Single-residue changes in the C-terminal disulfide-bonded loop of the Pseudomonas aeruginosa type IV pilin influence pilus assembly and twitching motility.
PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.
Harvey
Hanjeong
H
Department of Biochemistry and Biomedical Sciences and the Michael G DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada.
Habash
Marc
M
Aidoo
Francisca
F
Burrows
Lori L
LL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008958:Models, Molecular; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D021122:Protein Subunits; D011550:Pseudomonas aeruginosa / Q000378:metabolism*
D021122:Protein Subunits; D036781:Fimbriae Proteins