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Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases.

Vlahou G, Schmidt O, Wagner B, Uenlue H, Dersch P, Rivero F, Weissenmayer BA - BMC Microbiol. (2009)

Bottom Line: No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental.YopE also caused reduced uptake of yeast particles.We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Zentrum für Biochemie und Zentrum für Molekulare Medizin, Medizinische Fakultät, Universität Köln, Joseph-Stelzmann-Strasse 52, 50931 Köln, Germany. gvlahou@uni-koeln.de

ABSTRACT

Background: All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops).

Results: The Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH.

Conclusion: The phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

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Related in: MedlinePlus

YopE inhibits amoebial growth. Vegetative growth was measured in liquid cultures of cell lines with non-induced and induced expression of YopE, GFP-YopE, YopH, GFP-YopH, GFP-YopJ and GFP-YopM. Black squares: non-induced cell lines; grey circles: induced cell lines. For each growth curve, two independent cultures, each run in duplicate, were analyzed and averaged. Standard error bars are mostly smaller than symbol sizes.
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Figure 2: YopE inhibits amoebial growth. Vegetative growth was measured in liquid cultures of cell lines with non-induced and induced expression of YopE, GFP-YopE, YopH, GFP-YopH, GFP-YopJ and GFP-YopM. Black squares: non-induced cell lines; grey circles: induced cell lines. For each growth curve, two independent cultures, each run in duplicate, were analyzed and averaged. Standard error bars are mostly smaller than symbol sizes.

Mentions: First we tested whether growth of Dictyostelium in liquid culture was affected by in vivo expression of Yop effectors. Growth measurements over several days showed that the growth of YopE and GFP-YopE expressing cell lines was drastically reduced in comparison with non-induced cell lines (Fig. 2). At the beginning, growth of YopE expressing cells was significantly reduced, with generation times of about 62 hours in comparison with 12 hours of the non-induced controls. After 10 days, the cells of the same culture started to regrow, albeit slower than the control cells with generation times of 20 and 38 hours. Unlike YopE, growth of Dictyostelium cell lines expressing other Yops or their GFP-fusion derivatives showed no noticeable difference between induced and non-induced cell lines (Fig. 2). Comparable results were obtained when the cells were plated on Klebsiella lawns and the plaque numbers were counted after 4 days. Only the plaque numbers of YopE or GFP-YopE expressing cell lines were reduced in comparison with the non-induced cell line (not shown).


Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases.

Vlahou G, Schmidt O, Wagner B, Uenlue H, Dersch P, Rivero F, Weissenmayer BA - BMC Microbiol. (2009)

YopE inhibits amoebial growth. Vegetative growth was measured in liquid cultures of cell lines with non-induced and induced expression of YopE, GFP-YopE, YopH, GFP-YopH, GFP-YopJ and GFP-YopM. Black squares: non-induced cell lines; grey circles: induced cell lines. For each growth curve, two independent cultures, each run in duplicate, were analyzed and averaged. Standard error bars are mostly smaller than symbol sizes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724381&req=5

Figure 2: YopE inhibits amoebial growth. Vegetative growth was measured in liquid cultures of cell lines with non-induced and induced expression of YopE, GFP-YopE, YopH, GFP-YopH, GFP-YopJ and GFP-YopM. Black squares: non-induced cell lines; grey circles: induced cell lines. For each growth curve, two independent cultures, each run in duplicate, were analyzed and averaged. Standard error bars are mostly smaller than symbol sizes.
Mentions: First we tested whether growth of Dictyostelium in liquid culture was affected by in vivo expression of Yop effectors. Growth measurements over several days showed that the growth of YopE and GFP-YopE expressing cell lines was drastically reduced in comparison with non-induced cell lines (Fig. 2). At the beginning, growth of YopE expressing cells was significantly reduced, with generation times of about 62 hours in comparison with 12 hours of the non-induced controls. After 10 days, the cells of the same culture started to regrow, albeit slower than the control cells with generation times of 20 and 38 hours. Unlike YopE, growth of Dictyostelium cell lines expressing other Yops or their GFP-fusion derivatives showed no noticeable difference between induced and non-induced cell lines (Fig. 2). Comparable results were obtained when the cells were plated on Klebsiella lawns and the plaque numbers were counted after 4 days. Only the plaque numbers of YopE or GFP-YopE expressing cell lines were reduced in comparison with the non-induced cell line (not shown).

Bottom Line: No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental.YopE also caused reduced uptake of yeast particles.We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Zentrum für Biochemie und Zentrum für Molekulare Medizin, Medizinische Fakultät, Universität Köln, Joseph-Stelzmann-Strasse 52, 50931 Köln, Germany. gvlahou@uni-koeln.de

ABSTRACT

Background: All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops).

Results: The Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH.

Conclusion: The phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

Show MeSH
Related in: MedlinePlus