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Title:   Author  item
                                            Sharma, Vijayitem
                                            Bearson, Shawnitem
                                            Bearson, Bradley

                                                         - *Brad*
  Submitted to: Microbiology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 1/26/2010
                                    Publication  Date: 5/20/2010
                                    Citation: Sharma, V.K., Bearson, S.M., Bearson, B.L. 2010. Evaluation of the Effects of SDIA, a LUXR Homologue, on Adherence and Motility of Escherichia coli O157:H7. Microbiology. 156(5):1303-1312.Interpretive Summary: Escherichia coli O157:H7, a Shiga toxin-producing E. coli (STEC), is the most frequent cause of bloody diarrhea in humans. In certain age groups, O157:H7 infections may result in damage to the kidneys and nervous system, a potentially fatal form of the disease.  E. coli O157:H7 has been associated with the most important foodborne outbreaks in the USA and Canada. Cattle are considered the major reservoir for E. coli O157:H7 and these animals can secrete E. coli O157:H7 in their feces for extended periods of time. Most human infections result from consumption of undercooked ground beef, milk, produce and water that inadvertently become contaminated with cattle feces. Thus, an understanding of bacterial factors responsible for colonization and persistence of E. coli O157:H7 in cattle intestines is important for rational design of vaccines for reducing persistence and fecal shedding of E. coli O157:H7 in these animals. In the present study, we described that the presence or absence of SdiA, a quorum-sensing homologue of LuxR, had a significant effect on the adherence of E. coli O157:H7 to epithelial cells in tissue cultures. E. coli O157:H7 lacking SdiA showed increased adherence to epithelial cells and increased expression of curli fimbriae. This finding provides an opportunity to investigate if an increased expression of curli would enhance E. coli O157:H7 colonization of cattle intestine and cause increased fecal shedding in these animals. Practical applications that may result from the implication of curli in E. coli colonization will be based on the inclusion of culri fimbriae in vaccine preparations and testing the ability of these vaccines for reducing colonization of E. coli O157:H7 in cattle intestines and subsequently reducing the shedding of this foodborne human pathogen in cattle feces.
                                Technical Abstract: Quorum-sensing (QS) signaling pathways are important regulatory networks for controlling the expression of genes promoting adherence of Enterohemorrhagic Escherichia coli (EHEC) O157:H7 to epithelial cells. A recent study has shown that EHEC O157:H7 encodes a luxR homologue, called sdiA ̧ which  upon over-expression reduces the expression of genes encoding flagellar and LEE proteins, thus negatively impacting the motility and intimate adherence phenotypes, respectively. Here, we show that the deletion of sdiA from EHEC O157:H7 strain 86-24 and in a hha mutant (a negative regulator of ler) of this strain enhanced the bacterial adherence to Hep-2 cells of the sdiA mutant strains relative to the parent strain 86-24 and the hha mutant. Quantitative reverse transcription polymerase chain reaction (RT-PCR) showed that the expression of LEE-encoded genes ler, espA, and eae in the sdiA mutant were not significantly different from that of the parent strain. Similarly, the deletion of sdiA in the hha deletion mutant did not cause additional increases in the expression of LEE genes relative to that observed in the hha deletion mutant.  While the expression of fliC, which encodes flagellin, was enhanced in the sdiA mutant strain, the deletion of hha reduced the expression of fliC by several-fold irrespective of the presence or absence of sdiA, indicating that the genes sdiA and hha exert opposing effects on the expression of flagellar genes.  The strains with deletions in sdiA or hha showed enhanced expression of csgA, encoding curlin of the curli fimbriae, with the expression of csgA highest in the sdiA hha double mutant suggesting an additive effect of these two gene deletions on the expression of csgA. No significant differences were observed in the expression of the genes lpfA and fimA of the operons encoding long polar and type-1 fimbriae in the sdiA mutant strain. This data indicates that SdiA has no significant affect on LEE expression but it appears to act as a strong repressor of genes encoding flagella and curli fimbriae, and the alleviation of the SdiA-mediated repression of these genes in an EHEC O157:H7 sdiA mutant strain contributes to enhanced bacterial motility and increased adherence to HEp-2 cells.


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