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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

EhaJ mediates attachment to a range of ECM proteins in an ELISA-based binding assay. MS427 bound to MaxGel, collagen I, II, III and V, fibronectin, fibrinogen, and laminin when expressing ehaJ with or without co-expression of egtA [* indicates significant difference (P < 0.05) in comparison with BSA control]. EhaJ did not mediate adherence to collagen IV, elastin, heparin sulfate, or BSA.
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Figure 4: EhaJ mediates attachment to a range of ECM proteins in an ELISA-based binding assay. MS427 bound to MaxGel, collagen I, II, III and V, fibronectin, fibrinogen, and laminin when expressing ehaJ with or without co-expression of egtA [* indicates significant difference (P < 0.05) in comparison with BSA control]. EhaJ did not mediate adherence to collagen IV, elastin, heparin sulfate, or BSA.

Mentions: To study the adhesive properties of EhaJ we examined its ability to mediate binding to various cellular and extracellular targets. Initially, we tested the ability of EhaJ to mediate adherence to HeLa and Caco2 cells, however no binding was observed for E. coli OS56(pOMS2-kan; data not shown). Next, we tested for the ability of EhaJ to mediate adherence to MaxGel, a commercially available mixture of ECM components including collagens, laminin, fibronectin, tenascin, elastin, a number of proteoglycans, and glycosaminoglycans. Both E. coli MS427(pOMS2) and E. coli MS427(pOMS3) adhered equally well in this assay (Figure 4), and thus we examined binding in more detail by testing a range of different ECM proteins. E. coli MS427(pOMS2) and E. coli MS427(pOMS3) adhered strongly to collagen I, collagen II, collagen III, collagen V, fibronectin, fibrinogen, and laminin (Figure 4).


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)

EhaJ mediates attachment to a range of ECM proteins in an ELISA-based binding assay. MS427 bound to MaxGel, collagen I, II, III and V, fibronectin, fibrinogen, and laminin when expressing ehaJ with or without co-expression of egtA [* indicates significant difference (P < 0.05) in comparison with BSA control]. EhaJ did not mediate adherence to collagen IV, elastin, heparin sulfate, or BSA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: EhaJ mediates attachment to a range of ECM proteins in an ELISA-based binding assay. MS427 bound to MaxGel, collagen I, II, III and V, fibronectin, fibrinogen, and laminin when expressing ehaJ with or without co-expression of egtA [* indicates significant difference (P < 0.05) in comparison with BSA control]. EhaJ did not mediate adherence to collagen IV, elastin, heparin sulfate, or BSA.
Mentions: To study the adhesive properties of EhaJ we examined its ability to mediate binding to various cellular and extracellular targets. Initially, we tested the ability of EhaJ to mediate adherence to HeLa and Caco2 cells, however no binding was observed for E. coli OS56(pOMS2-kan; data not shown). Next, we tested for the ability of EhaJ to mediate adherence to MaxGel, a commercially available mixture of ECM components including collagens, laminin, fibronectin, tenascin, elastin, a number of proteoglycans, and glycosaminoglycans. Both E. coli MS427(pOMS2) and E. coli MS427(pOMS3) adhered equally well in this assay (Figure 4), and thus we examined binding in more detail by testing a range of different ECM proteins. E. coli MS427(pOMS2) and E. coli MS427(pOMS3) adhered strongly to collagen I, collagen II, collagen III, collagen V, fibronectin, fibrinogen, and laminin (Figure 4).

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