Limits...
The PagN protein of Salmonella enterica serovar Typhimurium is an adhesin and invasin.

Lambert MA, Smith SG - BMC Microbiol. (2008)

Bottom Line: S. enterica sv Typhimurium pagN mutants display a reduction in adhesion to and invasion of epithelial cells.Typhimurium.Finally PagN can be added to an ever-growing repertoire of factors that contribute to the pathogenesis of Salmonella.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Microbiology, Trinity College Dublin, St James's Hospital, Dublin 8, Ireland. malamber79@gmail.com

ABSTRACT

Background: The pagN gene of Salmonella enterica serovar Typhimurium is a PhoP-regulated gene that is up-regulated during growth within macrophages and in vivo in murine models of infection. The PagN protein displays similarity to the Hek and Tia invasins/adhesins of Escherichia coli. Thus far no function has been ascribed to the PagN protein.

Results: Here we show that the outer membrane located PagN protein mediates agglutination of red blood cells and that this can be masked by LPS. When expressed in Escherichia coli the PagN protein supports adhesion to and invasion of mammalian cells in a manner that is dependent on cytoskeletal rearrangements. S. enterica sv Typhimurium pagN mutants display a reduction in adhesion to and invasion of epithelial cells. Finally, we demonstrate that over-expression of PagN in a SPI-1 mutant can partially compensate for the lack of a functional invasasome.

Conclusion: PagN is an outer membrane protein that may contribute to the virulence of S. Typhimurium. This protein is a haemagglutinin and contributes to the adherence to mammalian cells. In addition, PagN can mediate high-level invasion of CHO-K1 cells. Previously,pagN mutants have been shown to be less competitive in vivo and thus this may be due to their lessened ability to interact with mammalian cells. Finally PagN can be added to an ever-growing repertoire of factors that contribute to the pathogenesis of Salmonella.

Show MeSH

Related in: MedlinePlus

Haemagglutination by E. coli or Salmonella expressing PagN. (A) Cultures of E. coli strain XL-1 Blue harboring plasmids pML1 (PagN+) or pTrc99a (PagN-) were induced with IPTG and then mixed with 3% human blood. Overnight cultures of E. coli harboring plasmids pHek6 (Hek+) and pBSKII+ (Hek-) were included as a positive control and vector control respectively. (B) Assays were carried out as described in Methods with cultures being incubated at 37°C or 70°C for 30 min prior to the assay. (C) The effect of LPS on PagN-promoted agglutination. S. Typhimurium strain LT-2 or CH133 harboring either pBSKII+ or pPagN2.3 were grown in MOPS minimal media adjusted to pH 5.8. Haemagglutination assays were performed as described in Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553418&req=5

Figure 1: Haemagglutination by E. coli or Salmonella expressing PagN. (A) Cultures of E. coli strain XL-1 Blue harboring plasmids pML1 (PagN+) or pTrc99a (PagN-) were induced with IPTG and then mixed with 3% human blood. Overnight cultures of E. coli harboring plasmids pHek6 (Hek+) and pBSKII+ (Hek-) were included as a positive control and vector control respectively. (B) Assays were carried out as described in Methods with cultures being incubated at 37°C or 70°C for 30 min prior to the assay. (C) The effect of LPS on PagN-promoted agglutination. S. Typhimurium strain LT-2 or CH133 harboring either pBSKII+ or pPagN2.3 were grown in MOPS minimal media adjusted to pH 5.8. Haemagglutination assays were performed as described in Methods.

Mentions: The Hek protein from E. coli K1 strain RS218 agglutinates erythrocytes in a heat-resistant manner [13,14]. PagN and Hek are ~54% similar, indicating that PagN may also promote erythrocyte agglutination. To test this hypothesis, a microtitre haemagglutination (HA) assay was performed as described in Methods. E. coli K-12 strain XL-1 Blue containing the pagN expression-vector pML1 or the empty vector control, pTrc99a, were incubated with human erythrocytes. The expression of PagN conferred upon recombinant E. coli the ability to agglutinate erythrocytes (Fig. 1A). Using 3% human blood, a bacterial HA titre of 16 was displayed by PagN-expressing E. coli, similar to that of E. coli expressing Hek (data not shown). Hek is a heat-resistant agglutinin; heating Hek-expressing cells to 70°C does not reduce agglutination activity [14]. We sought to determine if PagN was also a heat-resistant agglutinin. Unlike Hek, PagN displayed a decrease in HA titre after heating (Fig. 1B). Thus PagN is a heat-sensitive agglutinin.


The PagN protein of Salmonella enterica serovar Typhimurium is an adhesin and invasin.

Lambert MA, Smith SG - BMC Microbiol. (2008)

Haemagglutination by E. coli or Salmonella expressing PagN. (A) Cultures of E. coli strain XL-1 Blue harboring plasmids pML1 (PagN+) or pTrc99a (PagN-) were induced with IPTG and then mixed with 3% human blood. Overnight cultures of E. coli harboring plasmids pHek6 (Hek+) and pBSKII+ (Hek-) were included as a positive control and vector control respectively. (B) Assays were carried out as described in Methods with cultures being incubated at 37°C or 70°C for 30 min prior to the assay. (C) The effect of LPS on PagN-promoted agglutination. S. Typhimurium strain LT-2 or CH133 harboring either pBSKII+ or pPagN2.3 were grown in MOPS minimal media adjusted to pH 5.8. Haemagglutination assays were performed as described in Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553418&req=5

Figure 1: Haemagglutination by E. coli or Salmonella expressing PagN. (A) Cultures of E. coli strain XL-1 Blue harboring plasmids pML1 (PagN+) or pTrc99a (PagN-) were induced with IPTG and then mixed with 3% human blood. Overnight cultures of E. coli harboring plasmids pHek6 (Hek+) and pBSKII+ (Hek-) were included as a positive control and vector control respectively. (B) Assays were carried out as described in Methods with cultures being incubated at 37°C or 70°C for 30 min prior to the assay. (C) The effect of LPS on PagN-promoted agglutination. S. Typhimurium strain LT-2 or CH133 harboring either pBSKII+ or pPagN2.3 were grown in MOPS minimal media adjusted to pH 5.8. Haemagglutination assays were performed as described in Methods.
Mentions: The Hek protein from E. coli K1 strain RS218 agglutinates erythrocytes in a heat-resistant manner [13,14]. PagN and Hek are ~54% similar, indicating that PagN may also promote erythrocyte agglutination. To test this hypothesis, a microtitre haemagglutination (HA) assay was performed as described in Methods. E. coli K-12 strain XL-1 Blue containing the pagN expression-vector pML1 or the empty vector control, pTrc99a, were incubated with human erythrocytes. The expression of PagN conferred upon recombinant E. coli the ability to agglutinate erythrocytes (Fig. 1A). Using 3% human blood, a bacterial HA titre of 16 was displayed by PagN-expressing E. coli, similar to that of E. coli expressing Hek (data not shown). Hek is a heat-resistant agglutinin; heating Hek-expressing cells to 70°C does not reduce agglutination activity [14]. We sought to determine if PagN was also a heat-resistant agglutinin. Unlike Hek, PagN displayed a decrease in HA titre after heating (Fig. 1B). Thus PagN is a heat-sensitive agglutinin.

Bottom Line: S. enterica sv Typhimurium pagN mutants display a reduction in adhesion to and invasion of epithelial cells.Typhimurium.Finally PagN can be added to an ever-growing repertoire of factors that contribute to the pathogenesis of Salmonella.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Microbiology, Trinity College Dublin, St James's Hospital, Dublin 8, Ireland. malamber79@gmail.com

ABSTRACT

Background: The pagN gene of Salmonella enterica serovar Typhimurium is a PhoP-regulated gene that is up-regulated during growth within macrophages and in vivo in murine models of infection. The PagN protein displays similarity to the Hek and Tia invasins/adhesins of Escherichia coli. Thus far no function has been ascribed to the PagN protein.

Results: Here we show that the outer membrane located PagN protein mediates agglutination of red blood cells and that this can be masked by LPS. When expressed in Escherichia coli the PagN protein supports adhesion to and invasion of mammalian cells in a manner that is dependent on cytoskeletal rearrangements. S. enterica sv Typhimurium pagN mutants display a reduction in adhesion to and invasion of epithelial cells. Finally, we demonstrate that over-expression of PagN in a SPI-1 mutant can partially compensate for the lack of a functional invasasome.

Conclusion: PagN is an outer membrane protein that may contribute to the virulence of S. Typhimurium. This protein is a haemagglutinin and contributes to the adherence to mammalian cells. In addition, PagN can mediate high-level invasion of CHO-K1 cells. Previously,pagN mutants have been shown to be less competitive in vivo and thus this may be due to their lessened ability to interact with mammalian cells. Finally PagN can be added to an ever-growing repertoire of factors that contribute to the pathogenesis of Salmonella.

Show MeSH
Related in: MedlinePlus