GENETIC EXCHANGE OF DETERMINANTS FOR CAPSULAR POLYSACCHARIDE BIOSYNTHESIS BETWEEN KLEBSIELLA-PNEUMONIAE STRAINS EXPRESSING SEROTYPE-K2 AND SEROTYPE-K21A

The production of a capsular polysaccharide (CPS; K antigen) is charac teristic of Klebsiella pneumoniae, but CPS structure varies among stra ins, and many different serotypes are now known. In this study, cps ge ne clusters encoding the elements of capsular polysaccharide biosynthe sis were exchanged by homologous recombination between strains express ing different serotypes. The wild-type K. pneumoniae strains used for genetic exchange were KPA1 (cpsK2), expressing K2 CPS, and KPB1 (cpsK2 1a), expressing K21a CPS. Plasmid R68.45 was used to mobilize fragment s of chromosomal DNA from auxotrophic derivatives of donor strains. Au xotrophic his alleles introduced into recipient strains provided selec table markers to coinherit the adjacent cps gene clusters from donors expressing a heterologous CPS. Each of the capsule-switched recombinan ts, KPA5 (cpsK21a) and KPB20 (cpsK2), was shown to have a CPS that was immunologically identical to the serotype of the respective donor. Th e recombinants retained their respective recipient strain background, as evidenced by a genetic marker and demonstration of a distinctive re striction fragment length polymorphism in genomic DNA. KPB1 CPS contai ned a sequence (mannose-alpha-2-mannose) that binds to a macrophage le ctin and may be responsible for their higher susceptibility to macroph age binding and phagocytosis compared with KPA1, whose CPS lacked such sequences. The recombinant strains expressing heterologous cps genes inherited the macrophage-binding phenotype of the donor, thus confirmi ng that relative susceptibility to phagocytosis was determined by the capsule type expressed. KPA1 was highly virulent in a mouse lethality assay, which is a feature typical of K2 strains, whereas KPB1 was not virulent in mice. Recombinant KPA5 retained relatively high virulence in mice, even though it produced the heterologous K21a CPS, which sugg ests that a virulence factor other than capsule biosynthesis is encode d by the KPA genomic strain background. In contrast, KPB20 gained marg inal virulence in the mouse lethality assay through the inheritance an d expression of the K2 CPS from the virulent strain. Thus, pathogenesi s in K. pneumoniae may be multifactorial. Specific antibody was used t o stabilize the CPS on the surface of K. pneumoniae, and the structura l organization of the homologous and heterologous capsules was examine d by electron microscopy. Recombinant KPB20, expressing heterologous K 2 CPS, had a uniform layer of capsule surrounding the organism that wa s similar to that seen on the surfaces of the parental strains. Howeve r, KPA5, expressing the heterologous K21a CPS, was unusual in that the uniform capsular layer was physically separated from the cell wall by approximately 50 nm. Within this zone of separation, numerous and reg ularly spaced filaments of CPS were observed between the capsule and t he cell surface. These filaments may have their origins at specific po rts of CPS extrusion in the outer membrane and thus shed light on the mechanism of polymer export in gram-negative bacteria.