The phosphorylcholine epitope undergoes phase variation on a 43-kilodalton protein in Pseudomonas aeruginosa and on pili of Neisseria meningitidis and Neisseria gonorrhoeae

Abstract

Phosphorylcholine (ChoP) is a component of the teichoic acids of Streptococcus pneumoniae and has been recently identified on the lipopolysaccharide of Haemophilus influenzae, also a major pathogen of the human respiratory tract. Other gram-negative pathogens that frequently infect the human respiratory tract were surveyed for the presence of the ChoP epitope as indicated by binding to monoclonal antibodies (MAbs) recognizing this structure. The ChoP epitope was found on a 43-kDa protein on all clinical isolates of Pseudomonas aeruginosa examined and on several class I and II pili of Neisseria meningitidis. The specificity of the anti-ChoP MAb was demonstrated by the inhibition of binding in the presence of ChoP but not structural analogs. As in the case of H. influenzae, the expression of this epitope was phase variable on these species. In P. aeruginosa, this epitope was expressed at detectable levels only at lower growth temperatures. Expression of the ChoP epitope on piliated neisseriae displayed phase variation, both linked to pilus expression and independently of fully piliated bacteria.

FIG. 1

Relationship between growth temperature and expression of the ChoP epitope. P. aeruginosa PAO1 was grown to stationary phase at the temperature indicated, and equivalent numbers of cells in whole-cell lysates were examined by Western analysis on SDS-PAGE with MAb TEPC-15, which recognizes ChoP. Where indicated, following growth to stationary phase, the growth temperature was shifted for 4 h prior to the preparation of whole-cell lysates. Molecular sizes are in kilodaltons.

FIG. 2

Western blots showing the reactivity of anti-ChoP antibody with purified pilus preparations and with corresponding protein in whole-cell lysates of piliated meningococcal strains. Lanes: 1 and 3; class I piliated variants 3 and 16 of strain C311; 2 and 4, purified pili from these variants; 5 and 6, whole-cell lysates from nonpiliated variants of strain C311 (class I) or C114 (class II); 7 and 8, class II piliated variants of strains C319 and C114. Molecular sizes are in kilodaltons. Pilin migrations as detected with an anti-ChoP antibody are in agreement with previously known migrations of pilins of these strains and were confirmed by the use of MAbs against the pilin subunit (see Fig. 4). Note that the LPSs of neisseriae migrate farther than the 10-kDa molecular size marker. No reactivity of anti-ChoP antibody was detected in this region of the Western blot, even in the whole-cell lysates used in lanes 1, 3, and 5 to 8.

FIG. 3

Dot blot assay of whole-cell lysates of N. meningitidis (Nm) isolates to demonstrate the specificity of anti-ChoP antibodies. Blots contained whole-cell suspensions of class I piliated (B and E), class II piliated (A), and nonpiliated (C and D) strains of N. meningitidis. Blots in row F contained purified pili of strain C311. One nitrocellulose strip was reacted with polyclonal rabbit antiserum against strain C311 to show the presence of antigen in each dot (top). The stronger but equal reaction in rows D to F is due to the presence of homologous antigen in these dots. Other strips were reacted with MAb SM1 against class I pili, MAb AD211 against class II pili, or anti-ChoP MAb HAS. No reaction in any dots was observed when an irrelevant MAb (IgM class) was used as an isotype-matched control. Also, the alkaline phosphatase-conjugated secondary antibody against mouse IgM used in the experiment gave no reaction when used alone. The bottom three strips were subjected to urea treatment prior to antibody probing.

FIG. 4

Western blots demonstrating reactivity of pilins of N. gonorrhoeae with MAb TEPC-15 against ChoP. The top and bottom are photographs of the same Western blot developed sequentially. The blot was first developed by using an anti-ChoP antibody (top). After recording the reactivity shown at the top, we cut the lanes in half and reacted the nitrocellulose strips marked with asterisks with MAb SM1 against class I pilins. After development, the strips were combined and rephotographed (bottom composite blot) to show colocation of the ChoP epitope (unmarked strips) and pilins (strips marked with asterisks). Lanes: 1, purified pili of N. meningitidis C311 as a control; 2 to 6, piliated variants of gonococcal strain MS11; 7 and 8, piliated gonococcal strains R10 and SU95; 9, molecular size markers. Size markers of 30 kDa (large arrowheads) and 21.5 kDa (small arrowheads) are shown. Note that the pilins of one variant of strain MS11 (lane 3) do not contain the ChoP epitope.