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Title:   Author  item
                                            Sharma, Vijayitem
                                            Bearson, Bradley

                                                         - *Brad*
  Submitted to: Applied and Environmental Microbiology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 1/25/2013
                                    Publication  Date: 2/4/2013
                                    Citation: Sharma, V.K., Bearson, B.L. 2013. Hha controls Escherichia coli O157:H7 biofilm formation by differential regulation of global transcriptional regulators FlhDC and CsgD. Applied and Environmental Microbiology. 79(7):2384-2396. https://doi.org/10.1128/AEM.02998-12DOI: https://doi.org/10.1128/AEM.02998-12                                                              
                                Interpretive Summary: Escherichia coli O157:H7 bacteria are the frequent cause of bloody diarrhea in humans. In certain age groups, E. coli O157:H7 infections may result in damage to the kidneys and nervous system, a potentially fatal form of the disease.  Cattle are the primary source E. coli O157:H7 and cattle harboring these bacteria in the large intestine can secrete E. coli O157:H7 in their feces for extended periods of time. Most human infections result from the consumption of undercooked ground beef. In addition consumption of milk, produce, and water that inadvertently become contaminated with cattle feces enhances the risk of E. coli O157:H7 infections in humans. In order to develop strategies for reducing the risk of contamination of meats, milk, water, and produce, it is pertinent that a broader understanding of the mechanisms that enable E. coli O157:H7 to colonize cattle intestine resulting in fecal shedding of these bacteria is needed. With this objective as one of the major research focus of the E. coli group at NADC, we have extended our previous work on a specific genetic regulator (Hha) that we had shown previously to have an important effect on the production of protein factors essential for colonization of E. coli O157:H7 in cattle. In the current study, we have determined one of the mechanisms by which this genetic regulator (Hha) presumably controls the production of additional protein factors on the cell surface of E. coli O157:H7 bacteria. This knowledge is needed in the design of vaccines that would not only be more effective in mitigating the risk of colonization of E. coli O157:H7 in cattle, but might also be re-engineered to target other non-O157:H7 E. coli bacteria of concern to cattle and food industry.
                                Technical Abstract: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that produces a broad-spectrum of diarrheal illnesses in infected humans. Although molecular mechanisms enabling EHEC O157:H7 to produce characteristic adherence on epithelial cells are well characterized, regulatory mechanisms controlling biofilm formation on abiotic surfaces are not fully understood.  In this study, we demonstrate that the formation of biofilm in EHEC O157:H7 strain 86-24 is highly repressed compared to an isogenic strain with an hha deletion. The hha deletion mutant produced significantly large quantities of biofilm compared to the wild-type strain both at 30 degree C and 37 degree C. The complementation of the mutant with hha reduced the level of biofilm formation to that of the wild-type strain, indicating that Hha is a negative regulator of biofilm production in EHEC O157:H7. Further analysis revealed that the hha deletion mutant had significantly reduced bacterial motility and decreased expression of the flagellar gene fliC. On the other hand, there were significant increases in the expression of csgA, encoding curlin of curli fimbriae, and the ability to bind the Congo red dye. The expression of both fliC and csgA, and the phenotypes (motility and curli production, respectively) affected by these genes were restored to the wild-type levels in the complemented hha mutant strain. Curli fimbriae encoded by csgAB were determined as a major factor behind biofilm formation as deletion of csgA abolished biofilm formation both in the wild-type and hha deletion mutant strains and complementation with csgA restored biofilm formation to these strains. The regulatory effects of Hha on expression of flagellar and curli genes appear to occur via the induction and repression of the global regulators FlhDC and CsgD, as demonstrated by reduced flhD and increased csgD transcription in the hha mutant strain, respectively. By using gel-shift assays, we demonstrated that Hha interacts with flhDC and csgD promoters.  In conclusion, the data presented in this study provide strong evidence that Hha regulates biofilm formation in EHEC O157:H7 by differentially regulating expression of the flhDC and csgD genes that encode two important global regulators affecting bacterial motility and curli production by regulating the expression of the flagellar and curli operons, respectively.


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