Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a.
ABSTRACT: Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens.
Project description:Multiplex PCR assays were developed to identify Actinobacillus pleuropneumoniae serotypes 1, 2, and 8. Primers designed for the conserved capsular polysaccharide (CP) export region amplified a 489-bp DNA fragment from all serotypes. Primers specific to the CP biosynthesis regions of serotypes 1, 2, and 8 amplified fragments of 1.6 kb, 1.7 kb, and 970 bp from only their respective serotypes.
Project description:Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10(2) CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.
Project description:A DNA microarray system was prepared and shown to facilitate identification and typing of Actinobacillus pleuropneumoniae. The DNA microarray, composed of 18 DNA polymerase chain reaction (PCR) amplicons printed on glass slides and arranged in 3 subarrays, was developed. These target DNA included 1 or multiple fragments of the outer membrane lipoprotein, apx toxin, capsular polysaccharide, and disulfide bound formation protein E (dsbE)-like genes of A. pleuropneumoniae. These arrayed target DNA retained their expected hybridization properties. The hybridization signal intensities ranged from the least-intense to the most-intense, 4626 to 9789 arbitrary fluorescence units, respectively. Cy3-probes of A. pleuropneumoniae strains labeled with multiplex PCR were hybridized to the DNA microarray. A total of 51 different A. pleuropneumoniae strains representing serotype 1 to 12 reference strains and clinical isolates were detected and typed by the DNA microarray. Twelve reference serotypes produced 11 distinct target DNA hybridization patterns, and hybridization patterns of serotypes 1 (n = 7), 3 (n = 5), and 7 (n = 6) field isolates were identical to hybridization patterns of reference serotypes 1, 3, and 7, respectively. Non-serotyped isolates 4, 6, and 11 (out of 21) from diseased pigs had identical hybridization patterns to reference serotypes 3, 7, and 1, respectively. The results show that the DNA microarray system described in the present study is a valuable tool for identifying and typing reference strains and isolates of A. pleuropneumoniae, and enables relatively rapid identification of non-serotyped isolates.
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).
Project description:An antibody specific for a 16-kDa outer membrane protein of a rabbit strain of Pasteurella multocida was used to probe representatives of all 16 somatic serotypes of P. multocida, as well as the vaccine strains CU and M9, and all were shown to express the protein. The gene encoding this protein was cloned and sequenced and found to have extensive sequence homology with the gene encoding the P6 protein of Haemophilus influenzae. The protein in P. multocida has been designated P6-like. The gene encoding the P6-like protein was used to probe members of the family Pasteurellaceae and other gram-negative bacteria. Representatives of all 16 somatic serotypes (as well as the vaccine strains CU and M9) of P. multocida hybridized with the P6-like gene under conditions of high stringency. The DNA from H. influenzae hybridized weakly with the P6-like gene under these conditions, but Pasteurella haemolytica (representatives of A and T biotypes), Bordetella bronchiseptica, B. avium, Actinobacillus suis, A. suis-like, A. lignieresii, A. ureae, A. rossii, A. pleuropneumoniae, A. equuli, and various members of the family Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium) did not hybridize detectably. Under conditions of lower stringency, the P6-like gene also hybridized strongly with DNA from P. multocida, H. influenzae, and A. rossii but weakly with DNA from P. haemolytica and members of the genus Actinobacillus. These results suggest that the P6-like protein of P. multocida might be useful as an immunizing product to protect poultry from avian cholera. This suggestion stems from (i) our finding that the P6-like protein in P. multocida is widely distributed among all the somatic serotypes and (ii) the previous work of others demonstrating that the P6 protein of H. influenzae elicits a protective immune response in animal models of human disease.
Project description:We describe here isolation of genetically atypical serotype 6 Actinobacillus pleuropneumoniae in Japan indistinguishable by the multiplex PCR that can discriminate between immunologically cross-reactive serotypes 3, 6 and 8. Nucleotide sequence analysis of capsular export and biosynthesis genes revealed that the atypical isolates have capsular polysaccharide export and synthesis gene sequences that are distinct from those of the serotype 6 reference strain. The atypical strains contain a sequence that is identical with both serotype 3- and 6-specific primers, which causes cross-reactions in multiplex PCR.
Project description:A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames, cps5A, cps5B, cps5C, and cps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-D-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content of cps5ABC was substantially lower (28%) than that of cps5D and the rest of the A. pleuropneumoniae chromosome (42%). A 2.1-kb deletion spanning the cloned cps5ABC open reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating that cps5A, cps5B, and/or cps5C were involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD50) of strain J45. At six times the J45 LD50, J45-100 caused mild to moderate lung lesions but not death. Electroporation of cps5ABC into A. pleuropneumoniae serotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significantly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence of A. pleuropneumoniae.
Project description:The genetic organization of the gene involved in the capsular polysaccharide (CPS) biosynthesis of Actinobacillus pleuropneumoniae serotype 14 has been determined. The DNA region for the CPS biosynthesis of serotype 14 (cps14) comprised 9 open reading frames, designated as cps14AB1B2B3CDEFG genes, encoding Cps14A to Cps14G protein, respectively. Cps14A was similar to CpsA of A. pleuropneumoniae serotypes 1, 4 and 12; the Cps14B1 and Cps14B2 were similar to CpsB of A. pleuropneumoniae serotypes 1, 4 and 12, suggesting that CPS structure of A. pleuropneumoniae serotype 14 would belong to Group I including A. pleuropneumoniae serotypes 1, 4, 12 and 15. Surprisingly, the overall nucleotide sequence, deduced amino acid sequence, and the genetic organization of the cps14 were nearly identical to those of Actinobacillus suis. This study will provide the molecular basic knowledge for development of diagnostics and vaccine of A. pleuropneumoniae serotype 14.
Project description:Two Klebsiella bacteriophages K5-2 and K5-4, which are able to infect and grow on either capsular types K30/K69 and K5 or K8 and K5 of Klebsiella strains, were isolated and characterized. Each phage contained two open reading frames (ORFs), which encoded two putative capsule depolymerases, respectively. The first ORF encoded tail fiber proteins, which have K30/K69 depolymerase and K8 depolymerase activities. The second ORF encoded hypothetical proteins, which are almost identical in amino acid sequences, and have K5 depolymerase activity. Alcian blue staining of enzyme-treated capsular polysaccharides (CPS) showed that purified depolymerases can cleave purified Klebsiella CPS in vitro and liberate monosaccharaides. Capsule K5 deletion mutants were not lysed by either phage, suggesting that the capsule was essential for phage infection. Bacterial killing was observed when incubated Klebsiella strains with phages but not with purified depolymerases. Treatment with the K5-4 phage significantly increased the survival of mice infected with a K. pneumoniae K5 strain. In conclusion, two dual host-specific Klebsiella phages and their tailspikes exhibit capsule depolymerase activity were characterized. Each phage and phage-encoded depolymerase has specificity for capsular type K30/K69, K8 or K5, and could be used for the typing and treatment of K. pneumoniae infection.
Project description:Objective:Klebsiella pneumoniae, one of the clinical superbugs, causes diverse infections because of its variable capsular antigens. This study focused on K. pneumoniae and aimed to assess any correlation between capsular serotype, capsule-associated virulence genes, and evaluate its resistance to conventional antibiotics in order to gain insight into any regional differences. Materials and Methods:A total of 61 K. pneumoniae collected from various clinical specimens were confirmed genotypically. Clinical and demographic data for all patients were reviewed. All isolates were subjected to antimicrobial susceptibility tests. Capsular serotyping and capsule-associated virulence genes were studied using the molecular method. Results:All typeable isolates were typed into K5, K20, and K54 serotypes, and among them, K54 was observed to be predominant. The most common capsule-associated virulence genes comprised uge (93.4%), ycfM (91.8%), and wabG (88.5%), while wcaG (29.5%) and rmpA (21.3%) were noted at much lower prevalence rates. The gene wcaG was significantly associated with K54 positive isolates (p = 0.001), while rmpA was associated with K20 positive isolates (p = 0.01). Conclusion:Serotype K54 had a high frequency in isolates collected from patients with pulmonary diseases, while serotype K20 was associated with burn patients. Carbapenems and levofloxacin were the best therapeutic options for the treatment of infections with serotypes K20 and K54.