Deletion of sdhA upregulate LEE genes expression possibly via Lrp in enterohemorrhagic Escherichia coli O157: H7

  • 劉 書羽

Student thesis: Doctoral Thesis


Enterohemorrhagic Escherichia coli (EHEC) O157: H7 is a pathogen that can cause bloody diarrhea and hemolytic uremic syndrome (HUS) The cases mainly occur in the U S Canada Japan and other industrialized countries in Europe Because antibiotic treatment may enhance the severity of EHEC infection therefore it is important to discover new targets to develop new drugs In a previous study the feeding of EHEC ΔsdhA strain increased the survival rate of Caenorhabditis elegans compared with WT strain SdhA is a subunit of succinate dehydrogenase (SDH) involved in the central metabolism pathway TCA cycle and electron transport chain Thus disruption of TCA cycle will affect virulence factors expression and SdhA is therefore a promising target for drug development Interestingly the expression of the locus of enterocyte effacement (LEE) genes in ΔsdhA strain was upregulated in other studies Type three secretion system (T3SS) encoded by LEE pathogenicity island is an important factor for EHEC to adhere directly to host intestinal epithelium and to form attaching and effacing lesions (A/E lesions) In this study we try to clarify which major factor affect LEE genes expression in ΔsdhA strain First we used promoter activity assay trying to find a factor might play a major role that upregulates LEE genes expression in ΔsdhA strain We found that Lrp might play a role to upregulate LEE expression in ΔsdhA strain Lrp is a Leucine-responsive regulatory protein that senses butyrate and can also induce LEE genes upregulation through LeuO and Pch in EHEC We mutated lrp to test LEE genes expression in Δlrp strain will be identical in ΔsdhAΔlrp strain We confirmed that Δlrp strain and ΔΔlrp strain will downregulate expression of LEE genes while comparing with WT Because EHEC would not produce butyrate but Lrp still has activity So we wonder which metabolite affect Lrp Then we used M9 medium supplied with 2 5 mM succinate and fumarate respectively to check which supplement could affect lrp promoter activity in ΔsdhA strain We found out that fumarate will slightly affect the activity of lrp promoter but did not affect LEE1 promoter in ΔsdhA strain This indicates that fumarate is not the major role to regulate Lrp To further search we found that activity of leuO promoter is higher than WT In conclusion we know that deletion of sdhA will upregulate LEE genes expression and alter lrp and leuO expression The alteration is not because of fumarate or succinate So we suggest there must be other molecules affect Lrp upregulate in ΔsdhA strain Then upregulation of LEE genes expression is associated with Lrp-LeuO pathway in ΔsdhA strain
Date of Award2019
Original languageEnglish
SupervisorJenn-Wei Chen (Supervisor)

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