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

Syntaxin 10 localization to the chlamydial inclusion. (A) HeLa cells were transfected with 3XFLAG-syntaxin 10 (3XF-stx10) for 24 h prior to fixation and processing for imaging. The Golgi (outlined in white) was detected with a rabbit anti-giantin antibody; 3XF-Stx10 was detected using a mouse anti-FLAG M2 antibody. (B,C) HeLa or HEp2 cells transfected with 3XF-stx10 were infected for 16–18 h with C. trachomatis serovar L2, prior to fixation and processing for imaging. The inclusion membrane was detected using either a rabbit anti- IncA (B) (Additional images provided in Supplemental Figure 1) or IncG (C) antibody; 3XF-Stx10 was detected as above. To distinguish 3XF-Stx10 from surrounding cell structures, some samples were treated with brefeldin A (BFA) to collapse the surrounding Golgi. To examine if chlamydial protein synthesis was required for 3XF-Stx10 localization, infected monolayers were treated with 200 μg/ml chloramphenicol (Chlor) for an additional 24 h prior to fixation. In chloramphenicol treated cells, white asterisks indicate inclusions. All images were acquired using an Olympus Fluoview 1000 Laser Scanning Confocal Microscope with a 60X objective and 2x zoon. These results are representative of at least 3 independent experiments. White bars = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Syntaxin 10 localization to the chlamydial inclusion. (A) HeLa cells were transfected with 3XFLAG-syntaxin 10 (3XF-stx10) for 24 h prior to fixation and processing for imaging. The Golgi (outlined in white) was detected with a rabbit anti-giantin antibody; 3XF-Stx10 was detected using a mouse anti-FLAG M2 antibody. (B,C) HeLa or HEp2 cells transfected with 3XF-stx10 were infected for 16–18 h with C. trachomatis serovar L2, prior to fixation and processing for imaging. The inclusion membrane was detected using either a rabbit anti- IncA (B) (Additional images provided in Supplemental Figure 1) or IncG (C) antibody; 3XF-Stx10 was detected as above. To distinguish 3XF-Stx10 from surrounding cell structures, some samples were treated with brefeldin A (BFA) to collapse the surrounding Golgi. To examine if chlamydial protein synthesis was required for 3XF-Stx10 localization, infected monolayers were treated with 200 μg/ml chloramphenicol (Chlor) for an additional 24 h prior to fixation. In chloramphenicol treated cells, white asterisks indicate inclusions. All images were acquired using an Olympus Fluoview 1000 Laser Scanning Confocal Microscope with a 60X objective and 2x zoon. These results are representative of at least 3 independent experiments. White bars = 10 μm.

Mentions: Previous studies demonstrated that trans-Golgi SNARE proteins syntaxin 6 and VAMP4 localize to the chlamydial inclusion (Moore et al., 2011; Kabeiseman et al., 2013). We hypothesize that Chlamydia recruit specific SNARE proteins to help the chlamydial inclusion maintain an optimal growing environment for the pathogens. Missing from these previous analyses was an understanding of whether syntaxin 10, another trans-Golgi SNARE, localized to the chlamydial inclusion. We initially tried to visualize endogenous syntaxin 10 by indirect immunofluorescence, but commercially available antibodies were not suitable for this application. Therefore, for these studies, we transfected HeLa cells with a 3XFLAG-syntaxin 10 construct, which localized in vesicular-like structures throughout the cell and within the Golgi apparatus (Figure 1A). By confocal microscopy, exogenously expressed 3XFLAG-syntaxin 10 colocalized with two inclusion membrane markers: IncA and IncG (Figures 1B,C). What is apparent in these images is the vesicular nature of 3XFLAG-syntaxin 10 structures at the inclusion. 3XFLAG-syntaxin 10 does not form a distinct rim, as other eukaryotic proteins that localize to the chlamydial inclusion. Rather, it resembles a collection of vesicles, presumably Golgi-elements, since syntaxin 10 is strongly associated with the trans-Golgi network. Due to the localization pattern of syntaxin 10, the timing of the localization of 3XFLAG-syntaxin 10 at early time points post-infection is difficult to determine, but it likely occurs at some point between 8 and 14 h post-infection and remains associated with the inclusion beyond 36 h post-infection (Supplemental Figure 1). To distinguish 3XFLAG-syntaxin 10 that localized to the inclusion from surrounding cellular structures, cells were treated with 1 μg/ml of brefeldin A (BFA), which collapses the Golgi into the ER (Lippincott-Schwartz et al., 1989), for 2 h prior to fixation. BFA treatment did not eliminate the localization of 3XFLAG-syntaxin 10 with the inclusion, indicating that the localization of 3XFLAG-syntaxin 10 is not happenstance due to the inclusion's proximity with the Golgi (Figure 1C). As indicated in Figure 1C, association of these syntaxin 10 positive structures with the inclusion likely stabilizes the structures from the effects of BFA. Notably, inhibition of chlamydial protein synthesis at 18 h post-infection by chloramphenicol did not abolish the localization of 3XFLAG-syntaxin 10 to the chlamydial inclusion (Figure 1C, last row). These data indicate that once syntaxin 10 or syntaxin 10-positive structures are trafficked to the inclusion that the interaction is likely with a stable (i.e., low turnover) chlamydial protein. We were unable to determine if treatment of infected monolayers with chloramphenicol during early time points of infection inhibits localization of syntaxin 10 or syntaxin 10 positive structures with the inclusion (Supplemental Figure 1A).


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)

Syntaxin 10 localization to the chlamydial inclusion. (A) HeLa cells were transfected with 3XFLAG-syntaxin 10 (3XF-stx10) for 24 h prior to fixation and processing for imaging. The Golgi (outlined in white) was detected with a rabbit anti-giantin antibody; 3XF-Stx10 was detected using a mouse anti-FLAG M2 antibody. (B,C) HeLa or HEp2 cells transfected with 3XF-stx10 were infected for 16–18 h with C. trachomatis serovar L2, prior to fixation and processing for imaging. The inclusion membrane was detected using either a rabbit anti- IncA (B) (Additional images provided in Supplemental Figure 1) or IncG (C) antibody; 3XF-Stx10 was detected as above. To distinguish 3XF-Stx10 from surrounding cell structures, some samples were treated with brefeldin A (BFA) to collapse the surrounding Golgi. To examine if chlamydial protein synthesis was required for 3XF-Stx10 localization, infected monolayers were treated with 200 μg/ml chloramphenicol (Chlor) for an additional 24 h prior to fixation. In chloramphenicol treated cells, white asterisks indicate inclusions. All images were acquired using an Olympus Fluoview 1000 Laser Scanning Confocal Microscope with a 60X objective and 2x zoon. These results are representative of at least 3 independent experiments. White bars = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Syntaxin 10 localization to the chlamydial inclusion. (A) HeLa cells were transfected with 3XFLAG-syntaxin 10 (3XF-stx10) for 24 h prior to fixation and processing for imaging. The Golgi (outlined in white) was detected with a rabbit anti-giantin antibody; 3XF-Stx10 was detected using a mouse anti-FLAG M2 antibody. (B,C) HeLa or HEp2 cells transfected with 3XF-stx10 were infected for 16–18 h with C. trachomatis serovar L2, prior to fixation and processing for imaging. The inclusion membrane was detected using either a rabbit anti- IncA (B) (Additional images provided in Supplemental Figure 1) or IncG (C) antibody; 3XF-Stx10 was detected as above. To distinguish 3XF-Stx10 from surrounding cell structures, some samples were treated with brefeldin A (BFA) to collapse the surrounding Golgi. To examine if chlamydial protein synthesis was required for 3XF-Stx10 localization, infected monolayers were treated with 200 μg/ml chloramphenicol (Chlor) for an additional 24 h prior to fixation. In chloramphenicol treated cells, white asterisks indicate inclusions. All images were acquired using an Olympus Fluoview 1000 Laser Scanning Confocal Microscope with a 60X objective and 2x zoon. These results are representative of at least 3 independent experiments. White bars = 10 μm.
Mentions: Previous studies demonstrated that trans-Golgi SNARE proteins syntaxin 6 and VAMP4 localize to the chlamydial inclusion (Moore et al., 2011; Kabeiseman et al., 2013). We hypothesize that Chlamydia recruit specific SNARE proteins to help the chlamydial inclusion maintain an optimal growing environment for the pathogens. Missing from these previous analyses was an understanding of whether syntaxin 10, another trans-Golgi SNARE, localized to the chlamydial inclusion. We initially tried to visualize endogenous syntaxin 10 by indirect immunofluorescence, but commercially available antibodies were not suitable for this application. Therefore, for these studies, we transfected HeLa cells with a 3XFLAG-syntaxin 10 construct, which localized in vesicular-like structures throughout the cell and within the Golgi apparatus (Figure 1A). By confocal microscopy, exogenously expressed 3XFLAG-syntaxin 10 colocalized with two inclusion membrane markers: IncA and IncG (Figures 1B,C). What is apparent in these images is the vesicular nature of 3XFLAG-syntaxin 10 structures at the inclusion. 3XFLAG-syntaxin 10 does not form a distinct rim, as other eukaryotic proteins that localize to the chlamydial inclusion. Rather, it resembles a collection of vesicles, presumably Golgi-elements, since syntaxin 10 is strongly associated with the trans-Golgi network. Due to the localization pattern of syntaxin 10, the timing of the localization of 3XFLAG-syntaxin 10 at early time points post-infection is difficult to determine, but it likely occurs at some point between 8 and 14 h post-infection and remains associated with the inclusion beyond 36 h post-infection (Supplemental Figure 1). To distinguish 3XFLAG-syntaxin 10 that localized to the inclusion from surrounding cellular structures, cells were treated with 1 μg/ml of brefeldin A (BFA), which collapses the Golgi into the ER (Lippincott-Schwartz et al., 1989), for 2 h prior to fixation. BFA treatment did not eliminate the localization of 3XFLAG-syntaxin 10 with the inclusion, indicating that the localization of 3XFLAG-syntaxin 10 is not happenstance due to the inclusion's proximity with the Golgi (Figure 1C). As indicated in Figure 1C, association of these syntaxin 10 positive structures with the inclusion likely stabilizes the structures from the effects of BFA. Notably, inhibition of chlamydial protein synthesis at 18 h post-infection by chloramphenicol did not abolish the localization of 3XFLAG-syntaxin 10 to the chlamydial inclusion (Figure 1C, last row). These data indicate that once syntaxin 10 or syntaxin 10-positive structures are trafficked to the inclusion that the interaction is likely with a stable (i.e., low turnover) chlamydial protein. We were unable to determine if treatment of infected monolayers with chloramphenicol during early time points of infection inhibits localization of syntaxin 10 or syntaxin 10 positive structures with the inclusion (Supplemental Figure 1A).

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