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The trans-Golgi SNARE syntaxin 10 is required for optimal development of Chlamydia trachomatis.

Lucas AL, Ouellette SP, Kabeiseman EJ, Cichos KH, Rucks EA - Front Cell Infect Microbiol (2015)

Bottom Line: These defects in chlamydial development correlate with an overabundance of NBD-lipid retained by inclusions cultured in syntaxin 10 knockdown cells.Overall, loss of syntaxin 10 at the inclusion membrane negatively affects Chlamydia.Understanding host machinery involved in maintaining an optimal inclusion environment to support chlamydial growth and development is critical toward understanding the molecular signals involved in successful progression through the chlamydial developmental cycle.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota Vermillion, SD, USA.

ABSTRACT
Chlamydia trachomatis, an obligate intracellular pathogen, grows inside of a vacuole, termed the inclusion. Within the inclusion, the organisms differentiate from the infectious elementary body (EB) into the reticulate body (RB). The RB communicates with the host cell through the inclusion membrane to obtain the nutrients necessary to divide, thus expanding the chlamydial population. At late time points within the developmental cycle, the RBs respond to unknown molecular signals to redifferentiate into infectious EBs to perpetuate the infection cycle. One strategy for Chlamydia to obtain necessary nutrients and metabolites from the host is to intercept host vesicular trafficking pathways. In this study we demonstrate that a trans-Golgi soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE), syntaxin 10, and/or syntaxin 10-associated Golgi elements colocalize with the chlamydial inclusion. We hypothesized that Chlamydia utilizes the molecular machinery of syntaxin 10 at the inclusion membrane to intercept specific vesicular trafficking pathways in order to create and maintain an optimal intra-inclusion environment. To test this hypothesis, we used siRNA knockdown of syntaxin 10 to examine the impact of the loss of syntaxin 10 on chlamydial growth and development. Our results demonstrate that loss of syntaxin 10 leads to defects in normal chlamydial maturation including: variable inclusion size with fewer chlamydial organisms per inclusion, fewer infectious progeny, and delayed or halted RB-EB differentiation. These defects in chlamydial development correlate with an overabundance of NBD-lipid retained by inclusions cultured in syntaxin 10 knockdown cells. Overall, loss of syntaxin 10 at the inclusion membrane negatively affects Chlamydia. Understanding host machinery involved in maintaining an optimal inclusion environment to support chlamydial growth and development is critical toward understanding the molecular signals involved in successful progression through the chlamydial developmental cycle.

No MeSH data available.


Related in: MedlinePlus

Effect of syntaxin 10 siRNA knockdown on NBD-sphingomyelin trafficking to the chlamydial inclusion. (A) Syntaxin 10 (Stx10) or non-targeting (NT) siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 24 h, labeled with NBD-ceramide, and treated with back-exchange medium for an additional 3 h. Samples were imaged at 40X magnification with a 200 ms exposure time using an Axiovert 200 M Imager with the AxioCam HRm camera (Carl Zeiss Microscopy, LLC). These images are representative of 3 independent experiments. (B) Untreated HeLa cells were infected with C. trachomatis serovar L2 for 18 or 26 h, labeled with NBD-ceramide and back-exchanged for 1.5 h. In addition, Stx10 or NT siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 30 h, labeled and back-exchanged as 18- and 26-h samples. Samples from two independent experiments were imaged using 60X magnification Olympus BX 60 fluorescent scope and images taken with a Nikon DS-Qi1Mc camera, with a 40 ms exposure for 18- 26- and NT siRNA treated samples, and a 20 ms exposure time for syntaxin 10-treated samples, as described in Materials and Methods. Individual data points, including the mean and standard error of the mean, were graphed using GraphPad Prism 6 software. Statistical analysis included an ordinary One-Way ANOVA with a Tukey's multiple comparison test. Representative images used in the quantitation are provided in Supplemental Figure 4. (C) A representative Western blot to demonstrate efficient knockdown of Stx10 compared to the loading control GAPDH.
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Figure 5: Effect of syntaxin 10 siRNA knockdown on NBD-sphingomyelin trafficking to the chlamydial inclusion. (A) Syntaxin 10 (Stx10) or non-targeting (NT) siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 24 h, labeled with NBD-ceramide, and treated with back-exchange medium for an additional 3 h. Samples were imaged at 40X magnification with a 200 ms exposure time using an Axiovert 200 M Imager with the AxioCam HRm camera (Carl Zeiss Microscopy, LLC). These images are representative of 3 independent experiments. (B) Untreated HeLa cells were infected with C. trachomatis serovar L2 for 18 or 26 h, labeled with NBD-ceramide and back-exchanged for 1.5 h. In addition, Stx10 or NT siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 30 h, labeled and back-exchanged as 18- and 26-h samples. Samples from two independent experiments were imaged using 60X magnification Olympus BX 60 fluorescent scope and images taken with a Nikon DS-Qi1Mc camera, with a 40 ms exposure for 18- 26- and NT siRNA treated samples, and a 20 ms exposure time for syntaxin 10-treated samples, as described in Materials and Methods. Individual data points, including the mean and standard error of the mean, were graphed using GraphPad Prism 6 software. Statistical analysis included an ordinary One-Way ANOVA with a Tukey's multiple comparison test. Representative images used in the quantitation are provided in Supplemental Figure 4. (C) A representative Western blot to demonstrate efficient knockdown of Stx10 compared to the loading control GAPDH.

Mentions: We reasoned that defects to chlamydial maturation in the absence of syntaxin 10 might negatively correlate with chlamydial acquisition of host-derived nutrients. To examine this, we monitored sphingomyelin trafficking, a well-established marker of lipid trafficking in chlamydial infected cells, to inclusions by treating infected siRNA-treated monolayers with C6-NBD-ceramide and examined inclusions by live cell imaging. We observed that inclusions growing in syntaxin 10 siRNA-treated cells (Western blot analysis of knockdown efficiency shown in Figure 5C) retained substantially higher amounts of fluorescent lipid than in inclusions grown in NT siRNA-treated cells, when imaged at equivalent exposures (Figure 5A). To test if syntaxin 10 knockdown in HeLa cells caused overall retention of lipid within the Golgi and hence, by default, greater lipid retention within chlamydial inclusions, we examined uninfected cells by live cell imaging. After 4 h of back-exchange, there were no differences in remaining cell-associated fluorescence between non-targeting (control) and syntaxin 10 siRNA treated cells (data not shown). Additionally, TLC analysis of mock-infected HeLa cells treated with either syntaxin 10 or non-targeting siRNA demonstrated no difference in lipid retention (cell extracts)/trafficking (back-exchange medium); (data not shown); therefore, these results indicate that the increase in NBD-lipid in the chlamydial inclusion was not due to a general trafficking defect caused by syntaxin 10 knockdown. Further, we confirmed by TLC that the lipid species incorporated into purified chlamydial organisms was only NBD-sphingomyelin, indicating that the increase in fluorescence was not due to an alternative NBD-lipid product (Supplemental Figure 5).


The trans-Golgi SNARE syntaxin 10 is required for optimal development of Chlamydia trachomatis.

Lucas AL, Ouellette SP, Kabeiseman EJ, Cichos KH, Rucks EA - Front Cell Infect Microbiol (2015)

Effect of syntaxin 10 siRNA knockdown on NBD-sphingomyelin trafficking to the chlamydial inclusion. (A) Syntaxin 10 (Stx10) or non-targeting (NT) siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 24 h, labeled with NBD-ceramide, and treated with back-exchange medium for an additional 3 h. Samples were imaged at 40X magnification with a 200 ms exposure time using an Axiovert 200 M Imager with the AxioCam HRm camera (Carl Zeiss Microscopy, LLC). These images are representative of 3 independent experiments. (B) Untreated HeLa cells were infected with C. trachomatis serovar L2 for 18 or 26 h, labeled with NBD-ceramide and back-exchanged for 1.5 h. In addition, Stx10 or NT siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 30 h, labeled and back-exchanged as 18- and 26-h samples. Samples from two independent experiments were imaged using 60X magnification Olympus BX 60 fluorescent scope and images taken with a Nikon DS-Qi1Mc camera, with a 40 ms exposure for 18- 26- and NT siRNA treated samples, and a 20 ms exposure time for syntaxin 10-treated samples, as described in Materials and Methods. Individual data points, including the mean and standard error of the mean, were graphed using GraphPad Prism 6 software. Statistical analysis included an ordinary One-Way ANOVA with a Tukey's multiple comparison test. Representative images used in the quantitation are provided in Supplemental Figure 4. (C) A representative Western blot to demonstrate efficient knockdown of Stx10 compared to the loading control GAPDH.
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Related In: Results  -  Collection

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Show All Figures
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Figure 5: Effect of syntaxin 10 siRNA knockdown on NBD-sphingomyelin trafficking to the chlamydial inclusion. (A) Syntaxin 10 (Stx10) or non-targeting (NT) siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 24 h, labeled with NBD-ceramide, and treated with back-exchange medium for an additional 3 h. Samples were imaged at 40X magnification with a 200 ms exposure time using an Axiovert 200 M Imager with the AxioCam HRm camera (Carl Zeiss Microscopy, LLC). These images are representative of 3 independent experiments. (B) Untreated HeLa cells were infected with C. trachomatis serovar L2 for 18 or 26 h, labeled with NBD-ceramide and back-exchanged for 1.5 h. In addition, Stx10 or NT siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 30 h, labeled and back-exchanged as 18- and 26-h samples. Samples from two independent experiments were imaged using 60X magnification Olympus BX 60 fluorescent scope and images taken with a Nikon DS-Qi1Mc camera, with a 40 ms exposure for 18- 26- and NT siRNA treated samples, and a 20 ms exposure time for syntaxin 10-treated samples, as described in Materials and Methods. Individual data points, including the mean and standard error of the mean, were graphed using GraphPad Prism 6 software. Statistical analysis included an ordinary One-Way ANOVA with a Tukey's multiple comparison test. Representative images used in the quantitation are provided in Supplemental Figure 4. (C) A representative Western blot to demonstrate efficient knockdown of Stx10 compared to the loading control GAPDH.
Mentions: We reasoned that defects to chlamydial maturation in the absence of syntaxin 10 might negatively correlate with chlamydial acquisition of host-derived nutrients. To examine this, we monitored sphingomyelin trafficking, a well-established marker of lipid trafficking in chlamydial infected cells, to inclusions by treating infected siRNA-treated monolayers with C6-NBD-ceramide and examined inclusions by live cell imaging. We observed that inclusions growing in syntaxin 10 siRNA-treated cells (Western blot analysis of knockdown efficiency shown in Figure 5C) retained substantially higher amounts of fluorescent lipid than in inclusions grown in NT siRNA-treated cells, when imaged at equivalent exposures (Figure 5A). To test if syntaxin 10 knockdown in HeLa cells caused overall retention of lipid within the Golgi and hence, by default, greater lipid retention within chlamydial inclusions, we examined uninfected cells by live cell imaging. After 4 h of back-exchange, there were no differences in remaining cell-associated fluorescence between non-targeting (control) and syntaxin 10 siRNA treated cells (data not shown). Additionally, TLC analysis of mock-infected HeLa cells treated with either syntaxin 10 or non-targeting siRNA demonstrated no difference in lipid retention (cell extracts)/trafficking (back-exchange medium); (data not shown); therefore, these results indicate that the increase in NBD-lipid in the chlamydial inclusion was not due to a general trafficking defect caused by syntaxin 10 knockdown. Further, we confirmed by TLC that the lipid species incorporated into purified chlamydial organisms was only NBD-sphingomyelin, indicating that the increase in fluorescence was not due to an alternative NBD-lipid product (Supplemental Figure 5).

Bottom Line: These defects in chlamydial development correlate with an overabundance of NBD-lipid retained by inclusions cultured in syntaxin 10 knockdown cells.Overall, loss of syntaxin 10 at the inclusion membrane negatively affects Chlamydia.Understanding host machinery involved in maintaining an optimal inclusion environment to support chlamydial growth and development is critical toward understanding the molecular signals involved in successful progression through the chlamydial developmental cycle.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota Vermillion, SD, USA.

ABSTRACT
Chlamydia trachomatis, an obligate intracellular pathogen, grows inside of a vacuole, termed the inclusion. Within the inclusion, the organisms differentiate from the infectious elementary body (EB) into the reticulate body (RB). The RB communicates with the host cell through the inclusion membrane to obtain the nutrients necessary to divide, thus expanding the chlamydial population. At late time points within the developmental cycle, the RBs respond to unknown molecular signals to redifferentiate into infectious EBs to perpetuate the infection cycle. One strategy for Chlamydia to obtain necessary nutrients and metabolites from the host is to intercept host vesicular trafficking pathways. In this study we demonstrate that a trans-Golgi soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE), syntaxin 10, and/or syntaxin 10-associated Golgi elements colocalize with the chlamydial inclusion. We hypothesized that Chlamydia utilizes the molecular machinery of syntaxin 10 at the inclusion membrane to intercept specific vesicular trafficking pathways in order to create and maintain an optimal intra-inclusion environment. To test this hypothesis, we used siRNA knockdown of syntaxin 10 to examine the impact of the loss of syntaxin 10 on chlamydial growth and development. Our results demonstrate that loss of syntaxin 10 leads to defects in normal chlamydial maturation including: variable inclusion size with fewer chlamydial organisms per inclusion, fewer infectious progeny, and delayed or halted RB-EB differentiation. These defects in chlamydial development correlate with an overabundance of NBD-lipid retained by inclusions cultured in syntaxin 10 knockdown cells. Overall, loss of syntaxin 10 at the inclusion membrane negatively affects Chlamydia. Understanding host machinery involved in maintaining an optimal inclusion environment to support chlamydial growth and development is critical toward understanding the molecular signals involved in successful progression through the chlamydial developmental cycle.

No MeSH data available.


Related in: MedlinePlus