FIG. 2.

Pilins from strains Pa5196 and PA7 are modified with D-Araf and elicit antibodies that cross-react with mycobacterial lipoarabinomannan. (A) Strain PA7 produces functional pili when tested for twitching motility by agar subsurface assay, but it is less motile than strain PAO1 or Pa5196; for scale, the dish is 10 cm in diameter. See strain 5196NP in Fig. 3 for an example of a nonmotile strain. (B) SDS-PAGE and Western blot analyses of sheared surface pili demonstrate that Pa5196 and PA7 produce pilins with a larger mass than (nonglycosylated) PAO1, though PA7 produces fewer pili than Pa5196, corresponding with the reduced motility seen in panel A. Both Pa5196 and PA7 pilins react with anti-LAM and anti-PilA₅₁₉₆ antibodies, while the PAO1 pilins do not, confirming that PA7 pilins are also arabinosylated. The positions (in kilodaltons) of molecular mass markers (lane M) are shown to the left of the gel. (C) Immunization of rabbits with a sheared surface protein preparation from Pa5196 containing pilin and flagellin elicits antibodies that recognize cell wall material from Mycobacterium smegmatis (M.sm) of the same size range as anti-LAM sera. The flagellins of Pa5196 are not arabinosylated and do not react with anti-LAM sera. The positions of 15-kDa molecular mass markers (lane M) and of flagellin (F) and pilin (P) are shown to the left of the gel.