Increased adherence and actin pedestal formation by dam-deficient enterohaemorrhagic Escherichia coli O157:H7.
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.
Campellone
Kenneth G
KG
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Roe
Andrew J
AJ
Løbner-Olesen
Anders
A
Murphy
Kenan C
KC
Magoun
Loranne
L
Brady
Michael J
MJ
Donohue-Rolfe
Arthur
A
Tzipori
Saul
S
Gally
David L
DL
Leong
John M
JM
Marinus
M G
MG
D000199:Actins / Q000378:metabolism*; D018829:Adhesins, Bacterial / Q000032:analysis; D000818:Animals; D019677:Artificial Gene Fusion; D001422:Bacterial Adhesion; D002352:Carrier Proteins / Q000032:analysis; D004195:Disease Models, Animal; D004927:Escherichia coli Infections; D019453:Escherichia coli O157 / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000032:analysis; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D020411:Oligonucleotide Array Sequence Analysis; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000378:metabolism; D013552:Swine; D014158:Transcription, Genetic
D000199:Actins; D018829:Adhesins, Bacterial; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C489109:EspFU protein, E coli; D047908:Intracellular Signaling Peptides and Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; C485184:enhanced green fluorescent protein; C073819:eaeA protein, E coli; D049452:Green Fluorescent Proteins; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)