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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 siRNA knockdown of syntaxin 10 on inclusion development and chlamydial infectious progeny. (A) Syntaxin 10 (Stx10) or NT (non-targeting, control) siRNA-treated HeLa cells were infected at an MOI of 0.5 with C. trachomatis serovar L2 for 24, 44, or 67 h. Monolayers were lysed in dH2O, then serial dilutions were replated onto a fresh monolayer of cells. These cells were fixed and processed to enumerate inclusions. Inclusions were counted using a 20X objective and values are expressed as mean and standard error of the mean and then analyzed with an ordinary One-Way ANOVA with a Tukey's multiple comparison test in GraphPad Prism 6 software. Data are representative of 3 independent experiments. (B) Corresponding Western blot of cell lysates harvested at the time points described above to test for efficient knockdown of syntaxin 10 (stx10) compared to loading control GAPDH.
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Figure 2: Effect of siRNA knockdown of syntaxin 10 on inclusion development and chlamydial infectious progeny. (A) Syntaxin 10 (Stx10) or NT (non-targeting, control) siRNA-treated HeLa cells were infected at an MOI of 0.5 with C. trachomatis serovar L2 for 24, 44, or 67 h. Monolayers were lysed in dH2O, then serial dilutions were replated onto a fresh monolayer of cells. These cells were fixed and processed to enumerate inclusions. Inclusions were counted using a 20X objective and values are expressed as mean and standard error of the mean and then analyzed with an ordinary One-Way ANOVA with a Tukey's multiple comparison test in GraphPad Prism 6 software. Data are representative of 3 independent experiments. (B) Corresponding Western blot of cell lysates harvested at the time points described above to test for efficient knockdown of syntaxin 10 (stx10) compared to loading control GAPDH.

Mentions: The pattern of localization of syntaxin 10 to the chlamydial inclusion membrane suggested that this protein has a function for Chlamydia. To understand the relationship of syntaxin 10 with the Golgi, we initially used siRNA to knockdown syntaxin 10 and examined Golgi morphology (using Golgi protein, Giantin) around the chlamydial inclusion (Supplemental Figure 2). In control cells (cells transfected with non-targeting siRNA), the Golgi is condensed and encircles the chlamydial inclusion, as previously characterized (Heuer et al., 2009). In syntaxin 10 knockdown cells, the Golgi retains a discernable vesicular structure, but the tight association with the inclusion is partially lost. This suggests a potential role for syntaxin 10 and/or associated interacting protiens (i.e., Incs) in anchoring the Golgi to the inclusion. In the context of our working hypothesis, these data support a role for syntaxin 10 in contributing to an optimal environment for chlamydial development. We cannot readily distinguish between vesicular trafficking defects or “relaxed” Golgi effects on chlamydial development. However, previous studies have demonstrated that loss of Golgi morphology does not negatively impact chlamydial development (Hackstadt et al., 1996). As a first step in understanding the function of syntaxin 10 in chlamydial growth and development, we began by assessing the ability of organisms to produce infectious progeny 24, 44, and 67 h post-infection (Figure 2A). In a typical serovar L2 developmental cycle (organisms grown in HeLa or HEp2 cells), rapid division of RBs occurs between 8 and 16 h post-infection, with RB to EB differentiation occurring 24 to 36 h post-infection. Maximal RB to EB transition occurs between 42 and 48 h post-infection, with subsequent monolayer destruction due to maximal EB release occurring at or after 50+ h of infection (Ward, 1988; Dessus-Babus et al., 2008). Consistent with the progression of the normal chlamydial developmental cycle, there are low numbers of infectious progeny produced in both siRNA treatment groups (NT: 3.54 × 104 ± 3.97 × 103; Stx10: 2.41 × 104 ± 2.24 × 103) after 24 h of infection (mid-developmental cycle), indicating that chlamydial development is not altered due to depletion of syntaxin 10 at this time point post-infection. However, after 44 h of infection (late developmental cycle), there is a statistically and biologically significant ~10-fold decrease in infectious progeny obtained from Chlamydia grown in syntaxin 10 siRNA-treated cells (4.93 × 106 ± 4.85 × 105) vs. control cells (3.75 × 107 ± 1.62 × 106). Also apparent in these data is the 1000-fold increase of progeny produced in control cells between the 24 and 44 h time points vs. the 200-fold increase in progeny produced in syntaxin 10 knockdown cells within the same time frame. Therefore, organisms cultivated in the absence of syntaxin 10 demonstrated a 5-fold lower production rate of infectious progeny. We also assessed if a longer incubation of Chlamydia in syntaxin 10 siRNA-treated cells would yield more progeny. At 67 h post-infection, 9.78 × 106 ± 1.12 × 106 IFU/ml were recovered from syntaxin 10 siRNA-treated cells (Figure 2A). Infectious progeny are not included for NT siRNA-treated monolayers at 67 h post-infection because of the monolayer being destroyed by the end of the typical chlamydial developmental cycle; only ~21% of the monolayer remained intact (quantified by trypan blue exclusion assay, but also apparent in GAPDH levels, Figure 2B). The syntaxin 10 knockdown was 90.35% of control at 67 h post-infection (~113 h post-transfection), indicating that the slight increase (1.98-fold) in infectious progeny was not due to waning knockdown (Figure 2B). Notably, with an additional 23 h of culture, the organisms grown in syntaxin 10 siRNA-treated cells never displayed the same rate of growth or output levels as organisms grown in control cells. Further, syntaxin 10 knockdown monolayers, were largely intact at 67 h post-infection, in contrast to the control cells. These data support the notion that loss of syntaxin 10 results in a defect or delay in the chlamydial developmental cycle.


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 siRNA knockdown of syntaxin 10 on inclusion development and chlamydial infectious progeny. (A) Syntaxin 10 (Stx10) or NT (non-targeting, control) siRNA-treated HeLa cells were infected at an MOI of 0.5 with C. trachomatis serovar L2 for 24, 44, or 67 h. Monolayers were lysed in dH2O, then serial dilutions were replated onto a fresh monolayer of cells. These cells were fixed and processed to enumerate inclusions. Inclusions were counted using a 20X objective and values are expressed as mean and standard error of the mean and then analyzed with an ordinary One-Way ANOVA with a Tukey's multiple comparison test in GraphPad Prism 6 software. Data are representative of 3 independent experiments. (B) Corresponding Western blot of cell lysates harvested at the time points described above to test for efficient knockdown of syntaxin 10 (stx10) compared to loading control GAPDH.
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Figure 2: Effect of siRNA knockdown of syntaxin 10 on inclusion development and chlamydial infectious progeny. (A) Syntaxin 10 (Stx10) or NT (non-targeting, control) siRNA-treated HeLa cells were infected at an MOI of 0.5 with C. trachomatis serovar L2 for 24, 44, or 67 h. Monolayers were lysed in dH2O, then serial dilutions were replated onto a fresh monolayer of cells. These cells were fixed and processed to enumerate inclusions. Inclusions were counted using a 20X objective and values are expressed as mean and standard error of the mean and then analyzed with an ordinary One-Way ANOVA with a Tukey's multiple comparison test in GraphPad Prism 6 software. Data are representative of 3 independent experiments. (B) Corresponding Western blot of cell lysates harvested at the time points described above to test for efficient knockdown of syntaxin 10 (stx10) compared to loading control GAPDH.
Mentions: The pattern of localization of syntaxin 10 to the chlamydial inclusion membrane suggested that this protein has a function for Chlamydia. To understand the relationship of syntaxin 10 with the Golgi, we initially used siRNA to knockdown syntaxin 10 and examined Golgi morphology (using Golgi protein, Giantin) around the chlamydial inclusion (Supplemental Figure 2). In control cells (cells transfected with non-targeting siRNA), the Golgi is condensed and encircles the chlamydial inclusion, as previously characterized (Heuer et al., 2009). In syntaxin 10 knockdown cells, the Golgi retains a discernable vesicular structure, but the tight association with the inclusion is partially lost. This suggests a potential role for syntaxin 10 and/or associated interacting protiens (i.e., Incs) in anchoring the Golgi to the inclusion. In the context of our working hypothesis, these data support a role for syntaxin 10 in contributing to an optimal environment for chlamydial development. We cannot readily distinguish between vesicular trafficking defects or “relaxed” Golgi effects on chlamydial development. However, previous studies have demonstrated that loss of Golgi morphology does not negatively impact chlamydial development (Hackstadt et al., 1996). As a first step in understanding the function of syntaxin 10 in chlamydial growth and development, we began by assessing the ability of organisms to produce infectious progeny 24, 44, and 67 h post-infection (Figure 2A). In a typical serovar L2 developmental cycle (organisms grown in HeLa or HEp2 cells), rapid division of RBs occurs between 8 and 16 h post-infection, with RB to EB differentiation occurring 24 to 36 h post-infection. Maximal RB to EB transition occurs between 42 and 48 h post-infection, with subsequent monolayer destruction due to maximal EB release occurring at or after 50+ h of infection (Ward, 1988; Dessus-Babus et al., 2008). Consistent with the progression of the normal chlamydial developmental cycle, there are low numbers of infectious progeny produced in both siRNA treatment groups (NT: 3.54 × 104 ± 3.97 × 103; Stx10: 2.41 × 104 ± 2.24 × 103) after 24 h of infection (mid-developmental cycle), indicating that chlamydial development is not altered due to depletion of syntaxin 10 at this time point post-infection. However, after 44 h of infection (late developmental cycle), there is a statistically and biologically significant ~10-fold decrease in infectious progeny obtained from Chlamydia grown in syntaxin 10 siRNA-treated cells (4.93 × 106 ± 4.85 × 105) vs. control cells (3.75 × 107 ± 1.62 × 106). Also apparent in these data is the 1000-fold increase of progeny produced in control cells between the 24 and 44 h time points vs. the 200-fold increase in progeny produced in syntaxin 10 knockdown cells within the same time frame. Therefore, organisms cultivated in the absence of syntaxin 10 demonstrated a 5-fold lower production rate of infectious progeny. We also assessed if a longer incubation of Chlamydia in syntaxin 10 siRNA-treated cells would yield more progeny. At 67 h post-infection, 9.78 × 106 ± 1.12 × 106 IFU/ml were recovered from syntaxin 10 siRNA-treated cells (Figure 2A). Infectious progeny are not included for NT siRNA-treated monolayers at 67 h post-infection because of the monolayer being destroyed by the end of the typical chlamydial developmental cycle; only ~21% of the monolayer remained intact (quantified by trypan blue exclusion assay, but also apparent in GAPDH levels, Figure 2B). The syntaxin 10 knockdown was 90.35% of control at 67 h post-infection (~113 h post-transfection), indicating that the slight increase (1.98-fold) in infectious progeny was not due to waning knockdown (Figure 2B). Notably, with an additional 23 h of culture, the organisms grown in syntaxin 10 siRNA-treated cells never displayed the same rate of growth or output levels as organisms grown in control cells. Further, syntaxin 10 knockdown monolayers, were largely intact at 67 h post-infection, in contrast to the control cells. These data support the notion that loss of syntaxin 10 results in a defect or delay in the chlamydial developmental cycle.

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