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. 2013 Aug 7:13:185.
doi: 10.1186/1471-2180-13-185.

Chlamydia trachomatis homotypic inclusion fusion is promoted by host microtubule trafficking

Theresa S Richards  1 , Andrea E Knowlton, Scott S Grieshaber
Affiliations

Chlamydia trachomatis homotypic inclusion fusion is promoted by host microtubule trafficking

Theresa S Richards et al. BMC Microbiol. .

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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Figures

Figure 1
Figure 1
Inclusion fusion occurs at the centrosomes. HeLa cells were transfected with EB1-GFP to visualize centrosomes (arrow in A). Eighteen hours post-transfection, cells were infected with C. trachomatis at MOI = 20. During infection, cells were photographed every 10 minutes until 24 hpi. Times post infection are indicated in each corresponding image.
Figure 2
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).
Figure 3
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).
Figure 4
Figure 4
Inclusion fusion is delayed in cells with multiple unclustered centrosomes. HeLa cells (A) and neuroblastomas (B) were infected with C. trachomatis at MOI ~ 27 and fixed at 3 and 24 hpi. Cells were stained with anti-g-tubulin antibodies (green) and human sera (red). HeLa cells (C) and neuroblastomas (D) were infected with C. trachomatis at MOI ~ 3, 9 and 27 and fixed at 10, 12, 14, 16, 20, 22 and 24 hpi. Cells were stained with human sera and inclusions were enumerated.
Figure 5
Figure 5
Neuroblastomas are fusion competent and IncA localizes to the inclusion membrane during infection. HeLa cells (A) and neuroblastomas (B) were infected with C. trachomatis serovar G. At 40 hpi, cells were superinfected with C. trachomatis serovar L2 and fixed four hours after superinfection. Cells were stained with human sera (red) and anti-L2 MOMP antibodies (green). HeLa cells (C) and neuroblastomas (D) were infected with C. trachomatis serovar L2 at MOI ~ 9 and fixed 24 hpi. Cells were stained with human sera (blue) and anti-IncA antibodies (green).
Figure 6
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.
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