Author
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Sharma, Vijay |
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Zuerner, Richard |
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Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 7/30/2004 Publication Date: 11/20/2004 Citation: Sharma, V.K., Zuerner, R.L. 2004. Role of hha and ler in transcriptional regulation of the esp operon of enterohemorrhagic Escherichia coli O157:H7. Journal of Bacteriology. 186(21):7290-7301. Interpretive Summary: Escherichia coli O157:H7 is associated with severe gastrointestinal and systemic diseases in humans. Each year, about 73,000 estimated cases of diarrheal illness due to E. coli O157:H7 occur in the U.S. Most human E. coli O157:H7 infections are caused by consumption of contaminated foods (ground beef, milk, and produce) and water. E. coli O157:H7 colonizes the intestine of cattle and is secreted into the feces of these animals, where it can survive for months. Cattle feces, therefore, represents the major source for the transmission of E. coli O157:H7 to other cattle and humans. Understanding of the genetic factors of O157:H7 that enable it to colonize cattle will facilitate development of intervention strategies to reduce O157:H7 in cattle. The present study describes the role of one of the genetic factors (Hha) that directly influences the expression of O157:H7 genes involved in the colonization. In the presence of Hha, the colonization genes are expressed at very low levels as compared to O157:H7 lacking Hha. However, the nature of the pathway that regulates Hha synthesis is not known. Information generated in the current study will be pivotal in characterization of the pathway of Hha synthesis, particularly, understanding of the environmental factors that elevate the levels of Hha to a threshold sufficient enough to completely block the expression of colonization genes and inhibit the ability of O157:H7 to colonize cattle. This information will be useful to scientists, Food Safety and Inspection Service, veterinary biologics, and the pharmaceutical industry. Technical Abstract: One of the important virulence attributes of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is its ability to produce attaching and effacing (A/E) lesions on intestinal cells. Formation of A/E lesions is mediated by the locus of enterocyte effacement (LEE), a horizontally acquired segment of DNA containing five major operons. Expression of LEE operons is regulated by ler (LEE-encoded regulator), a gene whose transcription is induced by the integration host factor (IHF) and a quorum-sensing pathway. However, genes exerting negative affect on the transcription of ler are not known. In this study, we describe the role of hha in the expression of ler. A transcriptional fusion (esp::lac) was constructed in EHEC O157:H7 strain 86-24 by cloning a lac cassette downstream of a ler-activatable esp promoter. Determination of b-galactosidase activity of esp::lac fusion revealed low levels of growth-dependent expression of esp genes. However, a transpositional insertion in hha resulted in 17-fold increase in b-galactosidase activity, suggesting that hha down-regulates the expression of esp genes. No binding of Hha was detected to the esp promoter in gel shift assays. Transpositional mutagenesis of Dhha derivative of 86-24 (esp::lac), expressing high levels of b-galactosidase, resulted in isolation of clones producing reduced amounts of b-galactosidase. Analysis of the DNA sequences at the transposon insertion site resulted in the identification of an ORF with 100% homology to ler. The purified Hha bound to the ler-promoter, and RT-PCR analysis of the RNA from 86-24 lacking hha showed higher amounts of ler-, esp-, escR-, and tir-eae-specific amplicons. These results indicate that hha down-regulates the expression of LEE-encoded genes by reducing the transcription of ler. |
