Genetic characterization of Escherichia coli type 1 pilus adhesin mutants and identification of a novel binding phenotype.
Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31 degrees C but not at 42 degrees C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42 degrees C but sensitive after growth at 31 degrees C. The ts mutant thus binds macrophages with one receptor specificity at 31 degrees C and another at 42 degrees C.
Hamrick
T S
TS
Department of Microbiology, Pathology and Parasitology, College of Veterinary Medicine, North Carolina State University, Raleigh 27606, USA.
Harris
S L
SL
Spears
P A
PA
Havell
E A
EA
Horton
J R
JR
Russell
P W
PW
Orndorff
P E
PE
D018829:Adhesins, Bacterial / Q000235:genetics*; D018830:Adhesins, Escherichia coli; D000818:Animals; D001422:Bacterial Adhesion / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D004912:Erythrocytes / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics* / Q000472:pathogenicity; D036781:Fimbriae Proteins; D006168:Guinea Pigs; D006386:Hemagglutination Tests; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008969:Molecular Sequence Data; D010641:Phenotype; D017354:Point Mutation
D018829:Adhesins, Bacterial; D018830:Adhesins, Escherichia coli; C058769:fimH protein, E coli; D036781:Fimbriae Proteins