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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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Transfection with EB1.84-GFP disrupts inclusion fusion. HeLa cells were transfected with EB1.84-GFP or mock transfected. They were then infected with C. trachomatis. Twenty-four hours postinfection, cells were fixed and stained with human sera and inclusions per infected cell were enumerated. The distribution in the number of inclusions per infected cell is shown for the EB1.84-GFP transfected and mock transfected cells in A and B, respectively. Mock transfected cells were also stained with anti-g-tubulin antibodies (green). Representative transfected and mock transfected cells shown in C and D, respectively.
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Figure 6: Transfection with EB1.84-GFP disrupts inclusion fusion. HeLa cells were transfected with EB1.84-GFP or mock transfected. They were then infected with C. trachomatis. Twenty-four hours postinfection, cells were fixed and stained with human sera and inclusions per infected cell were enumerated. The distribution in the number of inclusions per infected cell is shown for the EB1.84-GFP transfected and mock transfected cells in A and B, respectively. Mock transfected cells were also stained with anti-g-tubulin antibodies (green). Representative transfected and mock transfected cells shown in C and D, respectively.

Mentions: Chlamydial inclusion fusion occurs at host centrosomes and is delayed when extra centrosomes are present. Inclusion migration is unidirectional resulting in the chlamydial inclusion residing at the cell centrosome for its entire intracellular growth phase. In the cell, the centrosome acts as the organizing center that anchors the majority of microtubule minus ends. We hypothesize that inclusion fusion is promoted by inclusion crowding at the anchored minus ends of microtubules. To determine if fusion is dependent on microtubule minus end anchoring, we transfected HeLa cells with the GFP tagged EB1 mutant, EB1.84-GFP. Cells expressing EB1.84-GFP have defects in microtubule organization and centrosomal anchoring resulting in unanchored free microtubule minus ends [12]. When we compared inclusion fusion in the cells that had been mock transfected to cells transfected with EB1.84-GFP, the EB1.84 producing cells were markedly delayed in inclusion fusion. At 24 hpi, transfected cells averaged 1.7 inclusions per infected cell while mock transfected cells averaged one inclusion per infected cell (P < 0.001). We also quantitated the distribution of inclusion numbers in these cells, slightly under half of the cells transfected with EB1.84-GFP contained one inclusion (46%) while the majority of mock transfected cells (92%) had a single inclusion (Figure 6A and B, respectively). Additionally, many of the EB1.85 transfected cells had four or more inclusions per cell, while mock transfected cells never had more than two inclusion per cell (Figure 6A and B, respectively). Representative images of inclusions in transfected and mock transfected cells are shown in Figure 6C and D, respectively.


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

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

Transfection with EB1.84-GFP disrupts inclusion fusion. HeLa cells were transfected with EB1.84-GFP or mock transfected. They were then infected with C. trachomatis. Twenty-four hours postinfection, cells were fixed and stained with human sera and inclusions per infected cell were enumerated. The distribution in the number of inclusions per infected cell is shown for the EB1.84-GFP transfected and mock transfected cells in A and B, respectively. Mock transfected cells were also stained with anti-g-tubulin antibodies (green). Representative transfected and mock transfected cells shown in C and D, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Transfection with EB1.84-GFP disrupts inclusion fusion. HeLa cells were transfected with EB1.84-GFP or mock transfected. They were then infected with C. trachomatis. Twenty-four hours postinfection, cells were fixed and stained with human sera and inclusions per infected cell were enumerated. The distribution in the number of inclusions per infected cell is shown for the EB1.84-GFP transfected and mock transfected cells in A and B, respectively. Mock transfected cells were also stained with anti-g-tubulin antibodies (green). Representative transfected and mock transfected cells shown in C and D, respectively.
Mentions: Chlamydial inclusion fusion occurs at host centrosomes and is delayed when extra centrosomes are present. Inclusion migration is unidirectional resulting in the chlamydial inclusion residing at the cell centrosome for its entire intracellular growth phase. In the cell, the centrosome acts as the organizing center that anchors the majority of microtubule minus ends. We hypothesize that inclusion fusion is promoted by inclusion crowding at the anchored minus ends of microtubules. To determine if fusion is dependent on microtubule minus end anchoring, we transfected HeLa cells with the GFP tagged EB1 mutant, EB1.84-GFP. Cells expressing EB1.84-GFP have defects in microtubule organization and centrosomal anchoring resulting in unanchored free microtubule minus ends [12]. When we compared inclusion fusion in the cells that had been mock transfected to cells transfected with EB1.84-GFP, the EB1.84 producing cells were markedly delayed in inclusion fusion. At 24 hpi, transfected cells averaged 1.7 inclusions per infected cell while mock transfected cells averaged one inclusion per infected cell (P < 0.001). We also quantitated the distribution of inclusion numbers in these cells, slightly under half of the cells transfected with EB1.84-GFP contained one inclusion (46%) while the majority of mock transfected cells (92%) had a single inclusion (Figure 6A and B, respectively). Additionally, many of the EB1.85 transfected cells had four or more inclusions per cell, while mock transfected cells never had more than two inclusion per cell (Figure 6A and B, respectively). Representative images of inclusions in transfected and mock transfected cells are shown in Figure 6C and D, 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