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Chlamydia trachomatis homotypic inclusion fusion is promoted by host microtubule trafficking.

Richards TS, Knowlton AE, Grieshaber SS - BMC Microbiol. (2013)

Bottom Line: The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins.The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion.Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL 32610, USA.

ABSTRACT

Background: The developmental cycle of the obligate intracellular pathogen Chlamydia is dependant on the formation of a unique intracellular niche termed the chlamydial inclusion. The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins. A unique property of the inclusion is its propensity for homotypic fusion. The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion. The chlamydial protein IncA is required for fusion, however the host process involved are uncharacterized.

Results: Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion. We established that factors involved in trafficking were required for efficient fusion as both disruption of the microtubule network and inhibition of microtubule trafficking reduced the efficiency of fusion. Additionally, fusion occurred at multiple sites in the cell and was delayed when the microtubule minus ends were either no longer anchored at a single MTOC or when a cell possessed multiple MTOCs.

Conclusions: The data presented demonstrates that efficient homotypic fusion requires the inclusions to be in close proximity and that this proximity is dependent on chlamydial microtubule trafficking to the minus ends of microtubules.

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Related in: MedlinePlus

Chlamydial inclusion trafficking and fusion is dynein dependent. Cos7 cells were infected with C. trachomatis serovar L2 following micro-injection with anti-dynein antibodies. Uninjected cells were infected in parallel. Twenty-four hours postinfection, cells were fixed and stained with human sera (red) and the appropriate secondary for the anti-dynein antibody (green). Representative picture of anti-dynein injected cells at 6 and 24 hpi (A and B, respectively). Inclusions per infected cell were enumerated for injected and uninjected cells at 24 hpi, P < 0.0001 (C).
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Figure 3: Chlamydial inclusion trafficking and fusion is dynein dependent. Cos7 cells were infected with C. trachomatis serovar L2 following micro-injection with anti-dynein antibodies. Uninjected cells were infected in parallel. Twenty-four hours postinfection, cells were fixed and stained with human sera (red) and the appropriate secondary for the anti-dynein antibody (green). Representative picture of anti-dynein injected cells at 6 and 24 hpi (A and B, respectively). Inclusions per infected cell were enumerated for injected and uninjected cells at 24 hpi, P < 0.0001 (C).

Mentions: Chlamydial microtubule trafficking is dependent on the host microtubule motor protein dynein. To investigate the role of dynein in inclusion fusion, we injected Cos7 cells with anti-dynein intermediate chain antibodies (DIC74.1). Following injection, cells were infected with C. trachomatis. Uninjected cells were infected in parallel. Cells were fixed at 6 and 24 hpi. In cells that had been injected with anti-dynein antibodies, inclusion clustering was decreased early in infection and inclusion fusion decreased (Figure 3A and B, respectively). At 24 hpi, there was a significant difference between injected and uninjected cells (P < 0.001); injected cells averaged three inclusions per infected cell while uninjected cells averaged one inclusion per infected cell (Figure 3C).


Chlamydia trachomatis homotypic inclusion fusion is promoted by host microtubule trafficking.

Richards TS, Knowlton AE, Grieshaber SS - BMC Microbiol. (2013)

Chlamydial inclusion trafficking and fusion is dynein dependent. Cos7 cells were infected with C. trachomatis serovar L2 following micro-injection with anti-dynein antibodies. Uninjected cells were infected in parallel. Twenty-four hours postinfection, cells were fixed and stained with human sera (red) and the appropriate secondary for the anti-dynein antibody (green). Representative picture of anti-dynein injected cells at 6 and 24 hpi (A and B, respectively). Inclusions per infected cell were enumerated for injected and uninjected cells at 24 hpi, P < 0.0001 (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750546&req=5

Figure 3: Chlamydial inclusion trafficking and fusion is dynein dependent. Cos7 cells were infected with C. trachomatis serovar L2 following micro-injection with anti-dynein antibodies. Uninjected cells were infected in parallel. Twenty-four hours postinfection, cells were fixed and stained with human sera (red) and the appropriate secondary for the anti-dynein antibody (green). Representative picture of anti-dynein injected cells at 6 and 24 hpi (A and B, respectively). Inclusions per infected cell were enumerated for injected and uninjected cells at 24 hpi, P < 0.0001 (C).
Mentions: Chlamydial microtubule trafficking is dependent on the host microtubule motor protein dynein. To investigate the role of dynein in inclusion fusion, we injected Cos7 cells with anti-dynein intermediate chain antibodies (DIC74.1). Following injection, cells were infected with C. trachomatis. Uninjected cells were infected in parallel. Cells were fixed at 6 and 24 hpi. In cells that had been injected with anti-dynein antibodies, inclusion clustering was decreased early in infection and inclusion fusion decreased (Figure 3A and B, respectively). At 24 hpi, there was a significant difference between injected and uninjected cells (P < 0.001); injected cells averaged three inclusions per infected cell while uninjected cells averaged one inclusion per infected cell (Figure 3C).

Bottom Line: The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins.The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion.Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL 32610, USA.

ABSTRACT

Background: The developmental cycle of the obligate intracellular pathogen Chlamydia is dependant on the formation of a unique intracellular niche termed the chlamydial inclusion. The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins. A unique property of the inclusion is its propensity for homotypic fusion. The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion. The chlamydial protein IncA is required for fusion, however the host process involved are uncharacterized.

Results: Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion. We established that factors involved in trafficking were required for efficient fusion as both disruption of the microtubule network and inhibition of microtubule trafficking reduced the efficiency of fusion. Additionally, fusion occurred at multiple sites in the cell and was delayed when the microtubule minus ends were either no longer anchored at a single MTOC or when a cell possessed multiple MTOCs.

Conclusions: The data presented demonstrates that efficient homotypic fusion requires the inclusions to be in close proximity and that this proximity is dependent on chlamydial microtubule trafficking to the minus ends of microtubules.

Show MeSH
Related in: MedlinePlus