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

Abstract

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 ⌬per 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 ⌬per 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 ⌬per 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 ⌬per 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.

Examination of the Genome-Wide Transcriptional Response of Escherichia coli O157:H7 to Cinnamaldehyde Exposure

“…Many enzymes involved in oxidative stress responses have been reported to reduce the intracellular NADPH level in E. coli, which not only lowers the reducing power of the cells but also inhibits cell multiplication by limiting its biosynthetic capability (46,(50)(51)(52). This may explain the observed repression of biosynthetic genes, such as genes for protein synthesis (as indicated by ribosomal and RNA synthesis) and O-antigen synthesis, which have been reported to be important for attachment to plants and for bovine colonization (53,54), as well as fimbrial synthesis. Furthermore, increased levels of tricarboxylic acid cycle enzymes, including fumC and acnA, that are resistant to oxidative stress (superoxide) (55), suggest that overexpression of these enzymes aids E. coli in combating effects of cinnamaldehyde-induced oxidative stress by increasing the reducing power of E. coli cells via reduction of NAD ϩ (46).…”

Chemical sensing in mammalian host–bacterial commensal associations

“…The genes for AE lesion formation are encoded within the locus of enterocyte effacement (LEE) (2). The LEE and AE lesion formations are also necessary for EHEC colonization of the recto-anal junction (RAJ) of cattle, facilitating its shedding to the environment (3)(4)(5). Whereas AE lesion formation leads to disease in humans, it is innocuous in adult cattle.…”

O-Antigen-NegativeSalmonella entericaSerovar Typhimurium Is Attenuated in Intestinal Colonization but Elicits Colitis in Streptomycin-Treated Mice

“…There are several other reports connecting LPS mutations with a loss in motility: in serovar Typhimurium, addition of a monoclonal anti-LPS antibody to a diluted bacterial culture led to instant paralysis of the bacteria (8), and in a Pseudomonas aeruginosa ⌬waaL strain, a severe impairment in motility was detected (2). Also, an Escherichia coli mutant lacking O antigen was reported to show a nonmotile phenotype (37).…”