An immunological study of the pili ofPseudomonas aeruginosa

Abstract

SUMMARYAn attempt was made to correlate serological relationships determined by the pili, the flagella and the O-somatic antigens ofPseudomonas aeruginosa, and to make a preliminary assessment of use of the pilus antigen as an epidemiological marker. A method is described for the preparation of antiserum specific for the ‘normal’ PSA pili ofP. aeruginosa.A high titre of pilus antibodies was obtained by immunizing rabbits with mutants whose pili had lost their ability to retract into the cell. The ‘normal’ form of the organism, with retractile pili, was poorly agglutinated by high-titre anti-pilus serum, but suspensions of it that had been treated with osmium tetroxide showed greatly increased agglutinability. Antibody labelling for electron microscopy was used to determine the serological relations of pili and of flagella forP. aeruginosastrains belonging to different serological groups as defined by O-somatic antigens. The distribution of pilar and flagellar antigens among strains was not correlated with the O-somatic serotype. A strain ofP. aeruginosacarrying a drug-resistance plasmid had fewer ‘normal’ PSA pili than the background strain.Pseudomonas aeruginosanormally possesses thin polar pili (fimbriae) which constitute one of the classes of heat-labile antigens of the organism. A previous study of the flagellar antigens (Pitt & Bradley, 1975), which are also heat-labile, was an essential preliminary to the development of a useful typing system based on heat-labile antigens. The present work extends this to the pilus antigens. The principle object is to define serological differences determined by pili, flagella, and heat-stable O-somatic antigens. In order to make this comparison, it has been necessary to develop a method for preparing specific antisera against pili, free from flagellar and O-somatic antibodies.Many studies have been made ofP. aeruginosapili; two basic types of pili have so far been established: (1) the ‘normal’ polar pili mentioned above (Bradley, 1966), to be called PSA pili for convenience and (2) the non-polar pili determined by the intergeneric P-group drug-resistance plasmid RP1, called RP1 pili (Bradley, 1974a; C. H. To & C. C. Brinton, in preparation). Non-polar pili have also been found in association with theP. aeruginosa-specific drug-resistance plasmid R130 (L. E. Bryan, University of Alberta, Canada, personal communication), but they are still under study and little is known about them. Since the present work is principally aimed at evaluating the usefulness of pilus antigens as serological markers, emphasis has been placed on PSA pili, though obviously the presence of R-factor-determined pili cannot be ignored.There are two ways of observing the serological relationships of bacterial appendages such as pili: either qualitatively by the direct observation of adsorbed antibodies in the electron microscope (Lawn, 1967), or quantitatively by conventional agglutination tests. In this work we have used the first method to define different serological types of pilus, and the second to study in detail the reactions of antibodies specific to the pilus.Although the injection into rabbits of formolized suspensions ofP. aeruginosaresults in the formation of flagellar antibody in high titre, the antibody response to the pili is poor (Pitt & Bradley, 1975). The pilus is in the form of a long thin filament consisting of polymerized pilus protein or pilin. It has been demonstrated (Bradley, 1972a) that under certain influences, such as chemical action or bacteriophage adsorption (see below), the filaments withdraw into the cell, the pilin probably being depolymerized by a mechanism at the base. CertainP. aeruginosamutants that lack the ability to retract their pili (Bradley, 1972a, 1974b) have many more filaments than do normal strains. We now show that these mutants are valuable in producing high-titre antiserum. Other mutants without pili have been isolated (Bradley, 1972b), and absorption of sera with these organisms removes antibodies to other components of the cell.Certain bacteriophages useP. aeruginosapili as receptors (Bradley, 1966, 1973a, b, 1974a; Bradley & Pitt, 1974). One aspect of the present work has been to determine whether the phage-sensitivity pattern varies with pili of different serological types.