TY - JOUR

T1 - Increased adherence and actin pedestal formation by dam-deficient enterohaemorrhagic Escherichia coli O157:H7

AU - Campellone, Kenneth G.

AU - Roe, Andrew J.

AU - Løbner-Olesen, Anders

AU - Murphy, Kenan C.

AU - Magoun, Loranne

AU - Brady, Michael J.

AU - Donohue-Rolfe, Arthur

AU - Tzipori, Saul

AU - Gally, David L.

AU - Leong, John M.

AU - Marinus, M. G.

PY - 2007

Y1 - 2007

N2 - Enterohaemorrhagic Escherichia coli (EHEC) are highly infectious pathogens capable of causing severe diarrhoeal illnesses. As a critical step during their colonization, EHEC adhere intimately to intestinal epithelial cells and generate F-actin 'pedestal' structures that elevate them above surrounding cell surfaces. Intimate adhesion and pedestal formation result from delivery of the EHEC type III secretion system (TTSS) effector proteins Tir and EspF(U) into the host cell and expression of the bacterial outer membrane adhesin, intimin. To investigate a role for DNA methylation during the regulation of adhesion and pedestal formation in EHEC, we deleted the dam (DNA adenine methyltransferase) gene from EHEC O157:H7 and demonstrate that this mutation results in increased interactions with cultured host cells. EHECDeltadam exhibits dramatically elevated levels of adherence and pedestal formation when compared with wild-type EHEC, and expresses significantly higher protein levels of intimin, Tir and EspF(U). Analyses of GFP fusions, Northern blotting, reverse transcription polymerase chain reaction, and microarray experiments indicate that the abundance of Tir in the dam mutant is not due to increased transcription levels, raising the possibility that Dam methylation can indirectly control protein expression by a post-transcriptional mechanism. In contrast to other dam-deficient pathogens, EHECDeltadam is capable of robust intestinal colonization of experimentally infected animals.

AB - Enterohaemorrhagic Escherichia coli (EHEC) are highly infectious pathogens capable of causing severe diarrhoeal illnesses. As a critical step during their colonization, EHEC adhere intimately to intestinal epithelial cells and generate F-actin 'pedestal' structures that elevate them above surrounding cell surfaces. Intimate adhesion and pedestal formation result from delivery of the EHEC type III secretion system (TTSS) effector proteins Tir and EspF(U) into the host cell and expression of the bacterial outer membrane adhesin, intimin. To investigate a role for DNA methylation during the regulation of adhesion and pedestal formation in EHEC, we deleted the dam (DNA adenine methyltransferase) gene from EHEC O157:H7 and demonstrate that this mutation results in increased interactions with cultured host cells. EHECDeltadam exhibits dramatically elevated levels of adherence and pedestal formation when compared with wild-type EHEC, and expresses significantly higher protein levels of intimin, Tir and EspF(U). Analyses of GFP fusions, Northern blotting, reverse transcription polymerase chain reaction, and microarray experiments indicate that the abundance of Tir in the dam mutant is not due to increased transcription levels, raising the possibility that Dam methylation can indirectly control protein expression by a post-transcriptional mechanism. In contrast to other dam-deficient pathogens, EHECDeltadam is capable of robust intestinal colonization of experimentally infected animals.

KW - Actins/metabolism

KW - Adhesins, Bacterial/analysis

KW - Animals

KW - Artificial Gene Fusion

KW - Bacterial Adhesion

KW - Carrier Proteins/analysis

KW - Disease Models, Animal

KW - Escherichia coli Infections

KW - Escherichia coli O157/enzymology

KW - Escherichia coli Proteins/analysis

KW - Gene Deletion

KW - Gene Expression Regulation, Bacterial

KW - Genes, Reporter

KW - Green Fluorescent Proteins/analysis

KW - HeLa Cells

KW - Humans

KW - Microscopy, Electron, Scanning

KW - Microscopy, Electron, Transmission

KW - Oligonucleotide Array Sequence Analysis

KW - RNA, Bacterial/analysis

KW - RNA, Messenger/analysis

KW - Receptors, Cell Surface/analysis

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics

KW - Swine

KW - Transcription, Genetic

U2 - 10.1111/j.1365-2958.2007.05602.x

DO - 10.1111/j.1365-2958.2007.05602.x

M3 - Journal article

C2 - 17302821

VL - 63

SP - 1468

EP - 1481

JO - Molecular Microbiology

JF - Molecular Microbiology

SN - 0950-382X

IS - 5

ER -