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Septins arrange F-actin-containing fibers on the Chlamydia trachomatis inclusion and are required for normal release of the inclusion by extrusion.

Volceanov L, Herbst K, Biniossek M, Schilling O, Haller D, Nölke T, Subbarayal P, Rudel T, Zieger B, Häcker G - MBio (2014)

Bottom Line: RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells.Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol.Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology and Hygiene, University Medical Center Freiburg, Freiburg, Germany.

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Related in: MedlinePlus

Septins are required for normal extrusion of the inclusion. (A) HeLa cells were transfected with siRNA directed against SEPT9 alone (top panel) or were transfected simultaneously with siRNAs targeting SEPT2, -7, and -9 (bottom panel), followed by infection with C. trachomatis about 4 h later. Extrusions released by host cells between 48 and 66 h p.i. were counted using fluorescence microscopy. Results were obtained from either 4 independent experiments (shown as means and SEM; P = 0.002) (siSeptin9, top panel) or 3 independent experiments (means and SEM; P = 0.0001) (siSeptin 2+7+9, bottom panel). (B) HeLa cells were transfected with siRNA targeting SEPT9, followed by infection with C. trachomatis about 4 h later. Culture supernatants containing extrusions released between 48 and 66 h p.i. were collected at 66 h p.i., extrusions were enriched by centrifugation and lysed, and bacteria were titrated on a fresh monolayer of HeLa cells. Twenty-five hours later, cells were fixed, and inclusions were counted using fluorescence microscopy. The IFU/ml of the siControl samples were set to 1. Fold change of results for siSEPT9 relative to those for siControl was calculated. Data represent means and SEM of 3 independent experiments; P = 0.0002.
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fig6: Septins are required for normal extrusion of the inclusion. (A) HeLa cells were transfected with siRNA directed against SEPT9 alone (top panel) or were transfected simultaneously with siRNAs targeting SEPT2, -7, and -9 (bottom panel), followed by infection with C. trachomatis about 4 h later. Extrusions released by host cells between 48 and 66 h p.i. were counted using fluorescence microscopy. Results were obtained from either 4 independent experiments (shown as means and SEM; P = 0.002) (siSeptin9, top panel) or 3 independent experiments (means and SEM; P = 0.0001) (siSeptin 2+7+9, bottom panel). (B) HeLa cells were transfected with siRNA targeting SEPT9, followed by infection with C. trachomatis about 4 h later. Culture supernatants containing extrusions released between 48 and 66 h p.i. were collected at 66 h p.i., extrusions were enriched by centrifugation and lysed, and bacteria were titrated on a fresh monolayer of HeLa cells. Twenty-five hours later, cells were fixed, and inclusions were counted using fluorescence microscopy. The IFU/ml of the siControl samples were set to 1. Fold change of results for siSEPT9 relative to those for siControl was calculated. Data represent means and SEM of 3 independent experiments; P = 0.0002.

Mentions: We tested for such a role in chlamydial extrusion by counting the extrusions released between 48 h and 66 h p.i. (11, 12). Using either siRNA directed against SEPT9 alone or the combinations of siRNA specific for SEPT2, -7, and -9, we found a 2- to 3-fold reduction in the numbers of extrusions when the septins were knocked down (Fig. 6A). Determination of infectious bacteria released by extrusion showed an about 4-fold reduction when SEPT9 was knocked down (Fig. 6B). Septins thus appear to be required for the normal organization of chlamydial exit by extrusion.


Septins arrange F-actin-containing fibers on the Chlamydia trachomatis inclusion and are required for normal release of the inclusion by extrusion.

Volceanov L, Herbst K, Biniossek M, Schilling O, Haller D, Nölke T, Subbarayal P, Rudel T, Zieger B, Häcker G - MBio (2014)

Septins are required for normal extrusion of the inclusion. (A) HeLa cells were transfected with siRNA directed against SEPT9 alone (top panel) or were transfected simultaneously with siRNAs targeting SEPT2, -7, and -9 (bottom panel), followed by infection with C. trachomatis about 4 h later. Extrusions released by host cells between 48 and 66 h p.i. were counted using fluorescence microscopy. Results were obtained from either 4 independent experiments (shown as means and SEM; P = 0.002) (siSeptin9, top panel) or 3 independent experiments (means and SEM; P = 0.0001) (siSeptin 2+7+9, bottom panel). (B) HeLa cells were transfected with siRNA targeting SEPT9, followed by infection with C. trachomatis about 4 h later. Culture supernatants containing extrusions released between 48 and 66 h p.i. were collected at 66 h p.i., extrusions were enriched by centrifugation and lysed, and bacteria were titrated on a fresh monolayer of HeLa cells. Twenty-five hours later, cells were fixed, and inclusions were counted using fluorescence microscopy. The IFU/ml of the siControl samples were set to 1. Fold change of results for siSEPT9 relative to those for siControl was calculated. Data represent means and SEM of 3 independent experiments; P = 0.0002.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Septins are required for normal extrusion of the inclusion. (A) HeLa cells were transfected with siRNA directed against SEPT9 alone (top panel) or were transfected simultaneously with siRNAs targeting SEPT2, -7, and -9 (bottom panel), followed by infection with C. trachomatis about 4 h later. Extrusions released by host cells between 48 and 66 h p.i. were counted using fluorescence microscopy. Results were obtained from either 4 independent experiments (shown as means and SEM; P = 0.002) (siSeptin9, top panel) or 3 independent experiments (means and SEM; P = 0.0001) (siSeptin 2+7+9, bottom panel). (B) HeLa cells were transfected with siRNA targeting SEPT9, followed by infection with C. trachomatis about 4 h later. Culture supernatants containing extrusions released between 48 and 66 h p.i. were collected at 66 h p.i., extrusions were enriched by centrifugation and lysed, and bacteria were titrated on a fresh monolayer of HeLa cells. Twenty-five hours later, cells were fixed, and inclusions were counted using fluorescence microscopy. The IFU/ml of the siControl samples were set to 1. Fold change of results for siSEPT9 relative to those for siControl was calculated. Data represent means and SEM of 3 independent experiments; P = 0.0002.
Mentions: We tested for such a role in chlamydial extrusion by counting the extrusions released between 48 h and 66 h p.i. (11, 12). Using either siRNA directed against SEPT9 alone or the combinations of siRNA specific for SEPT2, -7, and -9, we found a 2- to 3-fold reduction in the numbers of extrusions when the septins were knocked down (Fig. 6A). Determination of infectious bacteria released by extrusion showed an about 4-fold reduction when SEPT9 was knocked down (Fig. 6B). Septins thus appear to be required for the normal organization of chlamydial exit by extrusion.

Bottom Line: RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells.Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol.Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology and Hygiene, University Medical Center Freiburg, Freiburg, Germany.

Show MeSH
Related in: MedlinePlus