Transcriptome of Enterohemorrhagic Escherichia coli O157 Adhering to Eukaryotic Plasma Membranes

Abstract

Using a DNA microarray, we determined changes in enterohemorrhagic Escherichia coli O157:H7 gene expression during binding to plasma membranes. Analysis of the complete transcriptomes of the bound bacteria revealed increased levels of stress-associated mRNAs and decreased levels of mRNA encoding proteins involved in translation and type III secretion.Enterohemorrhagic Escherichia coli (EHEC) is an important cause of acute gastroenteritis in humans (26). EHEC causes a wide spectrum of illnesses ranging from mild diarrhea to severe diseases, such as hemorrhagic colitis and hemolytic-uremic syndrome; hemolytic-uremic syndrome is the leading cause of acute renal failure in children and is associated with the production of potent Shiga toxins (Stx) (26). Strains of EHEC belonging to serogroup O157 are most commonly associated with severe human disease.Adhesion of EHEC to intestinal epithelial cell results in induction of a histopathological feature known as attaching and effacing lesions (8), which are characterized by localized destruction of brush border microvilli and intimate attachment of the bacteria to actin-rich pedestal-like structures that are formed on the apical membrane underneath attached bacteria. The capacity to form attaching and effacing lesions is encoded on a pathogenicity island termed the locus of enterocyte effacement (LEE) (21). The LEE can be divided into three functional regions (6); the 5Ј end consists of three operons (LEE1 to LEE3) encoding a positive regulator, Ler (23), and the main structural components of the bacterial type III secretion system (TTSS). The central part of the LEE harbors an operon (LEE5) encoding the outer membrane adhesin intimin (15) and the translocated intimin receptor (Tir) (17). The 3Ј end of the LEE (LEE4) encodes additional TTSS structural proteins, translocators, and effectors.TTSSs are commonly found in pathogenic, gram-negative bacteria (13) and are used to transfer effector proteins directly into eukaryotic cells, where the normal cellular functions are subverted for the benefit of the pathogen. Both the organization and the composition of TTSSs are broadly conserved in different bacteria, and TTSSs contain homologues of many flagellar components (1). However, the TTSS of EHEC is unique in that, in addition to the conserved cell wall-associated needle complex, it contains a filamentous extension to the needle formed by one of the secreted proteins, EspA (20, 27).The three-dimensional structure of the EspA filament shows that it consists of a helical tube with an outer diameter of ca. 120 Å containing a hollow central channel that is ca. 25 Å in diameter and has a continuous wall, which is likely to contain the proteins within the channel during transfer (2). Evidence suggests that EspA is the major component of this filamentous structure, while EspD, in complex with a second translocator protein, EspB, is predicted to form a translocation pore in the host cell membrane that facilitates subsequent entry of effector proteins (Tir, EspF, Map, EspG, EspH, Cif,...

EspJ Is a Prophage-Carried Type III Effector Protein of Attaching and Effacing Pathogens That Modulates Infection Dynamics

“…This has shown that the majority of the LEE-genes were down-regulated in attached bacteria. In addition, decreased levels of two mRNAs of adjacent genes, Z3071 (Ecs2714) and Z3072 (Ecs2715), carried within prophage CP-933U, were also detected (8). Importantly, Z3072 (TccP/EspF U ) is present in EHEC but not in EPEC or C. rodentium (5,15), while Z3071 is present in EHEC, EPEC, and C. rodentium, and its product is homologous to the type III effector HopF of Pseudomonas syringae (43).…”

“…Recently, we monitored the global transcription profile of EHEC O157:H7 (Sakai strain) during attachment to eukaryotic plasma membranes (8). This has shown that the majority of the LEE-genes were down-regulated in attached bacteria.…”

Examination of animal and zoonotic pathogens using microarrays

“…This novel strategy of combining signature-tagged transposon mutagenesis and microarrays could identify mutants deficient in survival in cultured macrophages and identify components of SPI 2. The results were in conformity with Baro resistant/sensitive strains Identified factors that promote barotolerance [66] Mutants and varying O 2 concentrations Gene regulation during anaerobic growth [75,98] Zinc stress Transcriptional response of E. coli to zinc [61] Attachment to eukaryotic membrane Induction of stress associated transcripts in E. coli O157:H7 and down-regulation of genes involved in TTSS [31] Intraneutrophil Induction of genes regulated by OxyR [93] Deletion of fim gene cluster Expression of surface protein antigen 43 [90] P. multocida…”

“…A differential expression of fim genes was observed, while deletion of the fim gene cluster led to the expression of surface protein antigen 43 [90]. Another study demonstrated differential expression of E. coli O157 genes attached to the plasma membrane of red blood cells [31]. The stress-associated mRNA increased, while levels of mRNA involved in the type III secretion system were decreased.…”

Polarity of Enteropathogenic Escherichia coli EspA Filament Assembly and Protein Secretion

“…A prerequisite for intimate attachment is elimination of EspA filaments and the NC. Indeed, we have shown that expression of the espA gene (8) and the presence of EspA filaments (35) are gradually downregulated during a 6-h infection of cells in vitro.…”