• Home
  • Search Results
  • Golgi fragmentation and sphingomyelin transport to Chlamydia trachomatis during penicillin-induced persistence do not depend on the cytosolic presence of the chlamydial protease CPAF.

Golgi fragmentation and sphingomyelin transport to Chlamydia trachomatis during penicillin-induced persistence do not depend on the cytosolic presence of the chlamydial protease CPAF.

PloS one (2014-07-30)
Stephanie Dille, Katharina Herbst, Larisa Volceanov, Thilo Nölke, Oliver Kretz, Georg Häcker
ABSTRACT

Chlamydia grows inside a cytosolic vacuole (the inclusion) that is supplied with nutrients by the host through vesicular and non-vesicular transport. It is unclear in many respects how Chlamydia organizes this transport. One model posits that the Chlamydia-induced fragmentation of the Golgi-apparatus is required for normal transport processes to the inclusion and for chlamydial development, and the chlamydial protease CPAF has been controversially implicated in Golgi-fragmentation. We here use a model of penicillin-induced persistence of infection with Chlamydia trachomatis to test this link. Under penicillin-treatment the inclusion grew in size for the first 24 h but after that growth was severely reduced. Penicillin did not reduce the number of infected cells with fragmented Golgi-apparatus, and normal Golgi-fragmentation was found in a CPAF-deficient mutant. Surprisingly, sphingomyelin transport into the inclusion and into the bacteria, as measured by fluorescence accumulation upon addition of labelled ceramide, was not reduced during penicillin-treatment. Thus, both Golgi-fragmentation and transport of sphingomyelin to C. trachomatis still occurred in this model of persistence. The portion of cells in which CPAF was detected in the cytosol, either by immunofluorescence or by immune-electron microscopy, was drastically reduced in cells cultured in the presence of penicillin. These data argue against an essential role of cytosolic CPAF for Golgi-fragmentation or for sphingomyelin transport in chlamydial infection.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Sodium bicarbonate, powder, BioReagent, for molecular biology, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
Sodium bicarbonate, ACS reagent, ≥99.7%
Sigma-Aldrich
Urea, ACS reagent, 99.0-100.5%
Sigma-Aldrich
Urea, powder, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-15, ascites fluid
Sigma-Aldrich
Sodium bicarbonate, ReagentPlus®, ≥99.5%, powder
Sigma-Aldrich
Urea, ReagentPlus®, ≥99.5%, pellets
Sigma-Aldrich
Sodium bicarbonate, BioXtra, 99.5-100.5%
Sigma-Aldrich
Osmium tetroxide, ReagentPlus®, 99.8%
Supelco
Urea, 8 M (after reconstitution with 16 mL high purity water)
Sigma-Aldrich
Urea solution, BioUltra, ~8 M in H2O
Sigma-Aldrich
Osmium tetroxide, Sealed ampule.
Sigma-Aldrich
Urea, BioXtra, pH 7.5-9.5 (20 °C, 5 M in H2O)
Sigma-Aldrich
Sodium bicarbonate, anhydrous, free-flowing, Redi-Dri, ACS reagent, ≥99.7%
Sigma-Aldrich
Urea, suitable for electrophoresis
Sigma-Aldrich
Osmium tetroxide solution, suitable for electron microscopy, 4% in H2O
Sigma-Aldrich
Urea, puriss., meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%, 99.0-101.0% (calc. on dry substance)
Sigma-Aldrich
Nutrient Mixture F-12 Ham, Kaighn′s Modification, with L-glutamine, without sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
Urea, BioUltra, for molecular biology, ≥99% (T)
Sigma-Aldrich
Osmium tetroxide solution, 4 wt. % in H2O
Sigma-Aldrich
Osmium tetroxide solution, 2.5 wt. % in tert-butanol
Sigma-Aldrich
Urea, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99%
Sigma-Aldrich
Urea solution, 40 % (w/v) in H2O
Sigma-Aldrich
Sodium bicarbonate, puriss., meets analytical specification of Ph. Eur., BP, USP, FCC, E500, 99.0-100.5%, powder
Sigma-Aldrich
Urea, meets USP testing specifications
Sigma-Aldrich
Keratinocyte Growth Factor human, KGF, recombinant, expressed in E. coli, powder, suitable for cell culture
Sigma-Aldrich
Osmium tetroxide solution, suitable for electron microscopy, 2% in H2O
Supelco
Urea, analytical standard
Sigma-Aldrich
Sodium hydrogencarbonate, −40-+140 mesh, ≥95%