Rhein kills Actinobacillus pleuropneumoniae, reduces biofilm formation, and effectively treats bacterial lung infections in mice.

Background. Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae family, is known for its highly infectious nature and is the primary causative agent of infectious pleuropneumonia in pigs. This disease poses a considerable threat to the global pig industry and leads to substantial economic losses due to reduced productivity, increased mortality rates, and the need for extensive veterinary care and treatment. Due to the emergence of multi-drug-resistant strains, Chinese herbal medicine is considered one of the best alternatives to antibiotics due to its unique mechanism of action and other properties. As a type of Chinese herbal medicine, Rhein has the advantages of a wide antibacterial spectrum and is less likely to develop drug resistance, which can perfectly solve the limitations of current antibacterial treatments.Methods. The killing effect of Rhein on A. pleuropneumoniae was detected by fluorescence quantification of differential expression changes of key genes, and scanning electron microscopy was used to observe the changes in A. pleuropneumoniae status after Rhein treatment. Establishing a mouse model to observe the treatment of Rhein after A. pleuropneumoniae infection.Results. Here, in this study, we found that Rhein had a good killing effect on A. pleuropneumoniae and that the MIC was 25 µg ml-1. After 3 h of action, Rhein (4×MIC) completely kills A. pleuropneumoniae and Rhein has good stability. In addition, the treatment with Rhein (1×MIC) significantly reduced the formation of bacterial biofilms. Therapeutic evaluation in a murine model showed that Rhein protects mice from A. pleuropneumoniae and relieves lung inflammation. Quantitative RT-PCR (Quantitative reverse transcription polymerase chain reaction is a molecular biology technique that combines both reverse transcription and polymerase chain reaction methods to quantitatively detect the amount of a specific RNA molecule) results showed that Rhein treatment significantly downregulated the expression of the IL-18 (Interleukin refers to a class of cytokines produced by white blood cells), TNF-α, p65 and p38 genes. Along with the downregulation of genes such as IL-18, it means that Rhein has an inhibitory effect on the expression of these genes, thereby reducing the activation of inflammatory cells and the production of inflammatory mediators. This helps reduce inflammation and protects tissue from further damage.Conclusions. This study reports the activity of Rhein against A. pleuropneumoniae and its mechanism, and reveals the ability of Rhein to treat A. pleuropneumoniae infection in mice, laying the foundation for the development of new drugs for bacterial infections.

Antimicrobial susceptibility and resistome of Actinobacillus pleuropneumoniae in Taiwan: a next-generation sequencing analysis.

Actinobacillus pleuropneumoniae infection causes a high mortality rate in porcine animals. Antimicrobial resistance poses global threats to public health. The current study aimed to determine the antimicrobial susceptibilities and probe the resistome of A. pleuropneumoniae in Taiwan. Herein, 133 isolates were retrospectively collected; upon initial screening, 38 samples were subjected to next-generation sequencing (NGS). Over the period 2017-2022, the lowest frequencies of resistant isolates were found for ceftiofur, cephalexin, cephalothin, and enrofloxacin, while the highest frequencies of resistant isolates were found for oxytetracycline, streptomycin, doxycycline, ampicillin, amoxicillin, kanamycin, and florfenicol. Furthermore, most isolates (71.4%) showed multiple drug resistance. NGS-based resistome analysis revealed aminoglycoside- and tetracycline-related genes at the highest prevalence, followed by genes related to beta-lactam, sulfamethoxazole, florphenicol, and macrolide. A plasmid replicon (repUS47) and insertion sequences (IS10R and ISVAp11) were identified in resistant isolates. Notably, the multiple resistance roles of the insertion sequence IS10R were widely proposed in human medicine; however, this is the first time IS10R has been reported in veterinary medicine. Concordance analysis revealed a high consistency of phenotypic and genotypic susceptibility to florphenicol, tilmicosin, doxycycline, and oxytetracycline. The current study reports the antimicrobial characterization of A. pleuropneumoniae for the first time in Taiwan using NGS.

A kinetic model reveals the critical gating motifs for donor-substrate loading into Actinobacillus pleuropneumoniae N-glycosyltransferase.

Soluble N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) catalyzes the glycosylation of asparagine residues, and represents one of the most encouraging biocatalysts for N-glycoprotein production. Since the sugar tolerance of ApNGT is restricted to limited monosaccharides (e.g., Glc, GlcN, Gal, Xyl, and Man), tremendous efforts are devoted to expanding the substrate scope of ApNGT via enzyme engineering. However, rational design of novel NGT variants suffers from an elusive understanding of the substrate-binding process from a dynamic point of view. Here, by employing extensive all-atom molecular dynamics (MD) simulations integrated with a kinetic model, we reveal, at the atomic level, the complete donor-substrate binding process from the bulk solvent to the ApNGT active-site, and the key intermediate states of UDP-Glc during its loading dynamics. We are able to determine the critical transition event that limits the overall binding rate, which guides us to pinpoint the key ApNGT residues dictating the donor-substrate entry. The functional roles of several identified gating residues were evaluated through site-directed mutagenesis and enzymatic assays. Two single-point mutations, N471A and S496A, could profoundly enhance the catalytic activity of ApNGT. Our work provides deep mechanistic insights into the structural dynamics of the donor-substrate loading process for ApNGT, which sets a rational basis for design of novel NGT variants with desired substrate specificity.

Biological characteristics of mechanosensitive channels MscS and MscL in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

A Florfenicol-Resistant Plasmid Shuttling Between Actinobacillus pleuropneumoniae and Glaesserella parasuis.

Porcine contagious pleuropneumonia, caused by Actinobacillus pleuropneumoniae, has resulted in significant economic losses to the swine industry. Although antibiotics are commonly employed to control this disease, their widespread use or misuse can lead to the development of antibiotic resistance in A. pleuropneumoniae. Consequently, it is crucial to conduct antimicrobial susceptibility testing on clinical isolates. In our study, we identified one strain of A. pleuropneumoniae with resistance to florfenicol and extracted a 5919 bp plasmid named pAPPJY, which confers florfenicol resistance. Sequence analysis revealed that the plasmid contains four open reading frames, namely rep, antioxin vbha family protein, floR, and a partial copy of lysr. Although a few variations in gene position were observed, the plasmid sequence exhibits a high degree of similarity to other florfenicol-resistant plasmids found in Glaesserella parasuis and A. pleuropneumoniae. Therefore, it is possible that the pAPPJY plasmid functions as a shuttle, facilitating the spread of florfenicol resistance between G. parasuis and A. pleuropneumoniae. In addition, partial recombination may occur during bacterial propagation. In conclusion, this study highlights the horizontal transmission of antibiotic resistance among different bacterial species through plasmids, underscoring the need for increased attention to antibiotic usage.

Discovery of a Novel Integrative Conjugative Element ICEAplChn2 Related to SXT/R391 in Actinobacillus pleuropneumoniae.

Objective: The objective of this study was to characterize ICEAplChn2, a novel SXT/R391-related integration and conjugation element (ICE) carrying 19 drug resistance genes, in a clinical isolate of Actinobacillus pleuropneumoniae from swine. Methods: Whole genome sequencing (WGS) of A. pleuropneumoniae CP063424 strain was completed using a combination of third-generation PacBio and second-generation Illumina. The putative ICE was predicted by the online tool ICEfinder. ICEAplChn2 was analyzed by PCR, conjugation experiments, and bioinformatics tools. Results: A. pleuropneumoniae CP063424 strain exhibited high minimum inhibitory concentrations of clindamycin (1,024 mg/L). The WGS data revealed that ICEAplChn2, with a length of 167,870 bp and encoding 151 genes, including multiple antibiotic resistance genes such as erm(42), VanE, LpxC, dfrA1, golS, aadA3, EreA, dfrA32, tetR(C), tet(C), sul2, aph(3)″-lb, aph(6)-l, floR, dfrA, ANT(3″)-IIa, catB11, and VanRE, was found to be related to the SXT/R391 family on the chromosome of A. pleuronipneumoniae CP063424. The circular intermediate of ICEAplChn2 was detected by PCR, but conjugation experiments showed that it was not self-transmissible. Conclusions: To our knowledge, ICEAplChn2 is the longest member with the most resistance genes in the SXT/R391 family. Meanwhile, ATP-binding cassette superfamily was found to be inserted in the ICEAplChn2 and possessed a new insertion region, which is the first description in the SXT/R391 family.

Native ApxIIA secreted by Actinobacillus pleuropneumoniae induces apoptosis in porcine alveolar macrophages dependent on concentration and acylation.

Actinobacillus pleuropneumoniae is an important swine respiratory pathogen causing substantial economic losses to the global pig industry. The Apx toxins of A. pleuropneumoniae belong to the RTX toxin family and are major virulence factors. In addition to hemolysis and/or cytotoxicity via pore-forming activity, RTX toxins, such as ApxIA of A. pleuropneumoniae, have been reported to cause other effects on target cells, e.g., apoptosis. A. pleuropneumoniae ApxIIA is expressed by most serotypes and has moderate hemolytic and cytotoxic activities. In this study, porcine alveolar macrophages (3D4/21) were stimulated with different concentrations of purified native ApxIIA from the serotype 7 strain AP76 which only secretes ApxIIA. By observation of nuclear condensation via fluorescent staining and detection of apoptosis and necrosis by flow cytometry, it was found that high and low concentrations of native ApxIIA mainly caused necrosis or apoptosis of 3D4/21 cells, respectively. ApxIIA purified from an AP76 mutant with a deleted acetyltransferase gene (apxIIC) did not induce necrosis nor apoptosis. Western blot analysis using specific antibodies showed that a cleaved caspase 3 and activated capase 9 was detected after treatment of cells with a low concentration of native ApxIIA, while general or specific inhibitors of caspase 3, 8, 9 blocked these effects. ApxIIA-induced apoptosis of macrophages may be a mechanism of A. pleuropneumoniae to escape host immune clearance.

Characterization of Actinobacillus pleuropneumoniae isolated from pigs in Japan using whole genome sequencing.

We conducted whole-genome sequencing to investigate the serotypes, the presence of virulence and antimicrobial resistance genes, and the genetic relationships among isolates of Actinobacillus. pleuropneumoniae derived from diseased pigs. Serotype 2 (71.2%) was the most common, but the prevalence of serotypes 6 (13.6%) and 15 (6.8%) increased. Existing vaccines are considered ineffective on the isolates belonging to serotypes 6 and 15. The phylogenetic tree based on core genome single nucleotide polymorphisms showed that the isolates were clustered by serotype. Of the isolates, 62.5% did not have an antimicrobial resistance gene, including a florfenicol resistance gene, but 32.2% had a tetracycline resistance gene. The antimicrobial resistant phenotype and genotype were almost identical. The plasmid-derived contigs harbored resistance genes of aminoglycosides, tetracyclines, ß-lactams, phenicols, or sulfonamides. It has been suggested that isolates with different genetic properties from vaccine strains are circulating; however, antimicrobial resistance may not be widespread.

Characterization of nonencapsulated Actinobacillus pleuropneumoniae serovar K12:O3 isolates.

Three Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia were positive by capsular serovar 12-specific PCR assay, but not reactive to antiserum prepared against serovar 12 using the rapid slide agglutination (RSA) test. The isolates were positive for apxIICA, apxIIICA, apxIBD, apxIIIBD, and apxIVA in the PCR toxin gene assay, which is the profile seen in serovars 2, 4, 6, 8, and 15, and reacted with antisera against serovars 3, 6, 8, 15, and 17. Nucleotide sequence analysis revealed that genes involved in the biosynthesis of capsular polysaccharide of the 3 isolates were identical or nearly identical to those of serovar 12. However, genes involved in the biosynthesis of O-polysaccharide of the 3 isolates were highly similar to those of reference strains of serovars 3, 6, 8, 15, 17, and 19. In agreement with results from the RSA test, transmission electron microscopic analysis confirmed the absence of detectable capsular material in the 3 isolates. The existence of nonencapsulated A. pleuropneumoniae serovar K12:O3 would hamper precise serodetection.

TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

The morphology and metabolic changes of Actinobacillus pleuropneumoniae during its growth as a biofilm.

Actinobacillus pleuropneumoniae is an important swine respiratory pathogen. Previous studies have suggested that growth as a biofilm is a natural state of A. pleuropneumoniae infection. To understand the survival features involved in the biofilm state, the growth features, morphology and gene expression profiles of planktonic and biofilm A. pleuropneumoniae were compared. A. pleuropneumoniae in biofilms showed reduced viability but maintained the presence of extracellular polymeric substances (EPS) after late log-phase. Under the microscope, bacteria in biofilms formed dense aggregated structures that were connected by abundant EPS, with reduced condensed chromatin. By construction of Δpga and ΔdspB mutants, polymeric ß-1,6-linked N-acetylglucosamine and dispersin B were confirmed to be critical for normal biofilm formation. RNA-seq analysis indicated that, compared to their planktonic counterparts, A. pleuropneumoniae in biofilms had an extensively altered transcriptome. Carbohydrate metabolism, energy metabolism and translation were significantly repressed, while fermentation and genes contributing to EPS synthesis and translocation were up-regulated. The regulators Fnr (HlyX) and Fis were found to be up-regulated and their binding motifs were identified in the majority of the differentially expressed genes, suggesting their coordinated global role in regulating biofilm metabolism. By comparing the transcriptome of wild-type biofilm and Δpga, the utilization of oligosaccharides, iron and sulfur and fermentation were found to be important in adhesion and aggregation during biofilm formation. Additionally, when used as inocula, biofilm bacteria showed reduced virulence in mouse, compared with planktonic grown cells. Thus, these results have identified new facets of A. pleuropneumoniae biofilm maintenance and regulation.

Adh Promotes Actinobacillus pleuropneumoniae Survival in Porcine Alveolar Macrophages by Inhibiting CHAC2-Mediated Respiratory Burst and Inflammatory Cytokine Expression.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes porcine pleuropneumonia that seriously endangers pig's health. Adh, located in the head region of trimeric autotransporter adhesion of A. pleuropneumoniae, affects bacterial adhesion and pathogenicity. However, how Adh mediates A. pleuropneumoniae immune invasion is still unclear. Here, we established the A. pleuropneumoniae strain L20 or L20 ΔAdh-infected porcine alveolar macrophages (PAM) model, and applied protein overexpression, RNA interference, qRT-PCR, Western blot and immunoflourescence techniques to dissect the effects of Adh on PAM during A. pleuropneumoniae infection. We found that Adh could increase the A. pleuropneumoniae adhesion and intracellular survival in PAM. Gene chip analysis of piglet lungs further showed that Adh significantly induced cation transport regulatory-like protein 2 (CHAC2) expression, whose overexpression suppressed the phagocytic capacity of PAM. Furthermore, CHAC2 overexpression dramatically increased glutathione (GSH) expression, decreased reactive oxygen species (ROS), and promoted A. pleuropneumoniae survival in PAM, while the knockdown of CHAC2 reversed these phenomena. Meanwhile, CHAC2 silence activated the NOD1/NF-κB pathway, resulting in an increase in IL-1ß, IL-6, and TNF-α expression, whereas this effect was weakened by CHAC2 overexpression and addition of NOD1/NF-κB inhibitor ML130. Moreover, Adh enhanced the secretion of LPS of A. pleuropneumoniae, which regulated the expression of CHAC2 via TLR4. In conclusion, through a LPS-TLR4-CHAC2 pathway, Adh inhibits respiratory burst and inflammatory cytokines expression to promote A. pleuropneumoniae survival in PAM. This finding may provide a novel target for the prevention and treatment of A. pleuropneumoniae.

Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

Characterization of an atypical Actinobacillus pleuropneumoniae serovar 2 isolate with a rough-type lipopolysaccharide.

We describe phenotypic and genetic characterization of an atypical Japanese Actinobacillus pleuropneumoniae isolate OT761. Nucleotide sequence analysis revealed that gene clusters involved in capsular polysaccharide and O-polysaccharide (O-PS) biosynthesis of the isolate were nearly identical to those of serovar 2 reference strain. The main difference found between the O-PS loci is the shortening of 31 amino acids from the C terminus of WcaJ in the atypical isolate due to a 93 bp deletion at the 3' end of wcaJ gene. Immunoblot analysis revealed that this isolate could not produce O-PS. Taken together, our results showed that the C-terminal domain of the A. pleuropneumoniae WcaJ plays a critical role in enzyme function of WcaJ involved in the biosynthesis of O-PS.

Effects of OxyR regulator on oxidative stress, Apx toxin secretion and virulence of Actinobacillus pleuropneumoniae.

Introduction: Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, poses a significant threat to global swine populations due to its high prevalence, mortality rates, and substantial economic ramifications. Understanding the pathogen's defense mechanisms against host-produced reactive oxygen species is crucial for its survival, with OxyR, a conserved bacterial transcription factor, being pivotal in oxidative stress response. Methods: This study investigated the presence and role of OxyR in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis was conducted on an oxyR disruption mutant to delineate the biological activities influenced by OxyR. Additionally, specific assays were employed to assess urease activity, catalase expression, ApxI toxin secretion, as well as adhesion and invasion abilities of the oxyR disruption mutant on porcine 3D4/21 and PT cells. A mice challenge experiment was also conducted to evaluate the impact of oxyR inactivation on A. pleuropneumoniae virulence. Results: OxyR was identified as a conserved regulator present in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis revealed the involvement of OxyR in multiple biological activities. The oxyR disruption resulted in decreased urease activity, elevated catalase expression, enhanced ApxI toxin secretion-attributed to OxyR binding to the apxIBD promoter-and reduced adhesion and invasion abilities on porcine cells. Furthermore, inactivation of oxyR reduced the virulence of A. pleuropneumoniae in a mice challenge experiment. Discussion: The findings highlight the pivotal role of OxyR in influencing the virulence mechanisms of A. pleuropneumoniae. The observed effects on various biological activities underscore OxyR as an essential factor contributing to the pathogenicity of this bacterium.

CpxAR of Actinobacillus pleuropneumoniae Contributes to Heat Stress Response by Repressing Expression of Type IV Pilus Gene apfA.

Acute pleuropneumonia in swine, caused by Actinobacillus pleuropneumoniae, is characterized by a high and sustained fever. Fever creates an adverse environment for many bacteria, leading to reduced bacterial proliferation; however, most pathogenic bacteria can tolerate higher temperatures. CpxAR is a two-component regulation system, ubiquitous among Gram-negative bacteria, which senses and responds to envelope alterations that are mostly associated with protein misfolding in the periplasm. Our previous study showed that CpxAR is necessary for the optimal growth of Actinobacillus pleuropneumoniae under heat stress. Here, we showed that mutation of the type IV pilin gene apfA rescued the growth defect of the cpxAR deletion strain under heat stress. RNA sequencing (RNA-seq) analyses revealed that 265 genes were differentially expressed in the ΔcpxAR strains grown at 42°C, including genes involved in type IV pilus biosynthesis. We also demonstrated direct binding of the CpxR protein to the promoter of the apf operon by an electrophoretic mobility shift assay and identified the binding site by a DNase I footprinting assay. In conclusion, our results revealed the important role of CpxAR in A. pleuropneumoniae resistance to heat stress by directly suppressing the expression of ApfA. IMPORTANCE Heat acts as a danger signal for pathogens, especially those infecting mammalian hosts in whom fever indicates infection. However, some bacteria have evolved exquisite mechanisms to survive under heat stress. Studying the mechanism of resistance to heat stress is crucial to understanding the pathogenesis of A. pleuropneumoniae during the acute stage of infection. Our study revealed that CpxAR plays an important role in A. pleuropneumoniae resistance to heat stress by directly suppressing expression of the type IV pilin protein ApfA.

Outer Membrane Vesicles of Actinobacillus pleuropneumoniae Exert Immunomodulatory Effects on Porcine Alveolar Macrophages.

Outer membrane vesicles (OMVs) are spontaneously released by Gram-negative bacteria, including Actinobacillus pleuropneumoniae, which causes contagious pleuropneumonia in pigs and leads to considerable economic losses in the swine industry worldwide. A. pleuropneumoniae OMVs have previously been demonstrated to contain Apx toxins and proteases, as well as antigenic proteins. Nevertheless, comprehensive characterizations of their contents and interactions with host immune cells have not been made. Understanding the protein compositions and immunomodulating ability of A. pleuropneumoniae OMVs could help illuminate their biological functions and facilitate the development of OMV-based applications. In the current investigation, we comprehensively characterized the proteome of native A. pleuropneumoniae OMVs. Moreover, we qualitatively and quantitatively compared the OMV proteomes of a wild-type strain and three mutant strains, in which relevant genes were disrupted to increase OMV production and/or produce OMVs devoid of superantigen PalA. Furthermore, the interaction between A. pleuropneumoniae OMVs and porcine alveolar macrophages was also characterized. Our results indicate that native OMVs spontaneously released by A. pleuropneumoniae MIDG2331 appeared to dampen the innate immune responses by porcine alveolar macrophages stimulated by either inactivated or live parent cells. The findings suggest that OMVs may play a role in manipulating the porcine defense during the initial phases of the A. pleuropneumoniae infection. IMPORTANCE Owing to their built-in adjuvanticity and antigenicity, bacterial outer membrane vesicles (OMVs) are gaining increasing attention as potential vaccines for both human and animal use. OMVs released by Actinobacillus pleuropneumoniae, an important respiratory pathogen in pigs, have also been investigated for vaccine development. Our previous studies have shown that A. pleuropneumoniae secretes OMVs containing multiple immunogenic proteins. However, immunization of pigs with these vesicles was not able to relieve the pig lung lesions induced by the challenge with A. pleuropneumoniae, implying the elusive roles that A. pleuropneumoniae OMVs play in host-pathogen interaction. Here, we showed that A. pleuropneumoniae secretes OMVs whose yield and protein content can be altered by the deletion of the nlpI and palA genes. Furthermore, we demonstrate that A. pleuropneumoniae OMVs dampen the immune responses in porcine alveolar macrophages stimulated by A. pleuropneumoniae cells, suggesting a novel mechanism that A. pleuropneumoniae might use to evade host defense.

The Metabolic Adaptation in Response to Nitrate Is Critical for Actinobacillus pleuropneumoniae Growth and Pathogenicity under the Regulation of NarQ/P.

Nitrate metabolism is an adaptation mechanism used by many bacteria for survival in anaerobic environments. As a by-product of inflammation, nitrate is used by the intestinal bacterial pathogens to enable gut infection. However, the responses of bacterial respiratory pathogens to nitrate are less well understood. Actinobacillus pleuropneumoniae is an important bacterial respiratory pathogen of swine. Previous studies have suggested that adaptation of A. pleuropneumoniae to anaerobiosis is important for infection. In this work, A. pleuropneumoniae growth and pathogenesis in response to the nitrate were investigated. Nitrate significantly promoted A. pleuropneumoniae growth under anaerobic conditions in vitro and lethality in mice. By using narQ and narP deletion mutants and single-residue-mutated complementary strains of ΔnarQ, the two-component system NarQ/P was confirmed to be critical for nitrate-induced growth, with Arg50 in NarQ as an essential functional residue. Transcriptome analysis showed that nitrate upregulated multiple energy-generating pathways, including nitrate metabolism, mannose and pentose metabolism, and glycerolipid metabolism via the regulation of NarQ/P. Furthermore, narQ, narP, and its target gene encoding the nitrate reductase Nap contributed to the pathogenicity of A. pleuropneumoniae. The Nap inhibitor tungstate significantly reduced the survival of A. pleuropneumoniae in vivo, suggesting that Nap is a potential drug target. These results give new insights into how the respiratory pathogen A. pleuropneumoniae utilizes the alternative electron acceptor nitrate to overcome the hypoxia microenvironment, which can occur in the inflammatory or necrotic infected tissues.

Specific quantitative detection of Streptococcus suis and Actinobacillus pleuropneumoniae in co-infection and mixed biofilms.

Respiratory infections seriously affect the swine industry worldwide. Co-infections of two vital pathogenic bacteria Streptococcus suis (S. suis) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae), colonizing the respiratory tract often occurs in veterinary clinical practice. Moreover, our previous research found that S. suis and A. pleuropneumoniae can form biofilm in vitro. The formation of a mixed biofilm not only causes persistent infections, but also increases the multiple drug resistance of bacteria, which brings difficulties to disease prevention and control. However, the methods for detecting S. suis and A. pleuropneumoniae in co-infection and biofilm are immature. Therefore, in this study, primers and probes were designed based on the conservative sequence of S. suis gdh gene and A. pleuropneumoniae apxIVA gene. Then, a TaqMan duplex real-time PCR method for simultaneous detection of S. suis and A. pleuropneumoniae was successfully established via optimizing the reaction system and conditions. The specificity analysis results showed that this TaqMan real-time PCR method had strong specificity and high reliability. The sensitivity test results showed that the minimum detection concentration of S. suis and A. pleuropneumoniae recombinant plasmid was 10 copies/µL, which is 100 times more sensitive than conventional PCR methods. The amplification efficiencies of S. suis and A. pleuropneumoniae were 95.9% and 104.4% with R2 value greater than 0.995, respectively. The slopes of the calibration curves of absolute cell abundance of S. suis and A. pleuropneumoniae were 1.02 and 1.09, respectively. The assays were applied to cultivated mixed biofilms and approximately 108 CFUs per biofilm were quantified when 108 CFUs planktonic bacteria of either S. suis or A. pleuropneumoniae were added to biofilms. In summary, this study developed a TaqMan real-time PCR assay for specific, accurate quantification of S. suis or A. pleuropneumoniae in mixed biofilms, which may help for the detection, prevention and control of diseases caused by a bacterial mixed infection involving S. suis and A. pleuropneumoniae.

HtrA of Actinobacillus pleuropneumoniae is a virulence factor that confers resistance to heat shock and oxidative stress.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, which is a severe and often fatal disease that results in significant economic loss. The means by which A. pleuropneumoniae survives within the host are not clear. High temperature requirement A (HtrA) proteases have been shown to affect cell viability during stressful conditions and are virulence factors in many bacterial species. In this study, we examined the biological role of HtrA during A. pleuropneumoniae infection by analyzing the impact of htrA mutation on virulence-associated phenotypes. We found that htrA mutation had a dramatic impact on stress tolerance. The htrA mutant (ΔhtrA) displayed a lethal phenotype at elevated temperature (42 °C). Further, ΔhtrA exhibited increased susceptibility to H2O2-induced oxidative stress when compared to the parental strain (SLW01) and a complementation strain (ΔhtrA-Compl). Animal infection assays demonstrated that absence of HtrA led to decreased in vivo colonization ability, and ΔhtrA is less virulent in pigs relative to SLW01. Furthermore, pig competitive infection assays demonstrated fewer blood associated CFUs with ΔhtrA infection than with SLW01. These results demonstrate HtrA plays a significant role in the survival and growth of A. pleuropneumoniae during stressful conditions, and that immune escape and invasiveness are important to the process of A. pleuropneumoniae infection.