Limits...
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 expression of late chlamydial proteins: Hc1 and OmcB. Syntaxin 10 or non-targeting (NT) siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 24 or 44 h, prior to lysis in Laemmli sample buffer. (A) Equal amounts of protein were loaded onto SDS-PAGE gels and transferred prior to Western blot analysis using antibodies against chlamydial proteins Hc1, OmcB or chlamydial heat shock protein 60 (cHSP60) or eukaryotic proteins syntaxin 10 and GAPDH. The Western images shown are representative of two independent experiments. (B) Densitometry analysis was performed on the two independent experiments referred to in (A). These values [expressed in arbitrary units (AU)] were obtained using the Image Studio imaging software associated with the LI-COR Odyssey Infrared Imaging System and then graphed as mean and standard error of the mean. Statistical analysis included an ordinary One-Way ANOVA with Tukey's multiple comparison test in GraphPad Prism 6 software.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585193&req=5

Figure 3: Effect of siRNA knockdown of syntaxin 10 on expression of late chlamydial proteins: Hc1 and OmcB. Syntaxin 10 or non-targeting (NT) siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 24 or 44 h, prior to lysis in Laemmli sample buffer. (A) Equal amounts of protein were loaded onto SDS-PAGE gels and transferred prior to Western blot analysis using antibodies against chlamydial proteins Hc1, OmcB or chlamydial heat shock protein 60 (cHSP60) or eukaryotic proteins syntaxin 10 and GAPDH. The Western images shown are representative of two independent experiments. (B) Densitometry analysis was performed on the two independent experiments referred to in (A). These values [expressed in arbitrary units (AU)] were obtained using the Image Studio imaging software associated with the LI-COR Odyssey Infrared Imaging System and then graphed as mean and standard error of the mean. Statistical analysis included an ordinary One-Way ANOVA with Tukey's multiple comparison test in GraphPad Prism 6 software.

Mentions: To further examine the mechanism behind the decrease in chlamydial infectious progeny recovered from syntaxin 10 siRNA-treated cells, we examined the protein levels of two late developmental cycle proteins, Hc1 (hctA product) and OmcB by Western blot analysis (Figure 3). Consistent with the organisms being in mid-developmental cycle, at 24 h post-infection, there are no quantifiable differences in Hc1 or OmcB protein levels between organisms cultivated in NT or syntaxin 10 siRNA-treated cells. However, at 44 h post-infection, we observed an increase in protein levels of Hc1 and OmcB in NT siRNA-treated cells only, although only the difference in Hc1 levels was statistically significant (Figure 3B). We also tested transcript levels of the early gene euo by quantitative PCR, as a means to determine if we were seeing aberrant chlamydial development where euo transcript is elevated (see e.g., Ouellette et al., 2006). At 24 h post-infection, euo levels are approaching basal levels of transcription (Ouellette et al., 2014), and we found transcript levels increased by only 1.5 fold in organisms grown in syntaxin 10 siRNA-treated cells as compared to organisms grown in NT siRNA-treated cells (data not shown). While these data are statically significant, we do not consider these differences to be biologically significant and they indicate that the chlamydiae are not in a persistent growth state (see also Figure 4A and Supplemental Figure 3). We noticed a similar difference when examining omcB transcripts at 44-h post-infection (data not shown). Collectively, these data support that the developmental cycle of chlamydial organisms grown in syntaxin 10 knockdown cells is delayed or otherwise negatively impacted.


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 expression of late chlamydial proteins: Hc1 and OmcB. Syntaxin 10 or non-targeting (NT) siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 24 or 44 h, prior to lysis in Laemmli sample buffer. (A) Equal amounts of protein were loaded onto SDS-PAGE gels and transferred prior to Western blot analysis using antibodies against chlamydial proteins Hc1, OmcB or chlamydial heat shock protein 60 (cHSP60) or eukaryotic proteins syntaxin 10 and GAPDH. The Western images shown are representative of two independent experiments. (B) Densitometry analysis was performed on the two independent experiments referred to in (A). These values [expressed in arbitrary units (AU)] were obtained using the Image Studio imaging software associated with the LI-COR Odyssey Infrared Imaging System and then graphed as mean and standard error of the mean. Statistical analysis included an ordinary One-Way ANOVA with Tukey's multiple comparison test in GraphPad Prism 6 software.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585193&req=5

Figure 3: Effect of siRNA knockdown of syntaxin 10 on expression of late chlamydial proteins: Hc1 and OmcB. Syntaxin 10 or non-targeting (NT) siRNA-treated HeLa cells were infected with C. trachomatis serovar L2 for 24 or 44 h, prior to lysis in Laemmli sample buffer. (A) Equal amounts of protein were loaded onto SDS-PAGE gels and transferred prior to Western blot analysis using antibodies against chlamydial proteins Hc1, OmcB or chlamydial heat shock protein 60 (cHSP60) or eukaryotic proteins syntaxin 10 and GAPDH. The Western images shown are representative of two independent experiments. (B) Densitometry analysis was performed on the two independent experiments referred to in (A). These values [expressed in arbitrary units (AU)] were obtained using the Image Studio imaging software associated with the LI-COR Odyssey Infrared Imaging System and then graphed as mean and standard error of the mean. Statistical analysis included an ordinary One-Way ANOVA with Tukey's multiple comparison test in GraphPad Prism 6 software.
Mentions: To further examine the mechanism behind the decrease in chlamydial infectious progeny recovered from syntaxin 10 siRNA-treated cells, we examined the protein levels of two late developmental cycle proteins, Hc1 (hctA product) and OmcB by Western blot analysis (Figure 3). Consistent with the organisms being in mid-developmental cycle, at 24 h post-infection, there are no quantifiable differences in Hc1 or OmcB protein levels between organisms cultivated in NT or syntaxin 10 siRNA-treated cells. However, at 44 h post-infection, we observed an increase in protein levels of Hc1 and OmcB in NT siRNA-treated cells only, although only the difference in Hc1 levels was statistically significant (Figure 3B). We also tested transcript levels of the early gene euo by quantitative PCR, as a means to determine if we were seeing aberrant chlamydial development where euo transcript is elevated (see e.g., Ouellette et al., 2006). At 24 h post-infection, euo levels are approaching basal levels of transcription (Ouellette et al., 2014), and we found transcript levels increased by only 1.5 fold in organisms grown in syntaxin 10 siRNA-treated cells as compared to organisms grown in NT siRNA-treated cells (data not shown). While these data are statically significant, we do not consider these differences to be biologically significant and they indicate that the chlamydiae are not in a persistent growth state (see also Figure 4A and Supplemental Figure 3). We noticed a similar difference when examining omcB transcripts at 44-h post-infection (data not shown). Collectively, these data support that the developmental cycle of chlamydial organisms grown in syntaxin 10 knockdown cells is delayed or otherwise negatively impacted.

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