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In vitro Intestinal Mucosal Epithelial Responses to Wild-Type Salmonella Typhi and Attenuated Typhoid Vaccines.

Fiorentino M, Lammers KM, Levine MM, Sztein MB, Fasano A - Front Immunol (2013)

Bottom Line: Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure.We conclude that wild-type S.Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mucosal Biology Research Center, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
Typhoid fever, caused by S. Typhi, is responsible for approximately 200,000 deaths per year worldwide. Little information is available regarding epithelium-bacterial interactions in S. Typhi infection. We have evaluated in vitro the effects of wild-type S. Typhi, the licensed Ty21a typhoid vaccine and the leading strains CVD 908-htrA and CVD 909 vaccine candidates on intestinal barrier function and immune response. Caco2 monolayers infected with wild-type S. Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure. S. Typhi triggered the secretion of interleukin (IL)-8 and IL-6. Caco2 cells infected with the attenuated strains exhibited a milder pro-inflammatory response with minimal disruption of the barrier integrity. We conclude that wild-type S. Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

No MeSH data available.


Related in: MedlinePlus

Occludin is hyperphosphorylated on threonine and translocates into the cytoplasm. We analyzed the solubility of occludin as an indication of its association with tight junctions. Protein lysates in the form of X-100 Triton soluble (S) and insoluble (I) fractions were obtained from Caco2 cell monolayers after infection with wild-type S. Typhi, CVD 908-htrA, and CVD 909 for 4 and 22 h. Equal amounts were loaded on the gel, electrophoresed, transferred onto a membrane and blotted with antibodies. (A) Western blot of protein samples blotted with anti-occludin (upper panel), anti-phosphothreonine (middle panel), and anti-actin (lower panel) as loading control, 4 h following exposure to bacteria. (B,C) Quantification of the data shown in (A). (D) Western blot of protein samples blotted with anti-occludin (upper panel), anti-phosphothreonine (middle panel) and anti-actin (lower panel) 22 h post-infection with S. Typhi strains. (E,F). Quantification of the data shown in (D). Data have been normalized to the actin loading control. These results represent the average of two individual experiments.
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Figure 8: Occludin is hyperphosphorylated on threonine and translocates into the cytoplasm. We analyzed the solubility of occludin as an indication of its association with tight junctions. Protein lysates in the form of X-100 Triton soluble (S) and insoluble (I) fractions were obtained from Caco2 cell monolayers after infection with wild-type S. Typhi, CVD 908-htrA, and CVD 909 for 4 and 22 h. Equal amounts were loaded on the gel, electrophoresed, transferred onto a membrane and blotted with antibodies. (A) Western blot of protein samples blotted with anti-occludin (upper panel), anti-phosphothreonine (middle panel), and anti-actin (lower panel) as loading control, 4 h following exposure to bacteria. (B,C) Quantification of the data shown in (A). (D) Western blot of protein samples blotted with anti-occludin (upper panel), anti-phosphothreonine (middle panel) and anti-actin (lower panel) 22 h post-infection with S. Typhi strains. (E,F). Quantification of the data shown in (D). Data have been normalized to the actin loading control. These results represent the average of two individual experiments.

Mentions: After infection, equal protein amounts of cell lysates from the Triton X-100 soluble and insoluble fractions were resolved by SDS-PAGE and then analyzed by immunoblotting in parallel with uninfected controls (Figure 8). In the uninfected monolayer occludin was mainly localized in the insoluble fraction and is visible on western blots as a strong band of about 65 kDa [Low Molecular Weight (LMW); Figure 8A, upper panel]. Upon infection with wild-type S. Typhi, an additional band of about 72–79 kDa was detected in the insoluble membrane fraction at 4 h post-infection. This High Molecular Weight (HMW) band was also observed in both CVD mutants-treated cell lysates, although weaker than that observed with the wild-type S. Typhi (Figure 8A, upper panel; Figure 8B). At 22 h, we observed an increase in LMW occludin in the soluble fraction paralleled by a decrease of the HMW species in all bacteria samples (Figure 8D, upper panel; Figure 8E). The HMW species (72–79 kDa) has been previously shown to represent a hyperphosphorylated form of occludin and represents a sub-pool of this protein specifically associated with the functional sealing components of tight-junction (Sakakibara et al., 1997; Wong, 1997; Seth et al., 2007). Specifically it has been demonstrated that occludin undergoes dephosphorylation on Ser/Thr residues during the disruption of tight junctions by various insults. Analysis of the threonine phosphorylation status of our samples showed that occludin appears to be phosphorylated on Thr in the resting epithelium (uninfected controls, LMW band, Figures 8A,D, middle panels). Conversely, in 4 h wild-type S. Typhi-infected cell lysates, only the HMW band appeared phosphorylated and this represents the hyperphosphorylated form of occludin. We observed a similar, albeit milder, shift of the occludin phosphorylation status toward the HMW species in both CVD 909 and CVD 908-htrAS. Typhi mutants (Figure 8C). Analysis of lysates at 22 h post-infection revealed that S. Typhi induced both a loss of occludin and hyperphosphorylated occludin from the insoluble fraction and a shift of the signal to the soluble fraction (Figure 8D, middle panel; Figure 8F) in all Salmonella strain-treated samples. As in the uninfected control, the phosphorylation status of the 65 kDa band of the insoluble membrane fraction was apparently not affected.


In vitro Intestinal Mucosal Epithelial Responses to Wild-Type Salmonella Typhi and Attenuated Typhoid Vaccines.

Fiorentino M, Lammers KM, Levine MM, Sztein MB, Fasano A - Front Immunol (2013)

Occludin is hyperphosphorylated on threonine and translocates into the cytoplasm. We analyzed the solubility of occludin as an indication of its association with tight junctions. Protein lysates in the form of X-100 Triton soluble (S) and insoluble (I) fractions were obtained from Caco2 cell monolayers after infection with wild-type S. Typhi, CVD 908-htrA, and CVD 909 for 4 and 22 h. Equal amounts were loaded on the gel, electrophoresed, transferred onto a membrane and blotted with antibodies. (A) Western blot of protein samples blotted with anti-occludin (upper panel), anti-phosphothreonine (middle panel), and anti-actin (lower panel) as loading control, 4 h following exposure to bacteria. (B,C) Quantification of the data shown in (A). (D) Western blot of protein samples blotted with anti-occludin (upper panel), anti-phosphothreonine (middle panel) and anti-actin (lower panel) 22 h post-infection with S. Typhi strains. (E,F). Quantification of the data shown in (D). Data have been normalized to the actin loading control. These results represent the average of two individual experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 8: Occludin is hyperphosphorylated on threonine and translocates into the cytoplasm. We analyzed the solubility of occludin as an indication of its association with tight junctions. Protein lysates in the form of X-100 Triton soluble (S) and insoluble (I) fractions were obtained from Caco2 cell monolayers after infection with wild-type S. Typhi, CVD 908-htrA, and CVD 909 for 4 and 22 h. Equal amounts were loaded on the gel, electrophoresed, transferred onto a membrane and blotted with antibodies. (A) Western blot of protein samples blotted with anti-occludin (upper panel), anti-phosphothreonine (middle panel), and anti-actin (lower panel) as loading control, 4 h following exposure to bacteria. (B,C) Quantification of the data shown in (A). (D) Western blot of protein samples blotted with anti-occludin (upper panel), anti-phosphothreonine (middle panel) and anti-actin (lower panel) 22 h post-infection with S. Typhi strains. (E,F). Quantification of the data shown in (D). Data have been normalized to the actin loading control. These results represent the average of two individual experiments.
Mentions: After infection, equal protein amounts of cell lysates from the Triton X-100 soluble and insoluble fractions were resolved by SDS-PAGE and then analyzed by immunoblotting in parallel with uninfected controls (Figure 8). In the uninfected monolayer occludin was mainly localized in the insoluble fraction and is visible on western blots as a strong band of about 65 kDa [Low Molecular Weight (LMW); Figure 8A, upper panel]. Upon infection with wild-type S. Typhi, an additional band of about 72–79 kDa was detected in the insoluble membrane fraction at 4 h post-infection. This High Molecular Weight (HMW) band was also observed in both CVD mutants-treated cell lysates, although weaker than that observed with the wild-type S. Typhi (Figure 8A, upper panel; Figure 8B). At 22 h, we observed an increase in LMW occludin in the soluble fraction paralleled by a decrease of the HMW species in all bacteria samples (Figure 8D, upper panel; Figure 8E). The HMW species (72–79 kDa) has been previously shown to represent a hyperphosphorylated form of occludin and represents a sub-pool of this protein specifically associated with the functional sealing components of tight-junction (Sakakibara et al., 1997; Wong, 1997; Seth et al., 2007). Specifically it has been demonstrated that occludin undergoes dephosphorylation on Ser/Thr residues during the disruption of tight junctions by various insults. Analysis of the threonine phosphorylation status of our samples showed that occludin appears to be phosphorylated on Thr in the resting epithelium (uninfected controls, LMW band, Figures 8A,D, middle panels). Conversely, in 4 h wild-type S. Typhi-infected cell lysates, only the HMW band appeared phosphorylated and this represents the hyperphosphorylated form of occludin. We observed a similar, albeit milder, shift of the occludin phosphorylation status toward the HMW species in both CVD 909 and CVD 908-htrAS. Typhi mutants (Figure 8C). Analysis of lysates at 22 h post-infection revealed that S. Typhi induced both a loss of occludin and hyperphosphorylated occludin from the insoluble fraction and a shift of the signal to the soluble fraction (Figure 8D, middle panel; Figure 8F) in all Salmonella strain-treated samples. As in the uninfected control, the phosphorylation status of the 65 kDa band of the insoluble membrane fraction was apparently not affected.

Bottom Line: Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure.We conclude that wild-type S.Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mucosal Biology Research Center, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
Typhoid fever, caused by S. Typhi, is responsible for approximately 200,000 deaths per year worldwide. Little information is available regarding epithelium-bacterial interactions in S. Typhi infection. We have evaluated in vitro the effects of wild-type S. Typhi, the licensed Ty21a typhoid vaccine and the leading strains CVD 908-htrA and CVD 909 vaccine candidates on intestinal barrier function and immune response. Caco2 monolayers infected with wild-type S. Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure. S. Typhi triggered the secretion of interleukin (IL)-8 and IL-6. Caco2 cells infected with the attenuated strains exhibited a milder pro-inflammatory response with minimal disruption of the barrier integrity. We conclude that wild-type S. Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.

No MeSH data available.


Related in: MedlinePlus