Limits...
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 EhaJ-specific antiserum demonstrates the difference in size of EhaJ from MS427(pOMS2) and MS427(pOMS3). Molecular mass markers (M) are pre-stained Novex® Sharp Standard (Invitrogen). (B) SDS-PAGE analysis demonstrating (i) Coomassie blue staining and (ii) Glycoprofile III fluorescent staining of proteins prepared from whole cell lysates of MS427(pOMS2) and MS427(pOMS3). Staining of the 155-kDa EhaJ protein with Glycoprofile III was only observed in the presence of EgtA. A lower molecular weight band that also stained with Glycoprofile III and may represent a partially glycosylated form of EhaJ was also observed in MS427(pOMS2). Although this band is not visible on the western blot shown in (A), it was visible in other western blots that were allowed to develop for a longer time period. (C) Immunofluorescence microscopy demonstrating surface localization of EhaJ. Phase-contrast (left) and fluorescence (right) images of MS427(pBAD; top) and MS427(pOMS2; bottom). Strains were grown in the presence of 0.2% arabinose for all three panels. G-EhaJ indicates glycosylated EhaJ.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3108271&req=5

Figure 2: (A) Western blotting with EhaJ-specific antiserum demonstrates the difference in size of EhaJ from MS427(pOMS2) and MS427(pOMS3). Molecular mass markers (M) are pre-stained Novex® Sharp Standard (Invitrogen). (B) SDS-PAGE analysis demonstrating (i) Coomassie blue staining and (ii) Glycoprofile III fluorescent staining of proteins prepared from whole cell lysates of MS427(pOMS2) and MS427(pOMS3). Staining of the 155-kDa EhaJ protein with Glycoprofile III was only observed in the presence of EgtA. A lower molecular weight band that also stained with Glycoprofile III and may represent a partially glycosylated form of EhaJ was also observed in MS427(pOMS2). Although this band is not visible on the western blot shown in (A), it was visible in other western blots that were allowed to develop for a longer time period. (C) Immunofluorescence microscopy demonstrating surface localization of EhaJ. Phase-contrast (left) and fluorescence (right) images of MS427(pBAD; top) and MS427(pOMS2; bottom). Strains were grown in the presence of 0.2% arabinose for all three panels. G-EhaJ indicates glycosylated EhaJ.

Mentions: To demonstrate expression of the EhaJ protein, plasmids pOMS2 and pOMS3 were transformed into the previously described E. coli K-12 flu mutant strain MS427, respectively. E. coli MS427 is unable to mediate the classical cell aggregation and biofilm phenotypes associated with Ag43 expression (Reisner et al., 2003). EhaJ cell-surface expression was demonstrated by immunofluorescence microscopy (Figure 2) using a rabbit polyclonal antiserum targeting a region in the predicted N-terminal passenger domain of EhaJ. Western blot analysis employing the same EhaJ-specific antiserum detected a 155-kDa protein in whole cell lysates prepared from E. coli MS427(pOMS2) following induction with arabinose (Figure 2). When the same analysis was performed on E. coli MS427(pOMS3), a smaller protein of approximately 110 kDa was detected using the EhaJ-specific serum. The difference in the size of EhaJ in the presence and absence of the egtA gene provides evidence to suggest that EhaJ is glycosylated. Glycosylation of EhaJ was demonstrated by staining of SDS-PAGE separated proteins from whole cell lysates of MS427(pOMS2) with the Glycoprofile III stain. Staining of the 155-kDa EhaJ protein was only observed in the presence of EgtA (Figure 2). A lower molecular weight band that also stained with Glycoprofile III was also visible on this gel (only in the presence of EgtA); we suggest that this band may represent a partially glycosylated version of EhaJ.


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 EhaJ-specific antiserum demonstrates the difference in size of EhaJ from MS427(pOMS2) and MS427(pOMS3). Molecular mass markers (M) are pre-stained Novex® Sharp Standard (Invitrogen). (B) SDS-PAGE analysis demonstrating (i) Coomassie blue staining and (ii) Glycoprofile III fluorescent staining of proteins prepared from whole cell lysates of MS427(pOMS2) and MS427(pOMS3). Staining of the 155-kDa EhaJ protein with Glycoprofile III was only observed in the presence of EgtA. A lower molecular weight band that also stained with Glycoprofile III and may represent a partially glycosylated form of EhaJ was also observed in MS427(pOMS2). Although this band is not visible on the western blot shown in (A), it was visible in other western blots that were allowed to develop for a longer time period. (C) Immunofluorescence microscopy demonstrating surface localization of EhaJ. Phase-contrast (left) and fluorescence (right) images of MS427(pBAD; top) and MS427(pOMS2; bottom). Strains were grown in the presence of 0.2% arabinose for all three panels. G-EhaJ indicates glycosylated EhaJ.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: (A) Western blotting with EhaJ-specific antiserum demonstrates the difference in size of EhaJ from MS427(pOMS2) and MS427(pOMS3). Molecular mass markers (M) are pre-stained Novex® Sharp Standard (Invitrogen). (B) SDS-PAGE analysis demonstrating (i) Coomassie blue staining and (ii) Glycoprofile III fluorescent staining of proteins prepared from whole cell lysates of MS427(pOMS2) and MS427(pOMS3). Staining of the 155-kDa EhaJ protein with Glycoprofile III was only observed in the presence of EgtA. A lower molecular weight band that also stained with Glycoprofile III and may represent a partially glycosylated form of EhaJ was also observed in MS427(pOMS2). Although this band is not visible on the western blot shown in (A), it was visible in other western blots that were allowed to develop for a longer time period. (C) Immunofluorescence microscopy demonstrating surface localization of EhaJ. Phase-contrast (left) and fluorescence (right) images of MS427(pBAD; top) and MS427(pOMS2; bottom). Strains were grown in the presence of 0.2% arabinose for all three panels. G-EhaJ indicates glycosylated EhaJ.
Mentions: To demonstrate expression of the EhaJ protein, plasmids pOMS2 and pOMS3 were transformed into the previously described E. coli K-12 flu mutant strain MS427, respectively. E. coli MS427 is unable to mediate the classical cell aggregation and biofilm phenotypes associated with Ag43 expression (Reisner et al., 2003). EhaJ cell-surface expression was demonstrated by immunofluorescence microscopy (Figure 2) using a rabbit polyclonal antiserum targeting a region in the predicted N-terminal passenger domain of EhaJ. Western blot analysis employing the same EhaJ-specific antiserum detected a 155-kDa protein in whole cell lysates prepared from E. coli MS427(pOMS2) following induction with arabinose (Figure 2). When the same analysis was performed on E. coli MS427(pOMS3), a smaller protein of approximately 110 kDa was detected using the EhaJ-specific serum. The difference in the size of EhaJ in the presence and absence of the egtA gene provides evidence to suggest that EhaJ is glycosylated. Glycosylation of EhaJ was demonstrated by staining of SDS-PAGE separated proteins from whole cell lysates of MS427(pOMS2) with the Glycoprofile III stain. Staining of the 155-kDa EhaJ protein was only observed in the presence of EgtA (Figure 2). A lower molecular weight band that also stained with Glycoprofile III was also visible on this gel (only in the presence of EgtA); we suggest that this band may represent a partially glycosylated version of EhaJ.

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