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Tir Is Essential for the Recruitment of Tks5 to Enteropathogenic Escherichia coli Pedestals.

Jensen HH, Pedersen HN, Stenkjær E, Pedersen GA, Login FH, Nejsum LN - PLoS ONE (2015)

Bottom Line: The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells.EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5.Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Genetics and Interdiciplinary Nanoscience Center, Aarhus University, C. F. Moellers Allé 3, Aarhus, Denmark.

ABSTRACT
Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that infects the epithelial lining of the small intestine and causes diarrhea. Upon attachment to the intestinal epithelium, EPEC uses a Type III Secretion System to inject its own high affinity receptor Translocated intimin receptor (Tir) into the host cell. Tir facilitates tight adhesion and recruitment of actin-regulating proteins leading to formation of an actin pedestal beneath the infecting bacterium. The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells. Formation of podosomes is dependent upon the early podosome-specific scavenger protein Tks5, which is involved in actin recruitment. Although Tks5 is expressed in epithelial cells, and podosomes and EPEC pedestals share many components in their structure and mechanism of formation, the potential role of Tks5 in EPEC infections has not been studied. The aim of this study was to determine the subcellular localization of Tks5 in epithelial cells and to investigate if Tks5 is recruited to the EPEC pedestal. In an epithelial MDCK cell line stably expressing Tks5-EGFP, Tks5 localized to actin bundles. Upon infection, EPEC recruited Tks5-EGFP. Tir, but not Tir phosphorylation was essential for the recruitment. Time-lapse microscopy revealed that Tks5-EGFP was recruited instantly upon EPEC attachment to host cells, simultaneously with actin and N-WASp. EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5. Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation.

No MeSH data available.


Related in: MedlinePlus

The PX domain of Tks5 is not required for EPEC infection.Murine Embryonic Fibroblasts (MEFs) with wild-type Tks5 expression or expression of the deletion mutant ΔPX-Tks5 only were infected with WT EPEC for six hours. Cells and EPEC were then fixed and stained with Hoechst and phalloidin. A total of 60 cells of each type were imaged, and the number of bacteria infecting each cell was counted, and it was evaluated whether they formed pedestals. Outlier cells with more than 150 infecting EPEC were excluded from the analysis (six cells in total). A: Examples of MEFs infected by EPEC. Orange and white arrows indicate infecting bacteria with and without pedestal formation, respectively. Scale bars correspond to 20 μm for whole-cell images and 10 μm for inserts. Colors were adjusted to optimally visualize infecting bacteria. B: Number of infecting EPEC per cell with the average numbers indicated by a bar.
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pone.0141871.g005: The PX domain of Tks5 is not required for EPEC infection.Murine Embryonic Fibroblasts (MEFs) with wild-type Tks5 expression or expression of the deletion mutant ΔPX-Tks5 only were infected with WT EPEC for six hours. Cells and EPEC were then fixed and stained with Hoechst and phalloidin. A total of 60 cells of each type were imaged, and the number of bacteria infecting each cell was counted, and it was evaluated whether they formed pedestals. Outlier cells with more than 150 infecting EPEC were excluded from the analysis (six cells in total). A: Examples of MEFs infected by EPEC. Orange and white arrows indicate infecting bacteria with and without pedestal formation, respectively. Scale bars correspond to 20 μm for whole-cell images and 10 μm for inserts. Colors were adjusted to optimally visualize infecting bacteria. B: Number of infecting EPEC per cell with the average numbers indicated by a bar.

Mentions: As Tks5 in podosomes bind PI(3,4)P2 to initiate podosome formation, we hypothesizedthat the interaction between Tks5 and PI(3,4)P2 was necessary for pedestal formation. It has been shown that PI(3,4)P2 is synthesized in the membrane at the infection site [26], and Tks5 binds these via the PX domain [55]. To address the potential role of the PX domain a Murine Embryonic Fibroblast (MEF) cell line only expressing Tks5 isoforms lacking the PX domain (ΔPX-Tks5) was used [43]. WT MEFs and ΔPX-Tks5 MEFs were infected for six hours with equal numbers of WT EPEC, fixed and stained, and the number of infecting bacteria and their ability to form pedestals were evaluated. The number of EPEC bacteria infecting 59 WT MEFs and 56 ΔPX-Tks5 MEFs were 1414 and 1379, respectively (Fig 5A). As shown in Fig 5B the average number of infecting bacteria per cell was similar, however with high variance: 24.1 bacteria per WT MEF, and 27.8 bacteria per ΔPX-Tks5 MEF. Pedestals were observed at the infection site of 82.8% of bacteria infecting WT MEFs and 86.5% of bacteria infecting ΔPX-Tks5 MEFs. Thus, the infection efficiency on MEFs was not affected by the PX domain deletion; hence this domain of Tks5 is not essential for pedestal formation.


Tir Is Essential for the Recruitment of Tks5 to Enteropathogenic Escherichia coli Pedestals.

Jensen HH, Pedersen HN, Stenkjær E, Pedersen GA, Login FH, Nejsum LN - PLoS ONE (2015)

The PX domain of Tks5 is not required for EPEC infection.Murine Embryonic Fibroblasts (MEFs) with wild-type Tks5 expression or expression of the deletion mutant ΔPX-Tks5 only were infected with WT EPEC for six hours. Cells and EPEC were then fixed and stained with Hoechst and phalloidin. A total of 60 cells of each type were imaged, and the number of bacteria infecting each cell was counted, and it was evaluated whether they formed pedestals. Outlier cells with more than 150 infecting EPEC were excluded from the analysis (six cells in total). A: Examples of MEFs infected by EPEC. Orange and white arrows indicate infecting bacteria with and without pedestal formation, respectively. Scale bars correspond to 20 μm for whole-cell images and 10 μm for inserts. Colors were adjusted to optimally visualize infecting bacteria. B: Number of infecting EPEC per cell with the average numbers indicated by a bar.
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Related In: Results  -  Collection

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pone.0141871.g005: The PX domain of Tks5 is not required for EPEC infection.Murine Embryonic Fibroblasts (MEFs) with wild-type Tks5 expression or expression of the deletion mutant ΔPX-Tks5 only were infected with WT EPEC for six hours. Cells and EPEC were then fixed and stained with Hoechst and phalloidin. A total of 60 cells of each type were imaged, and the number of bacteria infecting each cell was counted, and it was evaluated whether they formed pedestals. Outlier cells with more than 150 infecting EPEC were excluded from the analysis (six cells in total). A: Examples of MEFs infected by EPEC. Orange and white arrows indicate infecting bacteria with and without pedestal formation, respectively. Scale bars correspond to 20 μm for whole-cell images and 10 μm for inserts. Colors were adjusted to optimally visualize infecting bacteria. B: Number of infecting EPEC per cell with the average numbers indicated by a bar.
Mentions: As Tks5 in podosomes bind PI(3,4)P2 to initiate podosome formation, we hypothesizedthat the interaction between Tks5 and PI(3,4)P2 was necessary for pedestal formation. It has been shown that PI(3,4)P2 is synthesized in the membrane at the infection site [26], and Tks5 binds these via the PX domain [55]. To address the potential role of the PX domain a Murine Embryonic Fibroblast (MEF) cell line only expressing Tks5 isoforms lacking the PX domain (ΔPX-Tks5) was used [43]. WT MEFs and ΔPX-Tks5 MEFs were infected for six hours with equal numbers of WT EPEC, fixed and stained, and the number of infecting bacteria and their ability to form pedestals were evaluated. The number of EPEC bacteria infecting 59 WT MEFs and 56 ΔPX-Tks5 MEFs were 1414 and 1379, respectively (Fig 5A). As shown in Fig 5B the average number of infecting bacteria per cell was similar, however with high variance: 24.1 bacteria per WT MEF, and 27.8 bacteria per ΔPX-Tks5 MEF. Pedestals were observed at the infection site of 82.8% of bacteria infecting WT MEFs and 86.5% of bacteria infecting ΔPX-Tks5 MEFs. Thus, the infection efficiency on MEFs was not affected by the PX domain deletion; hence this domain of Tks5 is not essential for pedestal formation.

Bottom Line: The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells.EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5.Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Genetics and Interdiciplinary Nanoscience Center, Aarhus University, C. F. Moellers Allé 3, Aarhus, Denmark.

ABSTRACT
Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that infects the epithelial lining of the small intestine and causes diarrhea. Upon attachment to the intestinal epithelium, EPEC uses a Type III Secretion System to inject its own high affinity receptor Translocated intimin receptor (Tir) into the host cell. Tir facilitates tight adhesion and recruitment of actin-regulating proteins leading to formation of an actin pedestal beneath the infecting bacterium. The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells. Formation of podosomes is dependent upon the early podosome-specific scavenger protein Tks5, which is involved in actin recruitment. Although Tks5 is expressed in epithelial cells, and podosomes and EPEC pedestals share many components in their structure and mechanism of formation, the potential role of Tks5 in EPEC infections has not been studied. The aim of this study was to determine the subcellular localization of Tks5 in epithelial cells and to investigate if Tks5 is recruited to the EPEC pedestal. In an epithelial MDCK cell line stably expressing Tks5-EGFP, Tks5 localized to actin bundles. Upon infection, EPEC recruited Tks5-EGFP. Tir, but not Tir phosphorylation was essential for the recruitment. Time-lapse microscopy revealed that Tks5-EGFP was recruited instantly upon EPEC attachment to host cells, simultaneously with actin and N-WASp. EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5. Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation.

No MeSH data available.


Related in: MedlinePlus