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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) Western blotting with rabbit antiserum specific for the passenger domain of demonstrates expression of EhaJ in wild-type EPEC strain MS455 and loss of EhaJ in MS455ehaJ. Molecular mass (kDa) for the protein markers (M) [Novex Sharp Standards, Invitrogen] is shown on the right. (B) Wild-type strain MS455 did not bind strongly to the MaxGel protein mixture in an ECM binding assay and inactivation of EhaJ in strain MS455ehaJ did not reduce binding to ECM proteins in this assay (P = 0.55). However, over-expression of ehaJ (but not egtA) in MS455ehaJ(pOMS3) lead to a small but reproducible increase in binding to the MaxGel protein mixture (P = 0.008). An unpaired t-test was used for statistical comparisons using GraphPad Prism 5.
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Figure 5: (A) Western blotting with rabbit antiserum specific for the passenger domain of demonstrates expression of EhaJ in wild-type EPEC strain MS455 and loss of EhaJ in MS455ehaJ. Molecular mass (kDa) for the protein markers (M) [Novex Sharp Standards, Invitrogen] is shown on the right. (B) Wild-type strain MS455 did not bind strongly to the MaxGel protein mixture in an ECM binding assay and inactivation of EhaJ in strain MS455ehaJ did not reduce binding to ECM proteins in this assay (P = 0.55). However, over-expression of ehaJ (but not egtA) in MS455ehaJ(pOMS3) lead to a small but reproducible increase in binding to the MaxGel protein mixture (P = 0.008). An unpaired t-test was used for statistical comparisons using GraphPad Prism 5.

Mentions: To determine whether EhaJ is expressed in wild-type EPEC MS455, we constructed an ehaJ deletion mutant employing λ-red mediated homologous recombination of linear DNA (referred to as MS455ehaJ). Examination of whole cell lysates prepared from MS455 and MS455ehaJ, following growth in LB broth, by SDS-PAGE and western blotting demonstrated a low level of expression of EhaJ in E. coli MS455 and an absence of EhaJ in MS455ehaJ (Figure 5). We then tested MS455 and MS455ehaJ for their ability to adhere to MaxGel. MS455 adhered poorly in this assay, and although MS455ehaJ adhered less well, the difference was not statistically significant. MS455ehaJ complemented with pOMS3 bound strongly to the MaxGel, consistent with results obtained when EhaJ was expressed under control of the araBAD promoter in the MS427 background (Figure 4 and data not shown). Similar results were also obtained with regards to biofilm formation in the microtiter plate assay (data not shown). Thus, while EhaJ is a newly identified AT protein expressed by E. coli MS455, further studies will be required to investigate the function of this protein in wild-type strains.


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) Western blotting with rabbit antiserum specific for the passenger domain of demonstrates expression of EhaJ in wild-type EPEC strain MS455 and loss of EhaJ in MS455ehaJ. Molecular mass (kDa) for the protein markers (M) [Novex Sharp Standards, Invitrogen] is shown on the right. (B) Wild-type strain MS455 did not bind strongly to the MaxGel protein mixture in an ECM binding assay and inactivation of EhaJ in strain MS455ehaJ did not reduce binding to ECM proteins in this assay (P = 0.55). However, over-expression of ehaJ (but not egtA) in MS455ehaJ(pOMS3) lead to a small but reproducible increase in binding to the MaxGel protein mixture (P = 0.008). An unpaired t-test was used for statistical comparisons using GraphPad Prism 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: (A) Western blotting with rabbit antiserum specific for the passenger domain of demonstrates expression of EhaJ in wild-type EPEC strain MS455 and loss of EhaJ in MS455ehaJ. Molecular mass (kDa) for the protein markers (M) [Novex Sharp Standards, Invitrogen] is shown on the right. (B) Wild-type strain MS455 did not bind strongly to the MaxGel protein mixture in an ECM binding assay and inactivation of EhaJ in strain MS455ehaJ did not reduce binding to ECM proteins in this assay (P = 0.55). However, over-expression of ehaJ (but not egtA) in MS455ehaJ(pOMS3) lead to a small but reproducible increase in binding to the MaxGel protein mixture (P = 0.008). An unpaired t-test was used for statistical comparisons using GraphPad Prism 5.
Mentions: To determine whether EhaJ is expressed in wild-type EPEC MS455, we constructed an ehaJ deletion mutant employing λ-red mediated homologous recombination of linear DNA (referred to as MS455ehaJ). Examination of whole cell lysates prepared from MS455 and MS455ehaJ, following growth in LB broth, by SDS-PAGE and western blotting demonstrated a low level of expression of EhaJ in E. coli MS455 and an absence of EhaJ in MS455ehaJ (Figure 5). We then tested MS455 and MS455ehaJ for their ability to adhere to MaxGel. MS455 adhered poorly in this assay, and although MS455ehaJ adhered less well, the difference was not statistically significant. MS455ehaJ complemented with pOMS3 bound strongly to the MaxGel, consistent with results obtained when EhaJ was expressed under control of the araBAD promoter in the MS427 background (Figure 4 and data not shown). Similar results were also obtained with regards to biofilm formation in the microtiter plate assay (data not shown). Thus, while EhaJ is a newly identified AT protein expressed by E. coli MS455, further studies will be required to investigate the function of this protein in wild-type strains.

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