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 (LD
50) of strain J45. At six times the J45 LD
50, 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.
Actinobacillus pleuropneumoniae is an encapsulated, gram-negative bacterium that causes swine pleuropneumonia, a frequently fatal and highly contagious respiratory disease. There are 12 recognized serotypes of
A. pleuropneumoniae that vary in virulence and geographic predominance (
33). The capsular polysaccharide (CP) is responsible for serotype specificity and is required for virulence (
19,
20). Nonencapsulated
A. pleuropneumoniae mutants obtained by chemical mutagenesis have been shown to be effective vaccine candidates in that they are safe and highly protective (
20). However, not all such mutants are stable, and the nature of the mutation(s) is unknown. Hence, characterization of the genes involved in CP export and biosynthesis would be desirable.We have previously reported cloning and sequencing an
A. pleuropneumoniae DNA region involved in export of the CP of serotype 5a. This region consists of four genes, designated
cpxABCD, which had a high degree of homology to the group II capsule export genes of
Haemophilus influenzae type b (
bexDCBA),
Neisseria meningitidis group B (
ctrABCD), and to a lesser extent,
Escherichia coli K5 (
kpsE and
kpsMT) (
54). This homology suggested that
A. pleuropneumoniae also synthesized a group II capsule, whose organization predicted that the biosynthesis region would be upstream of
cpxDCBA (
12). We now report cloning and sequencing of four genes upstream of the
cpx CP export region that correspond to capsular biosynthesis genes. A mutant with a deletion in three of these genes by allelic exchange was incapable of synthesizing CP and was avirulent in pigs.
trans expression of these three genes in serotype 1 resulted in a chimeric strain producing both serotype 1 and 5 CP. However, expression of serotype 5 CP genes resulted in diminished production of serotype 1 CP, as well as diminished virulence in pigs and mice.
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