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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.

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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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Inclusion fusion is delayed in HeLa cells treated with nocodazole. HeLa cells were infected with C. trachomatis at MOI ~ 9 in the presence and absence of nocodazole (Noc) and fixed at 10, 12, 14, 16, 20, 22 and 24 hpi. Cells were stained with human sera and anti-g-tubulin antibodies and inclusions were enumerated (A). Representative treated and untreated HeLa cells (B and C, respectively).
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Figure 2: Inclusion fusion is delayed in HeLa cells treated with nocodazole. HeLa cells were infected with C. trachomatis at MOI ~ 9 in the presence and absence of nocodazole (Noc) and fixed at 10, 12, 14, 16, 20, 22 and 24 hpi. Cells were stained with human sera and anti-g-tubulin antibodies and inclusions were enumerated (A). Representative treated and untreated HeLa cells (B and C, respectively).

Mentions: We demonstrated that fusion occurs at the centrosomes and we have previously reported that trafficking on microtubules is required for the localization of chlamydial inclusions at the centrosomes. We asked whether the microtubule network influenced inclusion fusion. HeLa cells were infected with C. trachomatis. Following infection, cells were incubated in the presence or absence of nocodazole and then fixed every two hours between 10 and 24 hpi. Inclusion fusion occurred at approximately 14 hpi for untreated cells (Figure 2A). In cells that had been treated with nocodazole, fusion was significantly delayed. Nocodazole-treated cells had an average of eight inclusions per cell at 24 hpi (Figure 2A). Fusion was not completely abolished by nocodazole treatment suggesting that the fusion machinery does not require microtubules but instead that the microtubules accelerate fusion. Representative pictures of nocodazole treated and untreated cells are shown in Figure 2B and C, respectively.


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

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

Inclusion fusion is delayed in HeLa cells treated with nocodazole. HeLa cells were infected with C. trachomatis at MOI ~ 9 in the presence and absence of nocodazole (Noc) and fixed at 10, 12, 14, 16, 20, 22 and 24 hpi. Cells were stained with human sera and anti-g-tubulin antibodies and inclusions were enumerated (A). Representative treated and untreated HeLa cells (B and C, respectively).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Inclusion fusion is delayed in HeLa cells treated with nocodazole. HeLa cells were infected with C. trachomatis at MOI ~ 9 in the presence and absence of nocodazole (Noc) and fixed at 10, 12, 14, 16, 20, 22 and 24 hpi. Cells were stained with human sera and anti-g-tubulin antibodies and inclusions were enumerated (A). Representative treated and untreated HeLa cells (B and C, respectively).
Mentions: We demonstrated that fusion occurs at the centrosomes and we have previously reported that trafficking on microtubules is required for the localization of chlamydial inclusions at the centrosomes. We asked whether the microtubule network influenced inclusion fusion. HeLa cells were infected with C. trachomatis. Following infection, cells were incubated in the presence or absence of nocodazole and then fixed every two hours between 10 and 24 hpi. Inclusion fusion occurred at approximately 14 hpi for untreated cells (Figure 2A). In cells that had been treated with nocodazole, fusion was significantly delayed. Nocodazole-treated cells had an average of eight inclusions per cell at 24 hpi (Figure 2A). Fusion was not completely abolished by nocodazole treatment suggesting that the fusion machinery does not require microtubules but instead that the microtubules accelerate fusion. Representative pictures of nocodazole treated and untreated cells are shown in Figure 2B and C, respectively.

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