REDUCED POLYSIALIC ACID CAPSULE EXPRESSION IN ESCHERICHIA-COLI K1 MUTANTS WITH CHROMOSOMAL DEFECTS IN KPSF

Neuroinvasive Escherichia coli K1 synthesizes and assembles a polysial ic acid capsule virulence factor on the external leaflet of the outer membrane. This capsule functions in pathogenesis by blocking nonimmune host defence mechanisms and acting as a relatively non-immunogenic mo lecular mimic of the polysialic acid chains found in high concentratio ns on neural cell adhesion molecules of the human embryo and neonate. The synthetic, regulatory and export components for capsule expression are encoded in three functionally distinct gene blocks or regions of the 20 kb kps 'pathogenicity island'. These regions are organized as t wo convergently transcribed operons inserted into the monocistronic tR NA gene, pheV. The six genes of the so-called region 1 operon are tran scribed in the same direction as pheV, and at least four of these gene s are required for polysialic acid export. Expression of this operon i s thermoregulated by transcriptional control of its first gene, kpsF. To investigate the function of region 1 further, two independent chrom osomal disruptions were engineered by inserting promoterless, terminat orless kanamycin or chloramphenicol resistance cassettes into the Hind III site of the kpsF coding sequence. The chromosomal insertions were regulated by temperature in the same way as the wild-type operon, demo nstrating that this control mechanism remained intact in these mutants . Chemical, immunological and ultrastructural microscopical methods de monstrated that full-length polysialic acid chains were synthesized bu t not exported by the kpsF mutants. This phenotype was correlated with decreased plaque diameter when the mutants were infected with the cap sule-specific bacteriophage K1F. The export defect could not be comple mented in trans with kpsF(+) containing its cia-regulatory region beca use of titration of an apparent positive regulator of region 1 express ion, whereas complementation was observed with a plasmid expressing kp sF from a physiologically irrelevant promoter, An N-terminal polyhisti dine peptide was attached to KpsF and used to purify the overproduced polypeptide, Antibodies raised against KpsF identified at least one of its paralogues in E. coli, GutQ, suggesting that KpsF and its homolog ues are membrane associated. The results indicate the requirement for a precise balance between region 1 components of the capsule export ma chinery, and that KpsF plays a positive role in the assembly, operatio n or regulation of this apparatus.