Article Figures & Data
Figures
- FIG. 1.
The mTOR kinase is not involved in chlamydial growth arrest. C. trachomatis-infected HeLa cells were treated with 5 mM Leu, Ile, Met, or Phe from 19 to 44 h p.i., with or without treatment with 100 nM rapamycin (Rapa) from 30 min before amino acid treatment until 44 h p.i. Cells were lysed 44 h p.i. and titrated onto fresh cells, and inclusions were counted 24 h later. Progeny infectivity is expressed as a percentage of the untreated control. Means standard errors of the means from triplicate experiments are shown. mTOR kinase inhibition did not alter chlamydial progeny infectivity in the absence or presence of Leu, Ile, Met, or Phe.
- FIG. 2.
Inhibition of host cell protein synthesis partially restores chlamydial growth. C. trachomatis-infected HeLa cells were treated with 10 mM Leu, Ile, Met, or Phe from 2 h p.i. until the end of the infection cycle, with or without 5 μg/ml cycloheximide. Infected cells were fixed 44 h p.i. and prepared for confocal immunofluorescence or transmission electron microscopy analysis. In the fluorescence micrographs, chlamydial inclusions were stained green, and cells were counterstained red with Evans blue. White bars = 20 μm. White arrows indicate RBs, black arrows indicate EBs, and black arrowheads indicate ABs. For better comparison, sections containing typical EBs and RBs were enlarged fourfold. Black bars = 2 μm. Chlamydial inclusion growth was suppressed by Leu, Ile, Met, or Phe and was partially restored by simultaneous addition of cycloheximide. Phe-induced growth suppression was more efficiently reversed than the antichlamydial effects induced by Leu, Ile, or Met.
- FIG. 3.
Valine addition reverses the abrogation of chlamydial growth induced by leucine, isoleucine, methionine, or phenylalanine. (A) HeLa cells were infected using an MOI of 0.5 and were treated with the indicated amino acids at a concentration of 5 mM from 2 h p.i. until the end of the infection cycle. The essential amino acid mixture (Ess. AAs) contained 0.5 mM Trp, 5 mM Leu, 5 mM Ile, 5 mM Met, 5 mM Phe, 5 mM Gln, and 5 mM His. Infected cells were fixed at 44 h p.i. and immunostained for chlamydial inclusions (green). Cells were counterstained red. Bars = 40 μm. (B) HeLa cells were infected and treated as described above for panel A. Cells were lysed at 44 h p.i. and titrated onto fresh cells, and inclusions were counted 24 h later. Progeny infectivity is expressed as a percentage of the untreated control. Means and standard errors of the means for triplicate experiments are shown. Chlamydial inclusion growth and progeny infectivity were restored upon addition of Val to infected cells treated with Leu, Ile, Met, or Phe.
- FIG. 4.
Valine supplementation does not inhibit chlamydial growth suppression induced by glycine, serine, or threonine. (A) HeLa cells were infected using an MOI of 0.5 and treated with the indicated amino acids at a concentration of 5 mM from 2 h p.i. until the end of the infection cycle. Cells were fixed at 44 h p.i. and immunostained for chlamydial inclusions (green). Cells were counterstained red. Bars = 40 μm. (B) HeLa cells were infected and treated as described above for panel A. Cells were lysed at 44 h p.i. and titrated onto fresh cells, and inclusions were counted 24 h later. Progeny infectivity is expressed as a percentage of the untreated control value. Means and standard errors of the means for triplicate experiments are shown. Val could not significantly restore chlamydial growth when it was added to cells treated with Thr, Ser, or Gly, as Val treatment increased neither the inclusion size nor the progeny infectivity.
- FIG. 5.
Valine concentrations in the host cytoplasm are not disturbed by addition of other amino acids. HeLa cells were cultured for 24 h in medium supplemented with the amino acid indicated at a concentration of 10 mM or for 2 h with Hanks' balanced salt solution for total amino acid starvation. After thorough washing, free intracellular amino acids were extracted from the monolayers using 5% TCA and analyzed by high-performance liquid chromatography. The remaining intact monolayers were dissolved in 0.1 M NaOH for subsequent protein determination. Intracellular Val concentrations were normalized to the total protein and were expressed as a percentage of the untreated control plus the standard error of the mean. Intracellular Val levels were not reduced upon treatment with other amino acids, indicating that cellular Val transport is not impaired by addition of Leu, Ile, Met, Phe, or Thr.
- FIG. 6.
CT554 is expressed throughout the entire chlamydial developmental cycle. HeLa cells were infected using an MOI of 1, and total RNA was prepared at 2, 8, 16, 24, and 44 h p.i. using Trizol for analysis by quantitative real-time RT-PCR. The expression of CT554 was normalized to chlamydial 16S rRNA, and the average mRNA levels relative to the level at 24 h p.i. are shown; the error bars indicate standard errors. Experiments were done in duplicate.
- FIG. 7.
Characterization of the chlamydial BrnQ homolog. (A) Hanes blot of Val transport. E. coli and C. trachomatis BrnQ homologs were expressed in E. coli B7634, and uptake of 14C-labeled Val was measured using substrate concentrations of 1 to 80 μM. Km and V max values were calculated directly from regression lines. The regression lines intercept the horizontal axis at −Km and have slopes of 1/V max. The results for one of three experiments with similar outcomes are shown. •, C. trachomatis BrnQ; ○, E. coli BrnQ. [Val], Val concentration; v, Val transport rate in μmol·min−1·mg of total protein−1. (B) [14C]valine uptake was measured in the presence of 5 mM competitive amino acid, 60 μM CCCP, or 0.1% methanol. The results for Val uptake were normalized to the total protein and expressed as percentages of the transported Val in the absence of inhibitors (untreated control). The uptake rates of the untreated controls were 247 and 848 nmol·min−1·mg of total protein−1 for the C. trachomatis and E. coli homologs, respectively. Asterisks indicate preparations in which Val transport was significantly suppressed compared to controls. Filled bars, C. trachomatis BrnQ; open bars, E. coli BrnQ. The bars indicate means, and the error bars indicate standard errors. The competition profiles show that there was complete inhibition of the chlamydial transporter by Leu, Ile, Met, and Phe. Met inhibited the E. coli transporter to a lesser extent.
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Fig. S1, cell viability is not decreased upon treatment with elevated amino acid concentrations
Zipped PDF document, 26K - Supplemental file 2
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Fig. S2, mTOR is activated upon C. trachomatis infection
Zipped PDF document, 31K. - Supplemental file 3
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Fig. S3, 100 nM rapamycin completely blocks mTOR activity
Zipped PDF document, 26K.
- Supplemental file 1
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Fig. S1, cell viability is not decreased upon treatment with elevated amino acid concentrations
