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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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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).
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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).

Mentions: In order to determine whether neuroblastomas were fusion competent, HeLa and neuroblastoma cells were serially infected with different C. trachomatis serovars. Cells were infected with C. trachomatis serovar G for 40 hours and then superinfected with C. trachomatis serovar L2 for four hours. In both HeLa cells and neuroblastomas, fusion occurred between inclusions containing G and L2 indicating that the inclusions in neuroblastoma cells are fusion competent (Figure 5A and 5B). The inclusion membrane protein IncA is required for inclusion fusion and delays in IncA membrane localization lead to delayed homotypic fusion [8,9,15]. Therefore, we assessed the location of IncA in the infected neuroblastoma cells. HeLa and neuroblastoma cells were infected with C. trachomatis serovar L2, fixed at 24 hpi and stained with antibodies to IncA. IncA was present on inclusion membranes in both HeLa and neuroblastoma cells (Figure 5C and 5D, respectively). Taken together, these data demonstrate that the delay in inclusion fusion observed in neuroblastoma cells is not due to differences in fusion competency or to differences in the presence of IncA. Additionally, when infected neuroblastomas were grown on fibronectin micropatterns to force centrosome clustering, inclusion fusion was restored (Additional file 2: Figure S1).


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

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

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).
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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).
Mentions: In order to determine whether neuroblastomas were fusion competent, HeLa and neuroblastoma cells were serially infected with different C. trachomatis serovars. Cells were infected with C. trachomatis serovar G for 40 hours and then superinfected with C. trachomatis serovar L2 for four hours. In both HeLa cells and neuroblastomas, fusion occurred between inclusions containing G and L2 indicating that the inclusions in neuroblastoma cells are fusion competent (Figure 5A and 5B). The inclusion membrane protein IncA is required for inclusion fusion and delays in IncA membrane localization lead to delayed homotypic fusion [8,9,15]. Therefore, we assessed the location of IncA in the infected neuroblastoma cells. HeLa and neuroblastoma cells were infected with C. trachomatis serovar L2, fixed at 24 hpi and stained with antibodies to IncA. IncA was present on inclusion membranes in both HeLa and neuroblastoma cells (Figure 5C and 5D, respectively). Taken together, these data demonstrate that the delay in inclusion fusion observed in neuroblastoma cells is not due to differences in fusion competency or to differences in the presence of IncA. Additionally, when infected neuroblastomas were grown on fibronectin micropatterns to force centrosome clustering, inclusion fusion was restored (Additional file 2: Figure S1).

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