Characterization of an Escherichia coli O157:H7 O-antigen deletion mutant and effect of the deletion on bacterial persistence in the mouse intestine and colonization at the bovine terminal rectal mucosa.
Escherichia coli O157:H7 causes hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome in humans and transiently colonizes healthy cattle at the terminal rectal mucosa. To investigate the role of the O antigen in persistence and colonization in the animal host, we generated an E. coli O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (per) in the rfb cluster. The lack of O antigen was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anti-O157 antibody. The growth rate and cell membrane permeability of the Deltaper mutant were similar to the growth rate and cell membrane permeability of the wild type. Changes in membrane and secreted proteins were observed, but the expression of intimin, EspA, and EspB, implicated in bacterial intestinal colonization, was not altered, as determined by immunoblotting and reverse transcription-PCR. Similar to other O-antigen deletion mutants, the Deltaper mutant was pleiotropic for autoaggregation and motility (it was FliC negative as determined by immunoblotting and flagellum negative as determined by electron microscopy). The abilities of the mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa were compared. Mice fed the Deltaper mutant shed lower numbers of bacteria (P < 0.05) over a shorter time than mice fed the wild-type or complemented strain. After rectal application in steers, lower numbers of the Deltaper mutant than of the wild type colonized the rectoanal junction mucosa, and the duration of the colonization was shorter (P < 0.05). Our previous work showed that flagella do not influence E. coli O157:H7 colonization at the bovine terminal rectal mucosa, so the current findings suggest that the O antigen contributes to efficient bovine colonization.
Sheng
Haiqing
H
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844-3052, USA.
Lim
Ji Youn
JY
Watkins
Maryann K
MK
Minnich
Scott A
SA
Hovde
Carolyn J
CJ
D018829:Adhesins, Bacterial / Q000378:metabolism; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000235:genetics*; D002417:Cattle; D002463:Cell Membrane Permeability / Q000502:physiology; D004927:Escherichia coli Infections / Q000382:microbiology*; D019453:Escherichia coli O157 / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D029968:Escherichia coli Proteins / Q000378:metabolism; D017353:Gene Deletion; D007413:Intestinal Mucosa / Q000382:microbiology*; D051379:Mice; D019081:O Antigens / Q000235:genetics*; D010641:Phenotype; D000637:Transaminases / Q000235:genetics*
D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; C087863:EaeB protein, E coli; D029968:Escherichia coli Proteins; C102051:EspA protein, E coli; D019081:O Antigens; C073819:eaeA protein, E coli; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase