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Characterization of EhaJ, a New Autotransporter Protein from Enterohemorrhagic and Enteropathogenic Escherichia coli.

Easton DM, Totsika M, Allsopp LP, Phan MD, Idris A, Wurpel DJ, Sherlock O, Zhang B, Venturini C, Beatson SA, Mahony TJ, Cobbold RN, Schembri MA - Front Microbiol (2011)

Bottom Line: However, deletion of ehaJ did not significantly alter its adherence or biofilm properties.In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC.Further studies are required to elucidate the function of EhaJ in colonization and virulence.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Science, The University of Queensland Gatton, QLD, Australia.

ABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathotypes of E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. While certain EHEC and EPEC virulence mechanisms have been extensively studied, the factors that mediate host colonization remain to be properly defined. Previously, we identified four genes (ehaA, ehaB, ehaC, and ehaD) from the prototypic EHEC strain EDL933 that encode for proteins that belong to the autotransporter (AT) family. Here we have examined the prevalence of these genes, as well as several other AT-encoding genes, in a collection of EHEC and EPEC strains. We show that the complement of AT-encoding genes in EHEC and EPEC strains is variable, with some AT-encoding genes being highly prevalent. One previously uncharacterized AT-encoding gene, which we have termed ehaJ, was identified in 12/44 (27%) of EHEC and 2/20 (10%) of EPEC strains. The ehaJ gene lies immediately adjacent to a gene encoding a putative glycosyltransferase (referred to as egtA). Western blot analysis using an EhaJ-specific antibody indicated that EhaJ is glycosylated by EgtA. Expression of EhaJ in a recombinant E. coli strain, revealed EhaJ is located at the cell surface and in the presence of the egtA glycosyltransferase gene mediates strong biofilm formation in microtiter plate and flow cell assays. EhaJ also mediated adherence to a range of extracellular matrix proteins, however this occurred independent of glycosylation. We also demonstrate that EhaJ is expressed in a wild-type EPEC strain following in vitro growth. However, deletion of ehaJ did not significantly alter its adherence or biofilm properties. In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC. Further studies are required to elucidate the function of EhaJ in colonization and virulence.

No MeSH data available.


Related in: MedlinePlus

(A) Glycosylated EhaJ [MS427(pOMS2)] mediates enhanced static biofilm formation in a microtiter plate assay at both 28°C and 37°C. Results are expressed as the average of six technical replicates within each plate; error bars indicate SD. These results are representative, confirmed by three independent experiments. (B) Glycosylated EhaJ mediates enhanced biofilm formation in a dynamic flow cell system at 28°C. Spatial distribution of dynamic biofilm formation by GFP-labeled E. coli strains OS56(pBAD-kan) and E. coli OS56(pOMS2-kan) was monitored by confocal scanning laser microscopy at 15 h (left) and 48 h (right) after inoculation. Shown are representative horizontal sections collected within each biofilm and vertical sections (to the right and below for each individual panel) representing the yz-plane and the xz-plane, respectively, at the positions indicated by the white lines.
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Figure 3: (A) Glycosylated EhaJ [MS427(pOMS2)] mediates enhanced static biofilm formation in a microtiter plate assay at both 28°C and 37°C. Results are expressed as the average of six technical replicates within each plate; error bars indicate SD. These results are representative, confirmed by three independent experiments. (B) Glycosylated EhaJ mediates enhanced biofilm formation in a dynamic flow cell system at 28°C. Spatial distribution of dynamic biofilm formation by GFP-labeled E. coli strains OS56(pBAD-kan) and E. coli OS56(pOMS2-kan) was monitored by confocal scanning laser microscopy at 15 h (left) and 48 h (right) after inoculation. Shown are representative horizontal sections collected within each biofilm and vertical sections (to the right and below for each individual panel) representing the yz-plane and the xz-plane, respectively, at the positions indicated by the white lines.

Mentions: To determine whether the EhaJ protein promotes biofilm formation, we examined the effect of EhaJ over-expression in E. coli MS427 in a static biofilm assay in a non-treated polystyrene microtiter plate model after growth in M9 minimal medium (Figure 3A). MS427(pOMS2), which produces glycosylated EhaJ, exhibited greatly enhanced biofilm production in comparison to both the negative control strain, MS427(pBAD) and the strain expressing EhaJ without the glycosyltransferase [MS427(pOMS3)]. The ability of glycosylated EhaJ to promote biofilm formation was further assessed by over-expression in E. coli OS56 (a GFP-tagged derivative of MS427) in dynamic conditions using the continuous flow chamber model, which permits monitoring of the bacterial distribution within an evolving biofilm at the single cell level due to the combination of GFP-tagged cells and scanning confocal laser microscopy. Glycosylated EhaJ [OS56(pOMS2)] promoted strong biofilm growth under these conditions and produced a structure with a depth of approximately 10 μm (Figure 3B).


Characterization of EhaJ, a New Autotransporter Protein from Enterohemorrhagic and Enteropathogenic Escherichia coli.

Easton DM, Totsika M, Allsopp LP, Phan MD, Idris A, Wurpel DJ, Sherlock O, Zhang B, Venturini C, Beatson SA, Mahony TJ, Cobbold RN, Schembri MA - Front Microbiol (2011)

(A) Glycosylated EhaJ [MS427(pOMS2)] mediates enhanced static biofilm formation in a microtiter plate assay at both 28°C and 37°C. Results are expressed as the average of six technical replicates within each plate; error bars indicate SD. These results are representative, confirmed by three independent experiments. (B) Glycosylated EhaJ mediates enhanced biofilm formation in a dynamic flow cell system at 28°C. Spatial distribution of dynamic biofilm formation by GFP-labeled E. coli strains OS56(pBAD-kan) and E. coli OS56(pOMS2-kan) was monitored by confocal scanning laser microscopy at 15 h (left) and 48 h (right) after inoculation. Shown are representative horizontal sections collected within each biofilm and vertical sections (to the right and below for each individual panel) representing the yz-plane and the xz-plane, respectively, at the positions indicated by the white lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3108271&req=5

Figure 3: (A) Glycosylated EhaJ [MS427(pOMS2)] mediates enhanced static biofilm formation in a microtiter plate assay at both 28°C and 37°C. Results are expressed as the average of six technical replicates within each plate; error bars indicate SD. These results are representative, confirmed by three independent experiments. (B) Glycosylated EhaJ mediates enhanced biofilm formation in a dynamic flow cell system at 28°C. Spatial distribution of dynamic biofilm formation by GFP-labeled E. coli strains OS56(pBAD-kan) and E. coli OS56(pOMS2-kan) was monitored by confocal scanning laser microscopy at 15 h (left) and 48 h (right) after inoculation. Shown are representative horizontal sections collected within each biofilm and vertical sections (to the right and below for each individual panel) representing the yz-plane and the xz-plane, respectively, at the positions indicated by the white lines.
Mentions: To determine whether the EhaJ protein promotes biofilm formation, we examined the effect of EhaJ over-expression in E. coli MS427 in a static biofilm assay in a non-treated polystyrene microtiter plate model after growth in M9 minimal medium (Figure 3A). MS427(pOMS2), which produces glycosylated EhaJ, exhibited greatly enhanced biofilm production in comparison to both the negative control strain, MS427(pBAD) and the strain expressing EhaJ without the glycosyltransferase [MS427(pOMS3)]. The ability of glycosylated EhaJ to promote biofilm formation was further assessed by over-expression in E. coli OS56 (a GFP-tagged derivative of MS427) in dynamic conditions using the continuous flow chamber model, which permits monitoring of the bacterial distribution within an evolving biofilm at the single cell level due to the combination of GFP-tagged cells and scanning confocal laser microscopy. Glycosylated EhaJ [OS56(pOMS2)] promoted strong biofilm growth under these conditions and produced a structure with a depth of approximately 10 μm (Figure 3B).

Bottom Line: However, deletion of ehaJ did not significantly alter its adherence or biofilm properties.In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC.Further studies are required to elucidate the function of EhaJ in colonization and virulence.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Science, The University of Queensland Gatton, QLD, Australia.

ABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathotypes of E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. While certain EHEC and EPEC virulence mechanisms have been extensively studied, the factors that mediate host colonization remain to be properly defined. Previously, we identified four genes (ehaA, ehaB, ehaC, and ehaD) from the prototypic EHEC strain EDL933 that encode for proteins that belong to the autotransporter (AT) family. Here we have examined the prevalence of these genes, as well as several other AT-encoding genes, in a collection of EHEC and EPEC strains. We show that the complement of AT-encoding genes in EHEC and EPEC strains is variable, with some AT-encoding genes being highly prevalent. One previously uncharacterized AT-encoding gene, which we have termed ehaJ, was identified in 12/44 (27%) of EHEC and 2/20 (10%) of EPEC strains. The ehaJ gene lies immediately adjacent to a gene encoding a putative glycosyltransferase (referred to as egtA). Western blot analysis using an EhaJ-specific antibody indicated that EhaJ is glycosylated by EgtA. Expression of EhaJ in a recombinant E. coli strain, revealed EhaJ is located at the cell surface and in the presence of the egtA glycosyltransferase gene mediates strong biofilm formation in microtiter plate and flow cell assays. EhaJ also mediated adherence to a range of extracellular matrix proteins, however this occurred independent of glycosylation. We also demonstrate that EhaJ is expressed in a wild-type EPEC strain following in vitro growth. However, deletion of ehaJ did not significantly alter its adherence or biofilm properties. In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC. Further studies are required to elucidate the function of EhaJ in colonization and virulence.

No MeSH data available.


Related in: MedlinePlus