The capsular polysaccharide of Actinobacillus pleuropneumoniae serotype 5A: Role in serum resistance and characterization of the genetic basis for expression

摘要

Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide (CP) that protects this bacterium from host defenses. In the presence of anti-CP IgG, encapsulated A. pleuropneumoniae K17 was killed in precolostral calf serum (PCS) but not in normal serum used as a complement source. In contrast, two capsule-deficient mutants were killed in normal serum. The CP of A. pleuropneumoniae contributed to serum-resistance by limiting the amount of C9, a component of the membrane attack complex, but not C3, that bound to the bacteria in PCS. A second mechanism of serum resistance was due to a lipopolysaccharide (LPS)-specific antibody present in the IgG fractions of normal swine serum, swine anti-K17 serum, and guinea pig anti-K17 LPS serum that blocked anti-CP IgG complement-mediated killing of A. pleuropneumoniae. This LPS-specific antibody prevented complement-mediated killing of K17 in the presence of potentially bactericidal anti-CP IgG by reducing the deposition of C9 onto A. pleuropneumoniae, and by directing the deposition of C9 to sites on the bacteria where the bound C9 was easily eluted. Thus, CP and anti-LPS antibody may act synergistically or at different stages of infection to limit the ability of complement to eliminate A. pleuropneumoniae.;Two overlapping regions of the A. pleuropneumoniae J45 capsulation locus were cloned and partially sequenced. One region was conserved among A. pleuropneumoniae serotypes and contained four open reading frames, cpxDCBA, that were highly homologous at both the nucleotide and amino acid levels to genes involved in the export of the CP of H. influenzae type b (bexDCBA), Neisseria meningitidis group B (ctrABCD), and to a lesser extent Escherichia coli K1 and K5 (kpsED, kpsMT). The J45 cpxDCBA gene cluster was able to partially complement kpsM::TnphoA or kpsT::TnphoA mutations within a plasmid-encoded E. coli K5 kps locus and restored sensitivity to a K5-specific bacteriophage, indicating that cpxDCBA functioned in capsular polysaccharide export. A DNA region adjacent to A. pleuropneumoniae J45 cpxDCBA was identified that was serotype-specific. This region contained two complete open reading frames (cpsA and cpsB), and a third partial open reading frame, cpsC. These genes may encode proteins involved in A. pleuropneumoniae J45 CP biosynthesis. A recombinant A. pleuropneumoniae J45 mutant in which the three serotype-specific genes, cpsABC, were partially or completely deleted was generated by allelic exchange. This mutant did not produce intracellular or extracellular CP, was serum-sensitive, and was attenuated in pigs. These studies demonstrated that CP contributed to the serum-resistance and virulence of A. pleuropneumoniae. This noncapsulated mutant will be evaluated as a potential live vaccine strain for the control of swine pleuropneumonia.