CLONING AND MUTAGENESIS OF A SEROTYPE-SPECIFIC DNA REGION INVOLVED INENCAPSULATION AND VIRULENCE OF ACTINOBACILLUS-PLEUROPNEUMONIAE SEROTYPE 5A - CONCOMITANT EXPRESSION OF SEROTYPE-5A AND SEROTYPE-1 CAPSULAR POLYSACCHARIDES IN RECOMBINANT A-PLEUROPNEUMONIAE SEROTYPE-1

A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 ca psular polysaccharide (CP) biosynthesis was identified and characteriz ed by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned fro m a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 g enomic DNA. DNA sequence analysis demonstrated that this region contai ned four complete open reading frames, cps5A, cps5B, cps5C, and cps5D, Cps5A, Cps5B, and Cps5C showed low homology with several bacterial gl ycosyltransferases 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-negat ive 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 fra mes was constructed and transferred into the J45 chromosome by homolog ous recombination with a kanamycin resistance cassette to produce muta nt J45-100. Multiplex PCR confirmed the deletion in this region of J45 -100 DNA. J45-100 did not produce intracellular or extracellular CP, i ndicating that cps5A, cps5B, and/or cps5C were involved in CP biosynth esis. However, biosynthesis of the Apr 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 sensiti ve 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 mod erate lung lesions but not death, Electroporation of cps5ABC into A. p leuropneumoniae 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 t o 4074. The recombinant strain produced significantly less total CP (s erotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significan tly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia i n pigs and mice following respiratory challenge than strain 4074, indi cating that virulence was affected by diminished capsule production. T hese results emphasize the importance of CP in the serum resistance an d virulence of A. pleuropneumoniae.