wild-type strain JS0135. These findings characterize the role of the TCST system CpxRA in H. parasuis conferring stress response tolerance and bactericidal resistance, which will deepen our understanding of the pathogenic mechanism in H. parasuis.

Transcriptional Control of Dual Transporters Involved in α-Ketoglutarate Utilization Reveals Their Distinct Roles in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) are the primary causative agents of urinary tract infections. Some UPEC isolates are able to infect renal proximal tubule cells, and can potentially cause pyelonephritis. We have previously shown that to fulfill their physiological roles renal proximal tubule cells accumulate high concentrations of α-ketoglutarate (KG) and that gene cluster c5032-c5039 contribute to anaerobic utilization of KG by UPEC str. CFT073, thereby promoting its in vivo fitness. Given the importance of utilizing KG for UPEC, this study is designed to investigate the roles of two transporters KgtP and C5038 in KG utilization, their transcriptional regulation, and their contributions to UPEC fitness in vivo. Our phylogenetic analyses support that kgtP is a widely conserved locus in commensal and pathogenic E. coli, while UPEC-associated c5038 was acquired through horizontal gene transfer. Global anaerobic transcriptional regulators Fumarate and nitrate reduction (FNR) and ArcA induced c5038 expression in anaerobiosis, and C5038 played a major role in anaerobic growth on KG. KgtP was required for aerobic growth on KG, and its expression was repressed by FNR and ArcA under anaerobic conditions. Analyses of FNR and ArcA binding sites and results of EMS assays suggest that FNR and ArcA likely inhibit kgtP expression through binding to the -35 region of kgtP promoter and occluding the occupancy of RNA polymerases. Gene c5038 can be specifically induced by KG, whereas the expression of kgtP does not respond to KG, yet can be stimulated during growth on glycerol. In addition, c5038 and kgtP expression were further shown to be controlled by different alternative sigma factors RpoN and RpoS, respectively. Furthermore, dual-strain competition assays in a murine model showed that c5038 mutant but not kgtP mutant was outcompeted by the wild-type strain during the colonization of murine bladders and kidneys, highlighting the importance of C5038 under in vivo conditions. Therefore, different transcriptional regulation led to distinct roles played by C5038 and KgtP in KG utilization and fitness in vivo. This study thus potentially expanded our understanding of UPEC pathobiology.

ClpP participates in stress tolerance and negatively regulates biofilm formation in Haemophilus parasuis.

Haemophilus parasuis is generally considered a commensal organism in the upper respiratory tract of pigs, where it evades the host immune system and survives the challenging host environment. In response to various host stresses, H. parasuis strains can adapt to the adverse conditions. However, the specific bacterial factors that participate in this process are poorly understood. Here, we investigated the role of ClpP in H. parasuis virulent strain CF7066 by generating a clpP deletion mutant (ΔclpP), as well as a complemented strain C-clpP. Our findings supported that ClpP is essential for stress tolerance of H. parasuis, by the demonstrations that the ΔclpP mutant showed decreased resistance to heat, oxidation, and osmotic pressure. Notably, we observed increased autoagglutination and biofilm formation in the ΔclpP mutant and the amount of polysaccharides and extracellular proteins, which are the main components of biofilm, were much higher in the ΔclpP mutant than the wild-type strain. Real-time PCR demonstrated that the transcriptional regulators csrA and rpoD, and a possible biofilm repressor luxS were significantly downregulated upon clpP deletion. Together, these observations suggest that ClpP plays an essential role in stress tolerance, and negatively regulates biofilm formation in H. parasuis.