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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.

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Characterization of pagN mutant strain ML6. (A) Colonies of strains SL1344 and ML6 were boiled and used as a template in a PCR reaction with primers PagNF2 and PagNR2 to amplify the pagN ORF. MW indicates molecular size markers. Lane 1 ML6, lane 2 SL1344. (B) Sarkosyl-extracted outer membrane proteins were electrophoresed through a 15% gel. Proteins were transferred to PVDF membrane and probed with an anti-PagN antibody. The position of PagN is arrowed. When expressed from a multicopy plasmid PagN was found to migrate as a doublet in common with the Hek protein. Lane 1, SL1344; Lane 2, ML6; Lane 3, ML6 with pBR322; Lane 4, ML6 with pML10.
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Figure 4: Characterization of pagN mutant strain ML6. (A) Colonies of strains SL1344 and ML6 were boiled and used as a template in a PCR reaction with primers PagNF2 and PagNR2 to amplify the pagN ORF. MW indicates molecular size markers. Lane 1 ML6, lane 2 SL1344. (B) Sarkosyl-extracted outer membrane proteins were electrophoresed through a 15% gel. Proteins were transferred to PVDF membrane and probed with an anti-PagN antibody. The position of PagN is arrowed. When expressed from a multicopy plasmid PagN was found to migrate as a doublet in common with the Hek protein. Lane 1, SL1344; Lane 2, ML6; Lane 3, ML6 with pBR322; Lane 4, ML6 with pML10.

Mentions: A pagN mutant strain ML6 (pagN-) was constructed as described in Methods. This mutant was verified by PCR (Fig. 4A) and the lack of PagN expression in the outer membrane of strain ML6 was verified by immunoblotting (Fig. 4B, lane 2). This mutation could be complemented by expressing PagN from plasmid pML10 (pagN+) (Fig. 4B, lane 4). The lack of PagN had no effect on the growth of S. Typhimurium with both wild-type SL1344 and pagN mutant strain ML6 having doubling times of ~45 min in MM 5.8 minimal media. After overnight culture in this medium these bacteria grew to a similar extent having optical densities of ~1.5 OD600 nm. Some pag gene products such as PagL have been associated with modification of LPS [21]. We thus compared the LPS profiles of SL1344 to ML6 (pagN-) and LT-2 to MLT-2 (pagN-) (Fig. 5). In each case there was no difference between the LPS profile of the wild-type and mutant.


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

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

Characterization of pagN mutant strain ML6. (A) Colonies of strains SL1344 and ML6 were boiled and used as a template in a PCR reaction with primers PagNF2 and PagNR2 to amplify the pagN ORF. MW indicates molecular size markers. Lane 1 ML6, lane 2 SL1344. (B) Sarkosyl-extracted outer membrane proteins were electrophoresed through a 15% gel. Proteins were transferred to PVDF membrane and probed with an anti-PagN antibody. The position of PagN is arrowed. When expressed from a multicopy plasmid PagN was found to migrate as a doublet in common with the Hek protein. Lane 1, SL1344; Lane 2, ML6; Lane 3, ML6 with pBR322; Lane 4, ML6 with pML10.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553418&req=5

Figure 4: Characterization of pagN mutant strain ML6. (A) Colonies of strains SL1344 and ML6 were boiled and used as a template in a PCR reaction with primers PagNF2 and PagNR2 to amplify the pagN ORF. MW indicates molecular size markers. Lane 1 ML6, lane 2 SL1344. (B) Sarkosyl-extracted outer membrane proteins were electrophoresed through a 15% gel. Proteins were transferred to PVDF membrane and probed with an anti-PagN antibody. The position of PagN is arrowed. When expressed from a multicopy plasmid PagN was found to migrate as a doublet in common with the Hek protein. Lane 1, SL1344; Lane 2, ML6; Lane 3, ML6 with pBR322; Lane 4, ML6 with pML10.
Mentions: A pagN mutant strain ML6 (pagN-) was constructed as described in Methods. This mutant was verified by PCR (Fig. 4A) and the lack of PagN expression in the outer membrane of strain ML6 was verified by immunoblotting (Fig. 4B, lane 2). This mutation could be complemented by expressing PagN from plasmid pML10 (pagN+) (Fig. 4B, lane 4). The lack of PagN had no effect on the growth of S. Typhimurium with both wild-type SL1344 and pagN mutant strain ML6 having doubling times of ~45 min in MM 5.8 minimal media. After overnight culture in this medium these bacteria grew to a similar extent having optical densities of ~1.5 OD600 nm. Some pag gene products such as PagL have been associated with modification of LPS [21]. We thus compared the LPS profiles of SL1344 to ML6 (pagN-) and LT-2 to MLT-2 (pagN-) (Fig. 5). In each case there was no difference between the LPS profile of the wild-type and mutant.

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