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.

Intranasal B5 promotes mucosal defence against Actinobacillus pleuropneumoniae via ameliorating early immunosuppression.

Actinobacillus pleuropneumoniae (APP) is an important pathogen of the porcine respiratory disease complex, which leads to huge economic losses worldwide. We previously demonstrated that Pichia pastoris-producing bovine neutrophil ß-defensin-5 (B5) could resist the infection by the bovine intracellular pathogen Mycobacterium bovis. In this study, the roles of synthetic B5 in regulating mucosal innate immune response and protecting against extracellular APP infection were further investigated using a mouse model. Results showed that B5 promoted the production of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and interferon (IFN)-ß in macrophages as well as dendritic cells (DC) and enhanced DC maturation in vitro. Importantly, intranasal B5 was safe and conferred effective protection against APP via reducing the bacterial load in lungs and alleviating pulmonary inflammatory damage. Furthermore, in the early stage of APP infection, we found that intranasal B5 up-regulated the secretion of TNF-α, IL-1ß, IL-17, and IL-22; enhanced the rapid recruitment of macrophages, neutrophils, and DC; and facilitated the generation of group 3 innate lymphoid cells in lungs. In addition, B5 activated signalling pathways associated with cellular response to IFN-ß and activation of innate immune response in APP-challenged lungs. Collectively, B5 via the intranasal route can effectively ameliorate the immune suppression caused by early APP infection and provide protection against APP. The immunization strategy may be applied to animals or human respiratory bacterial infectious diseases. Our findings highlight the potential importance of B5, enhancing mucosal defence against intracellular bacteria like APP which causes early-phase immune suppression.

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.

The cAMP receptor protein gene contributes to growth, stress resistance, and colonization of Actinobacillus pleuropneumoniae.

Porcine infectious pleuropneumonia (PCP) is a severe disease of porcine caused by Actinobacillus pleuropneumoniae (APP). The spread of PCP remains a threat to the porcine farms and has been known to cause severe economic losses. The cAMP receptor protein (CRP) serves as a pivotal player in helping bacteria adapt to shifts in their environment, particularly when facing the challenges posed by bacterial infections. In this study, we investigated the role of CRP in APP. Our results revealed that crp mutant (Δcrp) strains were more sensitive to acidic and osmotic stress resistance and had lower biofilm formation ability than wild-type (WT) strains. Furthermore, the Δcrp strains showed deficiencies in anti-phagocytosis, adhesion, and invasion upon interaction with host cells. Mice infected with the Δcrp strains demonstrated reduced bacterial loads in their lungs compared to those infected with the WT strains. This study reveals the pivotal role of crp gene expression in regulating pleuropneumonia growth, stress resistance, iron utilization, biofilm formation, phagocytosis, adhesion, invasion and colonization. Our discoveries offer novel perspectives on understanding the development and progression of APP infections.

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.

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.

Protective effect of lithium chloride on pulmonary injury caused by Actinobacillus pleuropneumoniae via inhibition of GSK-3ß-NF-κB-dependent pathway.

Porcine contagious pleuropneumonia (PCP) is a very serious respiratory disease which is difficult to prevent and treat. In this study, the therapeutic effects of lithium chloride (LiCl) on PCP were examined using a mouse model. A mouse model of PCP was established by intranasal infections with Actinobacillus pleuropneumoniae (App). Histopathological analysis was performed by routine paraffin sections and an H-E staining method. The inflammatory factors, TLR4 and CCL2 were analyzed by qPCR. The expression levels of p-p65 and pGSK-3ß were detected using the Western Blot Method. The death rates, clinical symptoms, lung injuries, and levels of TLR-4, IL-1ß, IL-6, TNF-α, and CCL2 were observed to decrease in the App-infected mice treated with LiCl. It was determined that the LiCl treatments had significantly reduced the mortality of the App-infected cells, as well as the expressions of p-p65 and pGSK-3ß. The results of this study indicated that LiCl could improve the pulmonary injuries of mice caused by App via the inhibition of the GSK-3ß-NF-κB-dependent pathways, and may potentially become an effective drug for improving pulmonary injuries caused by PCP.

Untargeted and targeted metabolomics reveal that adenosine nucleotides released in Actinobacillus pleuropneumoniae supernatant inhibit porcine reproductive and respiratory syndrome virus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses in the swine industry and causes major economic losses. To date, there has not been an effective antiviral treatment for the disease. We have shown in previous studies that culture supernatant of Actinobacillus pleuropneumoniae (App), the causative agent of porcine pleuropneumonia, possesses antiviral activity in vitro against PRRSV, and we have clearly established that the antiviral activity was mediated by small molecular weight (i.e., <1 kDa), heat resistant metabolites present in the App supernatant ultrafiltrates. However, the identity of those metabolites remains unknown. The objective of the current study was to identify the active metabolites using untargeted and targeted mass spectrometry-based metabolomics and test their respective antiviral activity against PRRSV in the Jude Porcine Lung Epithelial Cell Line (SJPL). The results presented reveal very significant antiviral activity of App supernatant ultrafiltrates against PRRSV in SJPL cells. Consequently, we identified and quantified several adenosine nucleotide metabolites present in App supernatant ultrafiltrates using mass spectrometry-based metabolomics, and the concentrations detected were very high. SJPL cells infected with PRRSV and treated with 2'-adenosine monophosphate (2-AMP), 3'-adenosine monophosphate (3-AMP) or 5'-adenosine monophosphate (5-AMP) significantly reduced PRRSV infection. Interestingly, many antiviral drugs or prodrugs are adenosine analogs, and the mechanism of action was previously elucidated. Currently marketed nucleoside analog drugs could potentially be used to treat PRRSV infection.

Identification of FtpA, a Dps-Like Protein Involved in Anti-Oxidative Stress and Virulence in Actinobacillus pleuropneumoniae.

Bacteria have evolved a variety of enzymes to eliminate endogenous or host-derived oxidative stress factors. The Dps protein, first identified in Escherichia coli, contains a ferroxidase center, and protects bacteria from reactive oxygen species damage. Little is known of the role of Dps-like proteins in bacterial pathogenesis. Actinobacillus pleuropneumoniae causes pleuropneumonia, a respiratory disease of swine. The A. pleuropneumoniae ftpA gene is upregulated during shifts to anaerobiosis, in biofilms and, as found in this study, in the presence of H2O2. An A. pleuropneumoniae ftpA deletion mutant (ΔftpA) had increased H2O2 sensitivity, decreased intracellular viability in macrophages, and decreased virulence in a mouse infection model. Expression of ftpA in an E. coli dps mutant restored wild-type H2O2 resistance. FtpA possesses a conserved ferritin domain containing a ferroxidase site. Recombinant rFtpA bound and oxidized Fe2+ reversibly. Under aerobic conditions, the viability of an ΔftpA mutant was reduced compared with the wild-type strain after extended culture, upon transition from anaerobic to aerobic conditions, and upon supplementation with Fenton reaction substrates. Under anaerobic conditions, the addition of H2O2 resulted in a more severe growth defect of ΔftpA than it did under aerobic conditions. Therefore, by oxidizing and mineralizing Fe2+, FtpA alleviates the oxidative damage mediated by intracellular Fenton reactions. Furthermore, by mutational analysis, two residues were confirmed to be critical for Fe2+ binding and oxidization, as well as for A. pleuropneumoniae H2O2 resistance. Taken together, the results of this study demonstrate that A. pleuropneumoniae FtpA is a Dps-like protein, playing critical roles in oxidative stress resistance and virulence. IMPORTANCE As a ferroxidase, Dps of Escherichia coli can protect bacteria from reactive oxygen species damage, but its role in bacterial pathogenesis has received little attention. In this study, FtpA of the swine respiratory pathogen A. pleuropneumoniae was identified as a new Dps-like protein. It facilitated A. pleuropneumoniae resistance to H2O2, survival in macrophages, and infection in vivo. FtpA could bind and oxidize Fe2+ through two important residues in its ferroxidase site and protected the bacteria from oxidative damage mediated by the intracellular Fenton reaction. These findings provide new insights into the role of the FtpA-based antioxidant system in the pathogenesis of A. pleuropneumoniae, and the conserved Fe2+ binding ligands in Dps/FtpA provide novel drug target candidates for disease prevention.

Production of microhomogeneous glycopeptide by a mutated NGT according FuncLib with unique sugar as substrate.

N-glycosylation is one of the most important post-translational modifications of proteins. Cytoplasmic soluble N-glycosyltransferase (NGT) exists in bacteria, which is able to transfer different monosaccharide from sugar nucleotide to the NXS/T(X ≠ Pro) consensus sequence in a polypeptide. At present, the NGT enzymes reported could transfer a variety of different sugars to protein, which will lead to the heterogeneity of the sugar chain and the complexity and instability of the structure and function of glycopeptides. According to the FuncLib algorithm, we obtained mutant ApNGT-P1 from ApNGT (the NGT from Actinobacillus pleuropneumoniae) with increased substrate specificity. Compared with the wild-type ApNGT, mutant ApNGT-P1 could only utilize UDP-Glc as sugar donors. The optimum temperature of ApNGT-P1 was about 40 °C and the optimum pH was 7.5-8.0 in PBS buffer. ApNGT-P1 exhibited better tolerance for K+, Mn2+, Ca2+, and Mg2+, but was strongly inhibited by Na+, Cu2+ and Zn2+. The mutant can be applied to the efficient production of glycosylated peptides or proteins with uniform glucose at their glycosylation sites. Besides, this work provided a feasible pathway for further studies on the improving donor substrates selectivity of NGTs.

Actinobacillus utilizes a binding protein-dependent ABC transporter to acquire the active form of vitamin B6.

Bacteria require high-efficiency uptake systems to survive and proliferate in nutrient-limiting environments, such as those found in host organisms. ABC transporters in the bacterial plasma membrane provide a mechanism for transport of many substrates. In this study, we examine an operon containing a periplasmic binding protein in Actinobacillus for its potential role in nutrient acquisition. The electron density map of 1.76 Å resolution obtained from the crystal structure of the periplasmic binding protein was best fit with a molecular model containing a pyridoxal-5'-phosphate (P5P/pyridoxal phosphate/the active form of vitamin B6) ligand within the protein's binding site. The identity of the P5P bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from Actinobacillus pleuropneumoniae into an Escherichia coli K-12 strain that was devoid of a key enzyme required for P5P synthesis. The growth of this strain at low levels of P5P supports the functional role of this operon in P5P uptake. This is the first report of a dedicated P5P bacterial uptake system, but through bioinformatics, we discovered homologs mainly within pathogenic representatives of the Pasteurellaceae family, suggesting that this operon exists more widely outside the Actinobacillus genus.

CopA Protects Actinobacillus pleuropneumoniae against Copper Toxicity.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium causing porcine pleuropneumonia and severe economic losses in the global swine industry. The toxic trace element copper is required for many physiological and pathological processes in organisms. However, CopA, one of the most well-characterized P-type ATPases contributing to copper resistance, has not been characterized in A. pleuropneumoniae. We used quantitative PCR analysis to examine expression of the copA gene in A. pleuropneumoniae and investigated sequence conservation among serotypes and other Gram-negative bacteria. Growth characteristics were determined using growth curve analyses and spot dilution assays of the wild-type strain and a △copA mutant. We also used flame atomic absorption spectrophotometry to determine intracellular copper content and examined the virulence of the △copA mutant in a mouse model. The copA expression was induced by copper, and its nucleotide sequence was highly conserved among different serotypes of A. pleuropneumoniae. The amino acid sequence of CopA shared high identity with CopA sequences reported from several Gram-negative bacteria. Furthermore, the △copA mutant exhibited impaired growth and had higher intracellular copper content compared with the wild-type strain when supplemented with copper. The mouse model revealed that CopA had no influence on the virulence of A. pleuropneumoniae. In conclusion, these results demonstrated that CopA is required for resistance of A. pleuropneumoniae to copper and protects A. pleuropneumoniae against copper toxicity via copper efflux.

Actinobacillus pleuropneumoniae exotoxin ApxI induces cell death via attenuation of FAK through LFA-1.

ApxI exotoxin is an important virulence factor derived from Actinobacillus pleuropneumoniae that causes pleuropneumonia in swine. Here, we investigate the role of lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), a member of the ß2 integrin family, and the involvement of the integrin signaling molecules focal adhesion kinase (FAK) and Akt in ApxI cytotoxicity. Using Western blot analysis, we found that ApxI downregulated the activity of FAK and Akt in porcine alveolar macrophages (AMs). Preincubation of porcine AMs with an antibody specific for porcine CD18 reduced ApxI-induced cytotoxicity as measured by a lactate dehydrogenase release assay and decreased ApxI-induced FAK and Akt attenuation, as shown by Western blot analysis. Pretreatment with the chemical compounds PMA and SC79, which activate FAK and Akt, respectively, failed to overcome the ApxI-induced attenuation of FAK and Akt and death of porcine AMs. Notably, the transfection experiments revealed that ectopic expression of porcine LFA-1 (pLFA-1) conferred susceptibility to ApxI in ApxI-insensitive cell lines, including human embryonic kidney 293T cells and FAK-deficient mouse embryonic fibroblasts (MEFs). Furthermore, ectopic expression of FAK significantly reduced ApxI cytotoxicity in pLFA-1-cotransfected FAK-deficient MEFs. These findings show for the first time that pLFA-1 renders cells susceptible to ApxI and ApxI-mediated attenuation of FAK activity via CD18, thereby contributing to subsequent cell death.

The Actinobacillus pleuropneumoniae sulfate-binding protein is required for the acquisition of sulfate and methionine, but is not essential for virulence.

Actinobacillus pleuropneumoniae is a Gram-negative pathogen that causes porcine pleuropneumonia, an infectious disease responsible for significant losses in the pig industry. Sulfur is an essential nutrient that is widely required by microorganisms; however, the mechanism involved in A. pleuropneumoniae sulfur transport is unknown. In this study, we showed that a periplasmic protein predicted to be involved in sulfur acquisition (sulfate-binding protein (Sbp)), is required for A. pleuropneumoniae growth in chemically defined medium (CDM) containing sulfate or methionine as the sole sulfur sources. However, utilization of glutathione and cysteine was not affected in the sbp-deletion mutant. The virulence of A. pleuropneumoniae in mice was not affected by the absence of Sbp. Moreover, we demonstrated that Sbp was not essential for the in vivo colonization of A. pleuropneumoniae in mice or pigs. Collectively, these findings reveal that A. pleuropneumoniae Sbp plays an important role in the acquisition of the sulfur nutrients, sulfate and methionine. The presence of other sulfur uptake systems suggests A. pleuropneumoniae has multiple functionally redundant pathways ensuring uptake of important nutrients during infection.

Enhancement of Apx Toxin Production in Actinobacillus pleuropneumoniae Serotypes 1, 2, and 5 by Optimizing Culture Condition.

Actinobacillus pleuropneumoniae (APP) is a causative agent of porcine pleuropneumonia. Therefore, the development of an effective vaccine for APP is necessary. Here, we optimized the culture medium and conditions to enhance the production yields of Apx toxins in APP serotype 1, 2, and 5 cultures. The use of Mycoplasma Broth Base (PPLO) medium improved both the quantity and quality of the harvested Apx toxins compared with Columbia Broth medium. Calcium chloride (CaCl2) was first demonstrated as a stimulation factor for the production of Apx toxins in APP serotype 2 cultures. Cultivation of APP serotype 2 in PPLO medium supplemented with 10 µg/ml of nicotinamide adenine dinucleotide (NAD) and 20 mM CaCl2 yielded the highest levels of Apx toxins. These findings suggest that the optimization of the culture medium and conditions increases the concentration of Apx toxins in the supernatants of APP serotype 1, 2, and 5 cultures and may be applied for the development of vaccines against APP infection.

An enzyme-based protocol for cell-free synthesis of nature-identical capsular oligosaccharides from Actinobacillus pleuropneumoniae serotype 1.

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-ß(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.

Basal-Level Effects of (p)ppGpp in the Absence of Branched-Chain Amino Acids in Actinobacillus pleuropneumoniae.

The (p)ppGpp-mediated stringent response (SR) is a highly conserved regulatory mechanism in bacterial pathogens, enabling adaptation to adverse environments, and is linked to pathogenesis. Actinobacillus pleuropneumoniae can cause damage to the lungs of pigs, its only known natural host. Pig lungs are known to have a low concentration of free branched-chain amino acids (BCAAs) compared to the level in plasma. We had investigated the role for (p)ppGpp in viability and biofilm formation of A. pleuropneumoniae Now, we sought to determine whether (p)ppGpp was a trigger signal for the SR in A. pleuropneumoniae in the absence of BCAAs. Combining transcriptome and phenotypic analyses of the wild type (WT) and an relA spoT double mutant [which does not produce (p)ppGpp], we found that (p)ppGpp could repress de novo purine biosynthesis and activate antioxidant pathways. There was a positive correlation between GTP and endogenous hydrogen peroxide content. Furthermore, the growth, viability, morphology, and virulence were altered by the inability to produce (p)ppGpp. Genes involved in the biosynthesis of BCAAs were constitutively upregulated, regardless of the existence of BCAAs, without accumulation of (p)ppGpp beyond a basal level. Collectively, our study shows that the absence of BCAAs was not a sufficient signal to trigger the SR in A. pleuropneumoniae (p)ppGpp-mediated regulation in A. pleuropneumoniae is different from that described for the model organism Escherichia coli Further work will establish whether the (p)ppGpp-dependent SR mechanism in A. pleuropneumoniae is conserved among other veterinary pathogens, especially those in the Pasteurellaceae family.IMPORTANCE (p)ppGpp is a key player in reprogramming transcriptomes to respond to nutritional challenges. Here, we present transcriptional and phenotypic differences of A. pleuropneumoniae grown in different chemically defined media in the absence of (p)ppGpp. We show that the deprivation of branched-chain amino acids (BCAAs) does not elicit a change in the basal-level (p)ppGpp, but this level is sufficient to regulate the expression of BCAA biosynthesis. The mechanism found in A. pleuropneumoniae is different from that of the model organism Escherichia coli but similar to that found in some Gram-positive bacteria. This study not only broadens the research scope of (p)ppGpp but also further validates the complexity and multiplicity of (p)ppGpp regulation in microorganisms that occupy different biological niches.

Comparison of metabolic adaptation and biofilm formation of Actinobacillus pleuropneumoniae field isolates from the upper and lower respiratory tract of swine with respiratory disease.

Most outbreaks of disease due to infection with Actinobacillus (A.) pleuropneumoniae are caused by pigs already pre-colonised in tonsillar tissue, where the pathogen is protected from exposure to antibiotic substances administered for treatment. As it has been shown recently under experimental conditions, A. pleuropneumoniae displays host tissue-specific metabolic adaptation. In this study, pairs of A. pleuropneumoniae field isolates were recovered from lung as well as from tonsillar and nasal tissue from 20 pigs suffering from acute clinical signs of pleuropneumonia and showing characteristic pathological lung alterations. Metabolic adaptation to the porcine lower and upper respiratory tract of 32 A. pleuropneumoniae serotype 2 field isolates was examined using Fourier transform infrared (FTIR) spectroscopy as a high resolution metabolic fingerprinting method. All strains showed metabolic adaptations to organ tissue reflected by hierarchical cluster analysis of FTIR spectra similar to those previously observed under experimental conditions. Notably, differences in antimicrobial resistance patterns and minimal inhibitory concentrations of isolates from different tissues in the same animal, but not in biofilm production capability in a microtiter plate assay were found. Overall, biofilm formation was observed for 71 % of the isolates, confirming that A. pleuropneumoniae field isolates are generally able to form biofilms, although rather in a serotype-specific than in an organ-specific manner. A. pleuropneumoniae serotype 6 isolates formed significantly more biofilm than the other serotypes. Furthermore, biofilm production was negatively correlated to the lung lesion scores and tonsillar isolates tended to be more susceptible to antimicrobial substances with high bioavailability than lung isolates.

Identification of genes regulated by the two-component system response regulator NarP of Actinobacillus pleuropneumoniae via DNA-affinity-purified sequencing.

Identifying the direct target genes of response regulators (RRs) of a bacterial two-component system (TCS) is critical to understand the roles of TCS in bacterial environmental adaption and pathogenesis. Actinobacillus pleuropneumoniae is an important respiratory bacterial pathogen that causes considerable economic losses to swine industry worldwide. The targets of A. pleuropneumoniae NarP (nitrate/nitrite RR), which is the cognate RR of the nitrate/nitrite sensor histidine kinase NarQ, are still unknown. In the present study, a DNA-affinity-purified sequencing (DAP-Seq) approach was established. The upstream regions of a total of 131 candidate genes from the genome of A. pleuropneumoniae were co-purified with the activated NarP protein. Electrophoretic mobility shift assay (EMSA) results confirmed the interactions of NarP with the promoter regions of five selected target genes, including dmsA, pgaA, ftpA, cstA and ushA. The EMSA-confirmed target genes were significantly up-regulated in the narP-deleted mutant in the presence of additional nitrate, whilst the transcriptional changes were restored in the complemented strain. The NarP binding motif in the upstream regions of the target genes dmsA and ftpA were further identified and confirmed by EMSA using the truncated binding motif. The NarP binding sites were present in a total of 25.2% of the DNA fragments captured by DAP-Seq. These results demonstrated that the established DAP-Seq method is effective for exploring the direct targets of RRs of bacterial TCSs and that the A. pleuropneumoniae NarP could be a repressor in response to nitrate.

Carbon source utilisation and evaluation of the Biolog system in the identification of Actinobacillus pleuropneumoniae.

Sixty-eight Actinobacillus pleuropneumoniae strains were isolated from porcine acute pleuropneumonia cases from different parts of Hungary between 2000 and 2014. A total of 41 isolates were identified as A. pleuropneumoniae bio-type I and 27 strains as biotype II based on cultural, morphological and biochemical characteristics. The aim of this study was to evaluate metabolic fingerprinting in the species-level identification of A. pleuropneumoniae isolates. Utilisation of carbon sources by these field isolates and six reference strains was characterised by the Biolog system (GN2 Microplate, MicroLog3 Version 4.20.05 software). Twenty-nine field strains were correctly identified by the Biolog system as A. pleuropneumoniae, 36 strains as A. lignieresii, two strains as H. paraphrohaemolyticus and one strain as A. equuli after 24 h of incubation. Among the six A. pleuropneumoniae reference strains the Biolog system identified one strain as A. pleuropneumoniae, four as A. lignieresii and one as H. paraphrohaemolyticus. There was no correlation between biotypes and serotypes of A. pleuropneumoniae and the carbon source utilisation pattern and species identification by the Biolog system. our data indicate that the efficacy of the Biolog system used here could be improved by including phenotypes of more A. pleuropneumoniae strains representing a wider geographical occurrence into the database.