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 - 2007/3
Y1 - 2007/3
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
SN - 0950-382X
VL - 63
SP - 1468
EP - 1481
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 5
ER -