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.

Prevalence and characteristics of Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) isolated from chicken meat in the province of Quebec, Canada.

This study was conducted to estimate the prevalence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) in retail chicken meat and broiler chickens from the Province of Quebec, Canada, and to characterize LA-MRSA isolates. A total of 309 chicken drumsticks and thighs were randomly selected in 2013 from 43 retail stores in the Monteregie. In addition, nasal swabs and caeca samples were collected in 2013-2014 from 200 broiler chickens of 38 different flocks. LA-MRSA was not detected in broiler chickens. Fifteen LA-MRSA isolates were recovered from four (1.3%) of the 309 chicken meat samples. Multi-Locus Sequence Typing (MLST) and SCCmec typing revealed two profiles (ST398-MRSA-V and ST8-MRSA-IVa), which were distinct using pulse-field gel electrophoresis (PFGE) and microarray (antimicrobial resistance and virulence genes) analyses. In addition to beta-lactam resistance, tetracycline and spectinomycin resistance was detected in all isolates from the 3 positive samples of the ST398 profile. Southern blot hybridization revealed that the resistance genes aad(D) and lnu(A), encoding resistances to aminoglycosides and lincosamides respectively, were located on plasmid. All isolates were able to produce biofilms, but biofilm production was not correlated with hld gene expression. Our results show the presence of two separate lineages of MRSA in retail chicken meat in Quebec, one of which is likely of human origin.

The Mastitis Pathogens Culture Collection.

The Mastitis Pathogen Culture Collection contains more than 16,000 mastitis-causing bacterial isolates from milk samples taken from cow quarters and bulk tanks in a national cohort of 91 dairy farms across Canada over a period of 2 years. These isolates are linked to demographic and production data that were recorded at the mammary gland, cow, and farm levels.

Coagulase-negative staphylococci species affect biofilm formation of other coagulase-negative and coagulase-positive staphylococci.

Coagulase-negative staphylococci (CNS) are considered to be commensal bacteria in humans and animals, but are now also recognized as etiological agents in several infections, including bovine mastitis. Biofilm formation appears to be an important factor in CNS pathogenicity. Furthermore, some researchers have proposed that CNS colonization of the intramammary environment has a protective effect against other pathogens. The mechanisms behind the protective effect of CNS have yet to be characterized. The aim of this study was to evaluate the effect of CNS isolates with a weak-biofilm phenotype on the biofilm formation of other staphylococcal isolates. We selected 10 CNS with a weak-biofilm phenotype and 30 staphylococcal isolates with a strong-biofilm phenotype for this study. We measured biofilm production by individual isolates using a standard polystyrene microtiter plate assay and compared the findings with biofilm produced in mixed cultures. We confirmed the results using confocal microscopy and a microfluidic system with low shear force. Four of the CNS isolates with a weak-biofilm phenotype (Staphylococcus chromogenes C and E and Staphylococcus simulans F and H) significantly reduced biofilm formation in approximately 80% of the staphylococcal species tested, including coagulase-positive Staphylococcus aureus. The 4 Staph. chromogenes and Staph. simulans isolates were also able to disperse pre-established biofilms, but to a lesser extent. We also performed a deferred antagonism assay and recorded the number of colony-forming units in the mixed-biofilm assays on differential or selective agar plates. Overall, CNS with a weak-biofilm phenotype did not inhibit the growth of isolates with a strong-biofilm phenotype. These results suggest that some CNS isolates can negatively affect the ability of other staphylococcal isolates and species to form biofilms via a mechanism that does not involve growth inhibition.

Biofilm formation and antimicrobial resistance genes of coagulase-negative staphylococci isolated from cows with mastitis in Argentina.

Mastitis affects the health and welfare of dairy cows worldwide. Coagulase-negative staphylococci (CNS) are known to form biofilms and are increasingly recognized as a cause of persistent bovine intramammary infections. A total of 90 CNS isolated from cows with clinical and subclinical mastitis in Argentina from 2008 to 2014 were identified by PCR-RFLP using the gap gene. Standard microtiter plate assays were used to assess CNS biofilm formation, and Staphylococcus haemolyticus species formed the strongest biofilms. The presence of biofilm-associated genes icaA, bap and aap was detected in a few isolates, while embP, fbe, atlE and eno were present in the majority of isolates. Genes encoding resistance to ß-lactams were detected among the isolates; blaZ, mecA and mecC were detected in 21, 4 and 1 isolate, respectively. Resistance to macrolides and lincosamides (n = 6) was attributable to ermB, ermC, mphC or mrsA or a combination of those genes. In this study, we identified CNS species involved in mastitis and provide information about pathogenicity and antimicrobial resistance, which is essential to design efficient strategies to control mastitis caused by CNS.

Actinobacillus pleuropneumoniae grows as aggregates in the lung of pigs: is it time to refine our in vitro biofilm assays?

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia and forms biofilms in vitro on abiotic surfaces; however, presence of biofilms during infections has not been documented. The aim of this study was to use a species-specific fluorescent oligonucleotide probe and confocal microscopy to localize A. pleuropneumoniae in the lungs of two naturally infected pigs. Actinobacillus pleuropneumoniae was detected by fluorescence in situ hybridization and observed to grow as aggregates (~30-45 µm) during a natural infection. As the A. pleuropneumoniae aggregates observed in porcine lungs differed from the biofilms grown on a solid surface obtained in vitro, we designed a new biofilm assay using agarose, a porous substrate, favouring the formation of aggregates. In this study, we described for the first time the mode of growth of A. pleuropneumoniae during a natural infection in pigs. We also propose an in vitro biofilm assay for A. pleuropneumoniae using a porous substrate which allows the formation of aggregates. This assay might be more representative of the in vivo situation, at least in terms of the size of the bacterial aggregates and the presence of a porous matrix, and could potentially be used to test the susceptibility of A. pleuropneumoniae aggregates to antibiotics and disinfectants.

Differential expression of putative adhesin genes of Actinobacillus suis grown in in vivo-like conditions.

Actinobacillus suis is an opportunistic pathogen that resides in the tonsils of the soft palate of swine. Unknown stimuli can cause this organism to invade the host, resulting in septicaemia and sequelae including death. To better understand its pathogenesis, the expression of several adhesin genes was evaluated by semi-quantitative real-time PCR in A. suis grown in conditions that mimic the host environment, including different nutrient and oxygen levels, exponential and stationary phases of growth, and in the presence of the stress hormone epinephrine. Fifty micromolar epinephrine did not affect the growth rate or expression of A. suis adhesin genes, but there was a significant growth phase effect for many genes. Most adhesin genes were also differentially expressed during anoxic static growth or aerobic growth, and in this study, all genes were differentially expressed in either exponential or stationary phase. Based on the time*treatment interactions observed in the anoxic study, a model of persistence of A. suis in the host environment in biofilm and planktonic states is proposed. Biofilm dynamics were further studied using wild type and isogenic mutants of the type IVb pilin (Δ flp1), the OmpA outer membrane protein (ΔompA), and the fibronectin-binding (ΔcomE1) genes. Disruption of these adhesin genes affected the early stages of biofilm formation, but in most cases, biofilm formation of the mutant strains was similar to that of the wild type by 24h of incubation. We postulate that other adhesins may have overlapping functions that can compensate for those of the missing adhesins.

Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation.

BACKGROUND: Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae. RESULTS: For the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure. CONCLUSIONS: In conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by cross-feeding, which enables A. pleuropneumoniae to grow and form multi-species biofilms.

Actinobacillus pleuropneumoniae induces SJPL cell cycle arrest in G2/M-phase and inhibits porcine reproductive and respiratory syndrome virus replication.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry and causes important economic losses. No effective antiviral drugs against it are commercially available. We recently reported that the culture supernatant of Actinobacillus pleuropneumoniae, the porcine pleuropneumonia causative agent, has an antiviral activity in vitro against PRRSV in SJPL cells. Objectives of this study were (i) to identify the mechanism behind the antiviral activity displayed by A. pleuropneumoniae and (ii) to characterize the active molecules present in the bacterial culture supernatant. METHODS: Antibody microarray analysis was used in order to point out cellular pathways modulated by the A. pleuropneumoniae supernatant. Subsequent, flow cytometry analysis and cell cycle inhibitors were used to confirm antibody microarray data and to link them to the antiviral activity of the A. pleuropneumoniae supernatant. Finally, A. pleuropneumoniae supernatant characterization was partially achieved using mass spectrometry. RESULTS: Using antibody microarray, we observed modulations in G2/M-phase cell cycle regulation pathway when SJPL cells were treated with A. pleuropneumoniae culture supernatant. These modulations were confirmed by a cell cycle arrest at the G2/M-phase when cells were treated with the A. pleuropneumoniae culture supernatant. Furthermore, two G2/M-phase cell cycle inhibitors demonstrated the ability to inhibit PRRSV infection, indicating a potential key role for PRRSV infection. Finally, mass spectrometry lead to identify two molecules (m/z 515.2 and m/z 663.6) present only in the culture supernatant. CONCLUSIONS: We demonstrated for the first time that A. pleuropneumoniae is able to disrupt SJPL cell cycle resulting in inhibitory activity against PRRSV. Furthermore, two putative molecules were identified from the culture supernatant. This study highlighted the cell cycle importance for PRRSV and will allow the development of new prophylactic or therapeutic approaches against PRRSV.

Biofilm formation by virulent and non-virulent strains of Haemophilus parasuis.

Haemophilus parasuis is a commensal bacterium of the upper respiratory tract of healthy pigs. It is also the etiological agent of Glässer's disease, a systemic disease characterized by polyarthritis, fibrinous polyserositis and meningitis, which causes high morbidity and mortality in piglets. The aim of this study was to evaluate biofilm formation by well-characterized virulent and non-virulent strains of H. parasuis. We observed that non-virulent strains isolated from the nasal cavities of healthy pigs formed significantly (p < 0.05) more biofilms than virulent strains isolated from lesions of pigs with Glässer's disease. These differences were observed when biofilms were formed in microtiter plates under static conditions or formed in the presence of shear force in a drip-flow apparatus or a microfluidic system. Confocal laser scanning microscopy using different fluorescent probes on a representative subset of strains indicated that the biofilm matrix contains poly-N-acetylglucosamine, proteins and eDNA. The biofilm matrix was highly sensitive to degradation by proteinase K. Comparison of transcriptional profiles of biofilm and planktonic cells of the non-virulent H. parasuis F9 strain revealed a significant number of up-regulated membrane-related genes in biofilms, and genes previously identified in Actinobacillus pleuropneumoniae biofilms. Our data indicate that non-virulent strains of H. parasuis have the ability to form robust biofilms in contrast to virulent, systemic strains. Biofilm formation might therefore allow the non-virulent strains to colonize and persist in the upper respiratory tract of pigs. Conversely, the planktonic state of the virulent strains might allow them to disseminate within the host.

Actinobacillus pleuropneumoniae possesses an antiviral activity against porcine reproductive and respiratory syndrome virus.

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (<1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon γ. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools.

Novel genes associated with biofilm formation of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a gram-negative bacterium and is the causative agent of swine pleuropneumonia, a highly contagious respiratory disease. Biofilm formation is an important ability possessed by numerous bacterial pathogens. The purpose of this study was to identify and characterize biofilm mutants of A. pleuropneumoniae serotype 1 strain S4074 created using a mini Tn-10 transposon. The transposon library was screened to identify mutants with a modified ability to form biofilms in polystyrene microtiter plates. A total of 1200 mutants were screened and the analysis identified 24 mutants that exhibited abnormal biofilm formation, at least 16 unique genes were identified. Most genes identified in the enhanced-biofilm mutants encoded proteins with unknown functions, whereas most genes identified in the biofilm-reduced mutants encoded proteins related to transport, protein synthesis and nucleic acid synthesis. Approximately 50% of genes, including hns, potD2, ptsI, tig and rpmF, identified in our screen have been previously associated with biofilm formation in A. pleuropneumoniae and other bacterial species, and thus validated the screening method. The rest of genes identified, such as APL_0049, APL_0637 and APL_1572, have not been previously associated with biofilm formation. Interestingly, gene APL_0049 was previously seen among the genes differentially expressed during a natural infection of pig lungs. Preliminary characterization of the mutants was also initiated by assessing their hydrophobicity, their biofilm matrix composition and their ability to adhere to a polystyrene surface or NPTr cells. Based on the preliminary characterization, some of the mutants identified appear to have deficiencies during the initial attachment or growth of the biofilm. In conclusion, transposon mutagenesis analysis allowed the identification of new genes associated with biofilm formation in A. pleuropneumoniae.

Effects of growth conditions on biofilm formation by Actinobacillus pleuropneumoniae.

Biofilm formation is an important virulence trait of many bacterial pathogens. It has been reported in the literature that only two of the reference strains of the swine pathogen Actinobacillus pleuropneumoniae, representing serotypes 5b and 11, were able to form biofilm in vitro. In this study, we compared biofilm formation by the serotype 1 reference strain S4074 of A. pleuropneumoniae grown in five different culture media. We observed that strain S4074 of A. pleuropneumoniae is able to form biofilms after growth in one of the culture conditions tested brain heart infusion (BHI medium, supplier B). Confocal laser scanning microscopy using a fluorescent probe specific to the poly-N-acetylglucosamine (PGA) polysaccharide further confirmed biofilm formation. In accordance, biofilm formation was susceptible to dispersin B, a PGA hydrolase. Transcriptional profiles of A. pleuropneumoniae S4074 following growth in BHI-B, which allowed a robust biofilm formation, and in BHI-A, in which only a slight biofilm formation was observed, were compared. Genes such as tadC, tadD, genes with homology to autotransporter adhesins as well as genes pgaABC involved in PGA biosynthesis and genes involved in zinc transport were up-regulated after growth in BHI-B. Interestingly, biofilm formation was inhibited by zinc, which was found to be more present in BHI-A (no or slight biofilm) than in BHI-B. We also observed biofilm formation in reference strains representing serotypes 3, 4, 5a, 12 and 14 as well as in 20 of the 37 fresh field isolates tested. Our data indicate that A. pleuropneumoniae has the ability to form biofilms under appropriate growth conditions and transition from a biofilm-positive to a biofilm-negative phenotype was reversible.

Mutation in the LPS outer core biosynthesis gene, galU, affects LPS interaction with the RTX toxins ApxI and ApxII and cytolytic activity of Actinobacillus pleuropneumoniae serotype 1.

Lipopolysaccharides (LPS) and Apx toxins are major virulence factors of Actinobacillus pleuropneumoniae, a pathogen of the respiratory tract of pigs. Here, we evaluated the effect of LPS core truncation in haemolytic and cytotoxic activities of this microorganism. We previously generated a highly attenuated galU mutant of A. pleuropneumoniae serotype 1 that has an LPS molecule lacking the GalNAc-Gal II-Gal I outer core residues. Our results demonstrate that this mutant exhibits wild-type haemolytic activity but is significantly less cytotoxic to porcine alveolar macrophages. However, no differences were found in gene expression and secretion of the haemolytic and cytotoxic toxins ApxI and ApxII, both secreted by A. pleuropneumoniae serotype 1. This suggests that the outer core truncation mediated by the galU mutation affects the toxins in their cytotoxic activities. Using both ELISA and surface plasmon resonance binding assays, we demonstrate a novel interaction between LPS and the ApxI and ApxII toxins via the core oligosaccharide. Our results indicate that the GalNAc-Gal II-Gal I trisaccharide of the outer core is fundamental to mediating LPS/Apx interactions. The present study suggests that a lack of binding between LPS and ApxI/II affects the cytotoxicity and virulence of A. pleuropneumoniae.

Isolation of an atypical strain of Actinobacillus pleuropneumoniae serotype 1 with a truncated lipopolysaccharide outer core and no O-antigen.

A field isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, was sent to the diagnostic laboratory for serotyping. The isolate presented a clear reaction, with both polyclonal antibodies against serotype 1 and monoclonal antibodies against the capsular polysaccharide of serotype 1. It also exhibited a PCR profile of Apx toxins expected for serotype 1. The isolate, however, failed to react with monoclonal antibodies against the O-antigen of serotype 1 lipopolysaccharide (LPS), suggesting a rough phenotype. The lipid A-core region of the isolate migrated faster than the corresponding region of the serotype 1 reference strain S4074 by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the presence of a truncated core. Sugar analysis and mass spectrometry analysis of the O-deacylated LPS from the field isolate were consistent with the absence of O-antigen and truncation of the outer core compared to the wild-type reference strain. Experimental infection of pigs confirmed the virulence of the isolate. This is the first report of an isolate of A. pleuropneumoniae serotype 1 with a truncated outer core and a rough LPS phenotype. Veterinary diagnostic laboratories should be vigilant, since infections caused by such an isolate will not be detected by serological tests based on LPS O-antigen.

Identification and preliminary characterization of a 75-kDa hemin- and hemoglobin-binding outer membrane protein of Actinobacillus pleuropneumoniae serotype 1.

The reference strains representing serotypes 1 to 12 of Actinobacillus pleuropneumoniae biotype 1 were examined for their ability to utilize porcine hemoglobin (Hb) or porcine hemin (Hm) as iron sources for growth. In a growth promotion assay, all of the reference strains were able to use porcine Hb, and all strains except 2 were able to use porcine Hm. Using a preliminary characterization procedure with Hm- or Hb-agarose, Hm- and Hb-binding outer membrane proteins (OMPs) of approximately 75 kDa were isolated from A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions. Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) analysis revealed a number of common tryptic peptides between the Hb-agarose- and Hm-agarose-purified 75 kDa OMPs, strongly suggesting that these peptides originate from the same protein. A database search of these peptide sequences revealed identities with proteins from various Gram-negative bacteria, including iron-regulated OMPs, transporter proteins, as well as TonB-dependent receptors. Taken together, our data suggest that A. pleuropneumoniae synthesizes potential Hm- and Hb-binding proteins that could be implicated in the iron uptake from porcine Hb and Hm.

Identification, cloning and characterization of rfaE of Actinobacillus pleuropneumoniae serotype 1, a gene involved in lipopolysaccharide inner-core biosynthesis.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and its lipopolysaccharides (LPS) have been identified as important adhesins involved in adherence to host cells. To better understand the role of LPS core in the virulence of this organism, the aim of the present study was to identify and clone genes involved in LPS core biosynthesis by complementation with Salmonella enterica serovar Typhimurium mutants (rfaC, rfaD, rfaE and rfaF). Complementation with an A. pleuropneumoniae 4074 genomic library was successful with Salmonella mutant SL1102. This Salmonella deep-rough LPS mutant is defective for the rfaE gene, which is an ADP-heptose synthase. Novobiocin was used to select transformants that had the smooth-LPS type, since Salmonella strains with wild-type smooth-LPS are less permeable, therefore more resistant to hydrophobic antibiotics like novobiocin. We obtained a clone that was able to restore the wild-type smooth-LPS Salmonella phenotype after complementation. The wild-type phenotype was confirmed using phage (Felix-O, P22c.2 and Ffm) susceptibility and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the open reading frames contained in the 3.3-kb insert in the plasmid encoded a 475-amino-acid protein with 71% identity and 85% similarity to the RfaE protein of S. enterica. We then attempted to generate an A. pleuropneumoniae rfaE mutant by gene replacement. The rfaE gene seems essential in A. pleuropneumoniae viability as we were unable to isolate a heptose-less knockout mutant.

Molecular cloning and characterization of the ferric hydroxamate uptake (fhu) operon in Actinobacillus pleuropneumoniae.

The bacterium Actinobacillus pleuropneumoniae, a swine pathogen, utilizes ferrichrome as an iron source. This study details the molecular cloning and sequencing of the genes involved in the uptake of this hydroxamate siderophore. Four ferric hydroxamate uptake (fhu) genes, fhuC, fhuD, fhuB and fhuA, were identified in a single operon, and these were found to encode proteins homologous to proteins of the fhu systems of several bacteria, including Escherichia coli. The fhuA gene encodes the 77 kDa outer-membrane protein (OMP) FhuA, the receptor for ferrichrome. FhuD is the 35.6 kDa periplasmic protein responsible for the translocation of ferric hydroxamate from the outer to the inner membrane. FhuC (28.5 kDa) and FhuB (69.4 kDa) are cytoplasmic-membrane-associated proteins that are components of an ABC transporter which internalizes the ferric hydroxamate. Reference strains of A. pleuropneumoniae that represented serotypes 1 to 12 of this organism all tested positive for the four fhu genes. When A. pleuropneumoniae FhuA was affinity-tagged with hexahistidine at its amino terminus and expressed in an E. coli host, the recombinant protein reacted with an mAb against E. coli FhuA, as well as with a polyclonal pig serum raised against an A. pleuropneumoniae infection. Hence, the authors conclude that fhuA is expressed in vivo by A. pleuropneumoniae. Three-dimensional modelling of the OMP FhuA was achieved by threading it to the X-ray crystallographic structure of the homologous protein in E. coli. FhuA from A. pleuropneumoniae was found to have the same overall fold as its E. coli homologue, i.e. it possesses an N-terminal cork domain followed by a C-terminal beta-barrel domain and displays 11 extracellular loops and 10 periplasmic turns.

Identification of genes involved in biosynthesis of Actinobacillus pleuropneumoniae serotype 1 O-antigen and biological properties of rough mutants.

Actinobacillus pleuropneumoniae is an important pathogen of swine. Lipopolysaccharide (LPS) has been identified as the major adhesin of A. pleuropneumoniae and it is involved in adherence to porcine respiratory tract cells. We previously generated seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn10 transposon mutagenesis system [Rioux S, Galarneau C, Harel J et al. Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1. Can J Microbiol 1999; 45: 1017-1026]. The purpose of the present study was to characterize these mutants in order to learn more about LPS O-antigen biosynthesis genes and their organization in A. pleuropneumoniae, and to determine the surface properties and virulence in pigs of these isogenic mutants. By mini-Tn10 insertions in rough mutants, four putative genes (ORF12, ORF16, ORF17, and ORF18) involved in O-antigen biosynthesis in A. pleuropneumoniae serotype 1 were found within a region of 18 ORFs. This region is homologous to the gene cluster of serotype-specific O-polysaccharide biosynthesis from A. actinomycetemcomitans strain Y4 (serotype b). Two mutants showed homology to a protein with identity to glycosyltransferases (ORF12); two others had the mini-Tn10 insertion localized in genes encoding for two distinct proteins with identity to rhamnosyltransferases (ORF16 and ORF17) and three showed homology to a protein which is known to initiate polysaccharide synthesis (ORF18). These four ORFs were also present in A. pleuropneumoniae serotypes 9 and 11 that express an O-antigen that serologically cross-reacts with serotype 1. Evaluation of some biological properties of rough mutants seems to indicate that the absence of O-chains does not appear to have an influence on the virulence of the bacteria in pigs and on the overall surface hydrophobicity, charge and hemoglobin-binding activity, or on LAL activation. An acapsular mutant was included in the present study in order to compare the influence of O-chains and capsule polysaccharides on different cell surface properties. Our data suggest that capsular polysaccharides and not O-chains polysaccharides have a major influence on surface properties of A. pleuropneumoniae serotype 1 and its virulence in pigs.