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Gut proteases target Yersinia invasin in vivo.

Trček J, Oellerich MF, Niedung K, Ebel F, Freund S, Trülzsch K - BMC Res Notes (2011)

Bottom Line: In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin.YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases.Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max von Pettenkofer Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität München, Germany. truelzsch@mvp.uni-muenchen.de.

ABSTRACT

Background: Yersinia enterocolitica is a common cause of food borne gastrointestinal disease. After oral uptake, yersiniae invade Peyer's patches of the distal ileum. This is accomplished by the binding of the Yersinia invasin to β1 integrins on the apical surface of M cells which overlie follicle associated lymphoid tissue. The gut represents a barrier that severely limits yersiniae from reaching deeper tissues such as Peyer's patches. We wondered if gut protease attack on invasion factors could contribute to the low number of yersiniae invading Peyer's patches.

Findings: Here we show that invasin is rapidly degraded in vivo by gut proteases in the mouse infection model. In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin. Protease treated yersiniae are shown to be less invasive in a cell culture model. YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases.

Conclusions: We demonstrate that gut proteases target important Yersinia virulence factors such as invasin and YadA in vivo. Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

No MeSH data available.


Related in: MedlinePlus

Gut protease treated yersiniae are less invasive in vitro. Y. enterocolitica WA-C or the invasin mutant WA-C-inv (Δinv) were digested with the supernatant of small intestinal luminal fluid (SI) or trypsin or mock digested with trypsin digestion buffer. Invasion (a) and adhesion (b) assays were performed using HeLa cells. Invasion assay was performed at 37°C, adhesion assay at 20°C. Percent yersiniae surviving gentamicin treatment is shown in (a), Percent yersiniae adhering to cells and SD is shown in (b). Statistical analysis was performed using Mann-Whitney test at the 0.05 significance level. Asteriks indicate a significant difference from WA-C.
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Figure 6: Gut protease treated yersiniae are less invasive in vitro. Y. enterocolitica WA-C or the invasin mutant WA-C-inv (Δinv) were digested with the supernatant of small intestinal luminal fluid (SI) or trypsin or mock digested with trypsin digestion buffer. Invasion (a) and adhesion (b) assays were performed using HeLa cells. Invasion assay was performed at 37°C, adhesion assay at 20°C. Percent yersiniae surviving gentamicin treatment is shown in (a), Percent yersiniae adhering to cells and SD is shown in (b). Statistical analysis was performed using Mann-Whitney test at the 0.05 significance level. Asteriks indicate a significant difference from WA-C.

Mentions: Yersiniae in Figure 2 lanes 11 and 12 were digested with 2,5 mg/ml trypsin in Tris-base pH 8.0, 11.5 mM CaCl2. This is higher than concentrations of trypsin found in intestinal fluid of humans [30]. Yersiniae in Figure 3 were digested with undiluted human ileal fluid. Yersiniae in Figures 4, 5 and 6 were digested with trypsin (23 μg/ml in Tris-base pH 8.0, 11.5 mM CaCl2), α-chymotrypsin (20 μg/ml in 100 mM Tris/Cl pH 7.8, 10 mM CaCl2), pancreatic elastase (10 μg/ml 100 mM NH4HCO3 pH 8.0) or carboxypeptidase A (50 μg/ml in 25 mM Tris/Cl pH 7.65). These concentrations are below physiological values which have been reported to be about 280 μg/g feces, 450 μg/g feces, and 200 μg/g feces respectively [30]. The pepsin digest in Figure 4a (4 mg/ml in 10 mM HCl) was performed using a concentration slightly higher than that reported for human gastric juice of 0.5 -1 mg/ml [31]. All proteases were obtained from Sigma-Aldrich.


Gut proteases target Yersinia invasin in vivo.

Trček J, Oellerich MF, Niedung K, Ebel F, Freund S, Trülzsch K - BMC Res Notes (2011)

Gut protease treated yersiniae are less invasive in vitro. Y. enterocolitica WA-C or the invasin mutant WA-C-inv (Δinv) were digested with the supernatant of small intestinal luminal fluid (SI) or trypsin or mock digested with trypsin digestion buffer. Invasion (a) and adhesion (b) assays were performed using HeLa cells. Invasion assay was performed at 37°C, adhesion assay at 20°C. Percent yersiniae surviving gentamicin treatment is shown in (a), Percent yersiniae adhering to cells and SD is shown in (b). Statistical analysis was performed using Mann-Whitney test at the 0.05 significance level. Asteriks indicate a significant difference from WA-C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3094372&req=5

Figure 6: Gut protease treated yersiniae are less invasive in vitro. Y. enterocolitica WA-C or the invasin mutant WA-C-inv (Δinv) were digested with the supernatant of small intestinal luminal fluid (SI) or trypsin or mock digested with trypsin digestion buffer. Invasion (a) and adhesion (b) assays were performed using HeLa cells. Invasion assay was performed at 37°C, adhesion assay at 20°C. Percent yersiniae surviving gentamicin treatment is shown in (a), Percent yersiniae adhering to cells and SD is shown in (b). Statistical analysis was performed using Mann-Whitney test at the 0.05 significance level. Asteriks indicate a significant difference from WA-C.
Mentions: Yersiniae in Figure 2 lanes 11 and 12 were digested with 2,5 mg/ml trypsin in Tris-base pH 8.0, 11.5 mM CaCl2. This is higher than concentrations of trypsin found in intestinal fluid of humans [30]. Yersiniae in Figure 3 were digested with undiluted human ileal fluid. Yersiniae in Figures 4, 5 and 6 were digested with trypsin (23 μg/ml in Tris-base pH 8.0, 11.5 mM CaCl2), α-chymotrypsin (20 μg/ml in 100 mM Tris/Cl pH 7.8, 10 mM CaCl2), pancreatic elastase (10 μg/ml 100 mM NH4HCO3 pH 8.0) or carboxypeptidase A (50 μg/ml in 25 mM Tris/Cl pH 7.65). These concentrations are below physiological values which have been reported to be about 280 μg/g feces, 450 μg/g feces, and 200 μg/g feces respectively [30]. The pepsin digest in Figure 4a (4 mg/ml in 10 mM HCl) was performed using a concentration slightly higher than that reported for human gastric juice of 0.5 -1 mg/ml [31]. All proteases were obtained from Sigma-Aldrich.

Bottom Line: In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin.YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases.Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max von Pettenkofer Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität München, Germany. truelzsch@mvp.uni-muenchen.de.

ABSTRACT

Background: Yersinia enterocolitica is a common cause of food borne gastrointestinal disease. After oral uptake, yersiniae invade Peyer's patches of the distal ileum. This is accomplished by the binding of the Yersinia invasin to β1 integrins on the apical surface of M cells which overlie follicle associated lymphoid tissue. The gut represents a barrier that severely limits yersiniae from reaching deeper tissues such as Peyer's patches. We wondered if gut protease attack on invasion factors could contribute to the low number of yersiniae invading Peyer's patches.

Findings: Here we show that invasin is rapidly degraded in vivo by gut proteases in the mouse infection model. In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin. Protease treated yersiniae are shown to be less invasive in a cell culture model. YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases.

Conclusions: We demonstrate that gut proteases target important Yersinia virulence factors such as invasin and YadA in vivo. Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

No MeSH data available.


Related in: MedlinePlus