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Differential Translocation of Host Cellular Materials into the Chlamydia trachomatis Inclusion Lumen during Chemical Fixation.

Kokes M, Valdivia RH - PLoS ONE (2015)

Bottom Line: However, we see little evidence of intraluminal localization of these organelles in live inclusions.These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy.Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Chlamydia trachomatis manipulates host cellular pathways to ensure its proliferation and survival. Translocation of host materials into the pathogenic vacuole (termed 'inclusion') may facilitate nutrient acquisition and various organelles have been observed within the inclusion, including lipid droplets, peroxisomes, multivesicular body components, and membranes of the endoplasmic reticulum (ER). However, few of these processes have been documented in living cells. Here, we survey the localization of a broad panel of subcellular elements and find ER, mitochondria, and inclusion membranes within the inclusion lumen of fixed cells. However, we see little evidence of intraluminal localization of these organelles in live inclusions. Using time-lapse video microscopy we document ER marker translocation into the inclusion lumen during chemical fixation. These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy. We speculate that the localization of a subset of organelles may be exaggerated during fixation. Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.

No MeSH data available.


Related in: MedlinePlus

ER structures are detected via immunofluorescence microscopy within inclusions after both formaldehyde and alcohol-based chemical fixation.HeLa cells were infected with C. trachomatis LGV L2. (A) Infected cells were transfected with ER-RFP, fixed at 30 hpi with different concentrations of either paraformaldehyde (PFA) or formaldehyde (Form), and assessed for the frequency of ER-RFP within inclusions. (B and C) Infected cells were transfected with ER-RFP as indicated. At 30 hpi, cells were fixed with PFA, methanol, or ethanol, and subsequently permeabilized with Triton X-100 (Tx100) as indicated. Treated cells were processed for immunofluorescence with an antibody to the ER protein PDI and assessed for the frequency of PDI-positive structures within inclusions. Scale bar represents 5 μm.
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pone.0139153.g004: ER structures are detected via immunofluorescence microscopy within inclusions after both formaldehyde and alcohol-based chemical fixation.HeLa cells were infected with C. trachomatis LGV L2. (A) Infected cells were transfected with ER-RFP, fixed at 30 hpi with different concentrations of either paraformaldehyde (PFA) or formaldehyde (Form), and assessed for the frequency of ER-RFP within inclusions. (B and C) Infected cells were transfected with ER-RFP as indicated. At 30 hpi, cells were fixed with PFA, methanol, or ethanol, and subsequently permeabilized with Triton X-100 (Tx100) as indicated. Treated cells were processed for immunofluorescence with an antibody to the ER protein PDI and assessed for the frequency of PDI-positive structures within inclusions. Scale bar represents 5 μm.

Mentions: To assess whether the organelle internalization process can occur during various fixation techniques used previously in the study of host cellular material within Chlamydia inclusions [12,14,16,28,29], we quantified the frequency of ER-RFP structures in inclusions after fixation of infected cells (Fig 4A). Fixation with different concentrations of formaldehyde prepared from either paraformaldehyde or liquid formalin (which contains small amounts of methanol) resulted in similarly high frequencies of inclusions with internal ER-RFP structures, indicating that formaldehyde concentrations and trace levels of methanol in formalin do not affect the frequency of internalization. Since gluteraldehyde fixation as used for electron microscopy causes high levels of autofluorescence across the visible light spectrum [38], we could not reliably distinguish any fluorescent marker or dye tested from background fluorescence under these conditions (data not shown). Furthermore, we could not assess the frequency of ER-RFP structures within inclusions after methanol fixation since mRFP fluorescence was quenched by this treatment.


Differential Translocation of Host Cellular Materials into the Chlamydia trachomatis Inclusion Lumen during Chemical Fixation.

Kokes M, Valdivia RH - PLoS ONE (2015)

ER structures are detected via immunofluorescence microscopy within inclusions after both formaldehyde and alcohol-based chemical fixation.HeLa cells were infected with C. trachomatis LGV L2. (A) Infected cells were transfected with ER-RFP, fixed at 30 hpi with different concentrations of either paraformaldehyde (PFA) or formaldehyde (Form), and assessed for the frequency of ER-RFP within inclusions. (B and C) Infected cells were transfected with ER-RFP as indicated. At 30 hpi, cells were fixed with PFA, methanol, or ethanol, and subsequently permeabilized with Triton X-100 (Tx100) as indicated. Treated cells were processed for immunofluorescence with an antibody to the ER protein PDI and assessed for the frequency of PDI-positive structures within inclusions. Scale bar represents 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591358&req=5

pone.0139153.g004: ER structures are detected via immunofluorescence microscopy within inclusions after both formaldehyde and alcohol-based chemical fixation.HeLa cells were infected with C. trachomatis LGV L2. (A) Infected cells were transfected with ER-RFP, fixed at 30 hpi with different concentrations of either paraformaldehyde (PFA) or formaldehyde (Form), and assessed for the frequency of ER-RFP within inclusions. (B and C) Infected cells were transfected with ER-RFP as indicated. At 30 hpi, cells were fixed with PFA, methanol, or ethanol, and subsequently permeabilized with Triton X-100 (Tx100) as indicated. Treated cells were processed for immunofluorescence with an antibody to the ER protein PDI and assessed for the frequency of PDI-positive structures within inclusions. Scale bar represents 5 μm.
Mentions: To assess whether the organelle internalization process can occur during various fixation techniques used previously in the study of host cellular material within Chlamydia inclusions [12,14,16,28,29], we quantified the frequency of ER-RFP structures in inclusions after fixation of infected cells (Fig 4A). Fixation with different concentrations of formaldehyde prepared from either paraformaldehyde or liquid formalin (which contains small amounts of methanol) resulted in similarly high frequencies of inclusions with internal ER-RFP structures, indicating that formaldehyde concentrations and trace levels of methanol in formalin do not affect the frequency of internalization. Since gluteraldehyde fixation as used for electron microscopy causes high levels of autofluorescence across the visible light spectrum [38], we could not reliably distinguish any fluorescent marker or dye tested from background fluorescence under these conditions (data not shown). Furthermore, we could not assess the frequency of ER-RFP structures within inclusions after methanol fixation since mRFP fluorescence was quenched by this treatment.

Bottom Line: However, we see little evidence of intraluminal localization of these organelles in live inclusions.These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy.Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Chlamydia trachomatis manipulates host cellular pathways to ensure its proliferation and survival. Translocation of host materials into the pathogenic vacuole (termed 'inclusion') may facilitate nutrient acquisition and various organelles have been observed within the inclusion, including lipid droplets, peroxisomes, multivesicular body components, and membranes of the endoplasmic reticulum (ER). However, few of these processes have been documented in living cells. Here, we survey the localization of a broad panel of subcellular elements and find ER, mitochondria, and inclusion membranes within the inclusion lumen of fixed cells. However, we see little evidence of intraluminal localization of these organelles in live inclusions. Using time-lapse video microscopy we document ER marker translocation into the inclusion lumen during chemical fixation. These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy. We speculate that the localization of a subset of organelles may be exaggerated during fixation. Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.

No MeSH data available.


Related in: MedlinePlus