Cloning, sequencing and expression of the transferrin-binding protein 1 gene from Actinobacillus pleuropneumoniae.
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ABSTRACT:
SUBMITTER: Daban M
PROVIDER: S-EPMC1217180 | biostudies-other | 1996 Apr
REPOSITORIES: biostudies-other
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Project description:Actinobacillus pleuropneumoniae can use porcine transferrin as the sole source of iron. Two proteins with molecular masses of approximately 60 kDa (TfbA) and 110 kDa have been shown to specifically bind porcine transferrin; from the TfbA protein, three isoforms from A. pleuropneumoniae serotypes 1, 5, and 7 have been identified and characterized by nucleotide sequence analysis. Here we defined the transferrin-binding region(s) of the TfbA protein of A. pleuropneumoniae serotype 7 by TnphoA mutagenesis, random mutagenesis, and peptide spot synthesis. The amino-terminal half of the TfbA molecule, which has only 36% amino acid sequence identity among the three isoforms, was shown to be responsible for transferrin binding by TnphoA mutagenesis. This result was confirmed by analysis of six random mutants with decreased transferrin binding affinity. The subsequent analysis of overlapping 16-mer peptides comprising the amino-terminal half of the TfbA molecule revealed three domains of 13 or 14 amino acids in length with transferrin-binding activity. They overlapped, or were very close to, point mutations decreasing transferrin-binding ability. The first and third domains were unique to the TfbA protein of A. pleuropneumoniae serotype 7. In contrast, the sequence of the second domain was present in almost identical forms (12 of 14 residues) in the TfbA proteins of A. pleuropneumoniae serotypes 1 and 5; in addition, a sequence consisting of functionally homologous amino acids was present in the otherwise completely distinct small transferrin-binding proteins of Neisseria gonorrhoeae (TbpB), N. meningitidis (Tbp2), and Haemophilus influenzae (Tbp2).
| S-EPMC173541 | biostudies-other
Project description:Bacteria can use mammalian hormones to modulate pathogenic processes that play essential roles in disease development. Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry globally. Stress is known to contribute to the outcome of A. pleuropneumoniae infection. To test whether A. pleuropneumoniae could respond to stress hormone catecholamines, gene expression profiles after epinephrine (Epi) and norepinephrine (NE) treatment were compared with those from untreated bacteria. The microarray results showed that 158 and 105 genes were differentially expressed in the presence of Epi and NE, respectively. These genes were assigned to various functional categories including many virulence factors. Only 18 genes were regulated by both hormones. These genes included apxIA (the ApxI toxin structural gene), pgaB (involved in biofilm formation), APL_0443 (an autotransporter adhesin) and genes encoding potential hormone receptors such as tyrP2, the ygiY-ygiX (qseC-qseB) operon and narQ-narP (involved in nitrate metabolism). Further investigations demonstrated that cytotoxic activity was enhanced by Epi but repressed by NE in accordance with apxIA gene expression changes. Biofilm formation was not affected by either of the two hormones despite pgaB expression being affected. Adhesion to host cells was induced by NE but not by Epi, suggesting that the hormones affect other putative adhesins in addition to APL_0443. This study revealed that A. pleuropneumoniae gene expression, including those encoding virulence factors, was altered in response to both catecholamines. The differential regulation of A. pleuropneumoniae gene expression by the two hormones suggests that this pathogen may have multiple responsive systems for the two catecholamines.
| S-EPMC3275570 | biostudies-literature
Project description:BACKGROUND:Actinobacillus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia, is an important pathogen of swine throughout the world. It must rapidly overcome the innate pulmonary immune defenses of the pig to cause disease. To better understand this process, the objective of this study was to identify genes that are differentially expressed in a medium that mimics the lung environment early in the infection process. METHODS AND PRINCIPAL FINDINGS:Since bronchoalveolar lavage fluid (BALF) contains innate immune and other components found in the lungs, we examined gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after a 30 min exposure to BALF, using DNA microarrays and real-time PCR. The functional classes of genes found to be up-regulated most often in BALF were those encoding proteins involved in energy metabolism, especially anaerobic metabolism, and in cell envelope, DNA, and protein biosynthesis. Transcription of a number of known virulence genes including apxIVA and the gene for SapF, a protein which is involved in resistance to antimicrobial peptides, was also up-regulated in BALF. Seventy-nine percent of the genes that were up-regulated in BALF encoded a known protein product, and of these, 44% had been reported to be either expressed in vivo and/or involved in virulence. CONCLUSIONS:The results of this study suggest that in early stages of infection, A. pleuropneumoniae may modulate expression of genes involved in anaerobic energy generation and in the synthesis of proteins involved in cell wall biogenesis, as well as established virulence factors. Given that many of these genes are thought to be expressed in vivo or involved in virulence, incubation in BALF appears, at least partially, to simulate in vivo conditions and may provide a useful medium for the discovery of novel vaccine or therapeutic targets.
| S-EPMC2700959 | biostudies-literature
Project description:The gene encoding the Actinobacillus pleuropneumoniae serotype 1 transferrin-binding protein (tfbA) was cloned, and the carboxy-terminal 70% of the protein was expressed as an aggregate protein in Escherichia coli. The nucleotide sequences of the tfbA genes from A. pleuropneumoniae serotypes 7 (G.-F. Gerlach, C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson, Infect. Immun. 60:892-898, 1992) and 1 were determined, and a comparison revealed that they had 65% sequence identity. The deduced amino acid sequences showed a sequence agreement of 55%, and both proteins possessed a lipoprotein-like signal sequence. The serotype 1 TfbA protein had a predicted molecular mass of 65 kDa, compared with 60 kDa for the serotype 7 TfbA protein, and both proteins were immunologically distinct as assessed in a competitive enzyme-linked immunosorbent assay. Southern hybridization and Western blot (immunoblot) analysis of the 13 A. pleuropneumoniae type strains revealed that serotypes 2, 3, 4, 8, 9, 10, and 11 encode and express a TfbA protein highly homologous to that of A. pleuropneumoniae serotype 7 whereas the TfbA proteins and the encoding genes of serotypes 6 and 12 were highly homologous to that found in A. pleuropneumoniae serotype 1. The tfbA genes of A. pleuropneumoniae serotypes 5A and 5B were recognized, under medium-stringency hybridization conditions, by the A. pleuropneumoniae serotype 1-derived tfbA probe, and the respective proteins were weakly reactive with the antibody raised against the A. pleuropneumoniae serotype 7 TfbA protein.
| S-EPMC257309 | biostudies-other
Project description:Urea transporters (UTs) effect rapid flux of urea across biological membranes. In the mammalian kidney, UT activity is essential for effective urine concentration. In bacteria, UT-mediated urea uptake permits intracellular urease to degrade urea to ammonia and CO(2), a process that either buffers acid loads or provides nutrient nitrogen. We have characterized the urea transport channel protein ApUT from Actinobacillus pleuropneumoniae. Kinetic analysis of bacterial inside-out membranes enriched in ApUT showed approximately 28-fold increase in urea permeability (3.3 +/- 0.4 x 10(-4) cm/s) compared with control vesicles (0.11 +/- 0.02 x 10(-4) cm/s). In addition to urea, ApUT also conducts water. Urea and water transport across the channel was phloretin and mercury inhibitable, and the site of inhibition may be located on the cytoplasmic side of the protein. Glycerol and urea analogs, such as methylamine, dimethylurea, formamide, acetamide, methylurea, propanamide, and ethylamine did not permeate across ApUT.
| S-EPMC2698602 | biostudies-literature
Project description:To study the role of Actinobacillus pleuropneumoniae-RTX-toxin III (ApxIII) in the pathogenesis of porcine pleuropneumonia, we cloned and characterized the gene encoding this toxin. For that purpose, we screened an expression library of genomic DNA of serotype 8 with an ApxIII-specific monoclonal antibody and isolated a 425-bp fragment of an immunoreactive clone. Using this fragment as a probe, we identified and cloned an overlapping chromosomal NsiI restriction fragment of 5.0 kbp. Escherichia coli cells that contained this fragment produced a protein similar to ApxIII. Like ApxIII, the protein had a molecular mass of approximately 120 kDa, was recognized by an ApxIII-specific antibody, killed porcine lung macrophages, and was not lytic for sheep erythrocytes. We concluded from these data that the 5.0-kbp NsiI fragment contained the ApxIII-coding gene. Nucleotide sequence analysis of the 5.0-kbp NsiI fragment revealed the presence of two genes, apxIIIC and apxIIIA. These genes coded for proteins ApxIIIC and ApxIIIA, respectively, which were 53 and 50% identical to the prototypic RTX proteins HlyC and HlyA of E. coli. We assumed that the apxIIIA gene coded for the structural RTX toxin and that the apxIIIC gene coded for its activator. In addition, we found that ApxIII could be secreted from E. coli by the heterologous RTX transporter proteins HlyB and HlyD. The deduced amino acid sequence of ApxIIIA was 50% identical to that of ApxIA and 41% identical to that of ApxIIA. We concluded that, beside ApxI and ApxII, ApxIII is the third RTX toxin produced by A. pleuropneumoniae.
| S-EPMC302824 | biostudies-other
Project description:The binding profile of Actinobacillus pleuropneumoniae serotypes 1 and 2 to various glycosphingolipids was evaluated by using thin-layer chromatogram overlay. A. pleuropneumoniae whole cells recognized glucosylceramide (Glcbeta1Cer), galactosylceramide (Galbeta1Cer) with hydroxy and nonhydroxy fatty acids, sulfatide (SO(3)-3Galbeta1Cer), lactosylceramide (Galbeta1-4Glcbeta1Cer), gangliotriaosylceramide GgO3 (GalNAcbeta1-4Galbeta1-4Glcbeta1Cer), and gangliotetraosylceramide GgO4 (Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1Cer) glycosphingolipids. We observed no binding to globoseries, globotriaosylceramide Gb3, globoside Gb4, or Forssman Gb5 glycosphingolipids or to gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, and GT1b. The A. pleuropneumoniae strains tested also failed to detect phosphatidylethanolamine or ceramide. Interestingly, extracted lipopolysaccharide (LPS) of serotype 1 and serotype 2 as well as detoxified LPS of serotype 1 showed binding patterns similar to that of whole bacterial cells. Binding to GlcCer, GalCer, sulfatide, and LacCer, but not to GgO3 and GgO4 glycosphingolipids, was inhibited after incubation of the bacteria with monoclonal antibodies against LPS O antigen. These findings indicate the involvement of LPS in recognition of three groups of glycosphingolipids: (i) GlcCer and LacCer, where glucose is probably an important saccharide sequence required for LPS binding; (ii) GalCer and sulfatide glycosphingolipids, where the sulfate group is part of the binding epitope of the isoreceptor; and (iii) GgO3 and GgO4, where GalNacbeta1-4Gal disaccharide represents the minimal common binding epitope. Taken together, our results indicate that A. pleuropneumoniae LPS recognize various saccharide sequences found in different glycosphingolipids, which probably represents a strong virulence attribute.
| S-EPMC96842 | biostudies-literature
Project description:LuxS is an enzyme involved in the activated methyl cycle and the by-product autoinducer 2 (AI-2) was a quorum sensing signal in some species. In our previous study, the functional LuxS in AI-2 production was verified in the porcine respiratory pathogen Actinobacillus pleuropneumoniae. Enhanced biofilm formation and reduced virulence were observed in the luxS mutant. To comprehensively understand the luxS function, in this study, the transcriptional profiles were compared between the A. pleuropneumoniae luxS mutant and its parental strain in four different growth phases using microarray. Many genes associated with infection were differentially expressed. The biofilm formation genes pgaABC in the luxS mutant were up-regulated in early exponential phase, while 8 genes associated with adhesion were down-regulated in late exponential phase. A group of genes involved in iron acquisition and metabolism were regulated in four growth phases. Further investigations on these virulence traits demonstrated that the luxS mutant showed enhanced biofilm formation and reduced adhesion ability and these effects were not due to lack of AI-2. But AI-2 could increase biofilm formation and adhesion of A. pleuropneumoniae independent of LuxS. Growth under iron restricted condition could be controlled by LuxS through AI-2 production. These results revealed pleiotropic roles of LuxS and AI-2 on A. pleuropneumoniae virulence traits.
2011-11-09 | GSE19056 | GEO
Project description:BACKGROUND: Actinobacillus pleuropneumoniae causes contagious pleuropneumonia, an economically important disease of commercially reared pigs throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Since bronchoalveolar fluid (BALF) contains many of the innate immune and other components found in the lungs, we examined the gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after exposure to concentrated BALF for 30 min. RESULTS: In reverse transcription PCR differential display (RT-PCR DD) experiments, A. pleuropneumoniae CM5 exposed to BALF up-regulated, among other genes, a gene predicted to encode LamB, an outer-membrane transport protein of the maltose regulon. To determine the role of the lamB and other genes of the maltose regulon in the pathogenesis of A. pleuropneumoniae, knockout mutations were created in the lamB and malT genes, the latter being the positive transcriptional regulator of the maltose regulon. Relative to the lamB mutant and the wild type, the malT mutant had a significant (P < 0.05) decrease in growth rate and an increased sensitivity to fresh porcine serum and high concentrations (more than 0.5 M) of sodium chloride. In DNA microarray experiments, the BALF-exposed malT mutant exhibited a gene-expression profile resembling that of a stringent type gene-expression profile seen in bacteria facing amino acid or carbon starvation. Genes encoding proteins for protein synthesis, energy metabolism, and DNA replication were down-regulated, while genes involved in stringent response (e.g., relA), amino acid and nucleotide biosynthesis, biofilm formation, DNA transformation, and stress response were up-regulated. CONCLUSION: These results suggest that MalT may be involved in protection against some stressors and in the transport of one or more essential nutrients in BALF. Moreover, if MalT is directly or indirectly linked to the stringent response, an important global mechanism of bacterial persistence and virulence in many bacterial pathogens, it might play a role in A. pleuropneumoniae pathogenesis.
| S-EPMC2752462 | biostudies-literature
Project description:The aim of this study was to evaluate the correlation between antimicrobial resistance (AMR) profiles of 96 clinical isolates of Actinobacillus pleuropneumoniae, an important porcine respiratory pathogen, and the identification of AMR genes in whole genome sequence (wgs) data. Susceptibility of the isolates to nine antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, sulfisoxazole, tetracycline, tilmicosin, trimethoprim, and tylosin) was determined by agar dilution susceptibility test. Except for the macrolides tested, elevated MICs were highly correlated to the presence of AMR genes identified in wgs data using ResFinder or BLASTn. Of the isolates tested, 57% were resistant to tetracycline [MIC ≥ 4 mg/L; 94.8% with either tet(B) or tet(H)]; 48% to sulfisoxazole (MIC ≥ 256 mg/L or DD = 6; 100% with sul2), 20% to ampicillin (MIC ≥ 4 mg/L; 100% with blaROB-1), 17% to trimethoprim (MIC ≥ 32 mg/L; 100% with dfrA14), and 6% to enrofloxacin (MIC ≥ 0.25 mg/L; 100% with GyrAS83F). Only 33% of the isolates did not have detectable AMR genes, and were sensitive by MICs for the antimicrobial agents tested. Although 23 isolates had MIC ≥ 32 mg/L for tylosin, all isolates had MIC ≤ 16 mg/L for both erythromycin and tilmicosin, and no macrolide resistance genes or known point mutations were detected. Other than the GyrAS83F mutation, the AMR genes detected were mapped to potential plasmids. In addition to presence on plasmid(s), the tet(B) gene was also found chromosomally either as part of a 56 kb integrative conjugative element (ICEApl1) in 21, or as part of a Tn7 insertion in 15 isolates. Our results indicate that, with the exception of macrolides, wgs data can be used to accurately predict resistance of A. pleuropneumoniae to the tested antimicrobial agents and provides added value for routine surveillance.
| S-EPMC5337627 | biostudies-literature