Competitive Inhibition of Amino Acid Uptake Suppresses Chlamydial Growth: Involvement of the Chlamydial Amino Acid Transporter BrnQ

Growth media, chemicals, and antibodies.RPMI medium and Hanks' balanced salt solution were purchased from Invitrogen GmbH (Karlsruhe, Germany), and fetal bovine serum (FBS) was purchased from Biochrom KG (Berlin, Germany). Cell growth medium (CGM) consisted of RPMI medium supplemented with 2 mM glutamine, 25 mM HEPES, 10% (vol/vol) FBS, and 10 μg/ml gentamicin, and infection medium (IM) consisted of RPMI medium supplemented with 2 mM glutamine, 25 mM HEPES, and 5% FBS. IM containing elevated concentrations of amino acids was prepared as described elsewhere (4).

Cycloheximide, sodium hydroxide, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), trichloroacetic acid (TCA), l-amino acids, d-lactate, U0126, Trizol, epidermal growth factor, and glutaraldehyde were provided by Sigma-Aldrich (Steinheim, Germany), Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany), or Fluka (Buchs, Germany). Rapamycin was obtained from LC Laboratories (Woburn, MA), and [U-¹⁴C]valine was purchased from Hartmann Analytic GmbH (Braunschweig, Germany). Scintillation fluid (Zinsser Aquasafe 300 plus) was purchased from Zinsser Analytic GmbH (Frankfurt, Germany), and cellulose nitrate filters with a pore size of 0.45 μm were obtained from Millipore (Eschborn, Germany). A BCA protein assay kit and an Imagen kit for chlamydial detection were purchased from Pearce (Rockford, IL) and Dako (Hamburg, Germany), respectively. The antibodies used in this study were rb-α-phospho-p70-S6K (catalog no. 9205; Cell Signaling), m-α-tubulin (catalog no. T-9026; Sigma-Aldrich), m-α-Hsp60 (catalog no. 804-071-R100; Alexis Biochemicals), and horseradish peroxidase-conjugated secondary antibodies donkey-α-rb and sheep-α-m (catalog no. NA934 and NA931, respectively; Amersham Biosciences).

Microarray experiments.HeLa cells were grown to 80% confluence in six-well plates and incubated in IM containing 10 mM Leu for 24 h at 37°C in the presence of 5% CO₂. As a control, HeLa cells were incubated in IM without supplements. RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) and was purified using an RNeasy RNA purification kit (Qiagen). The quality was verified both with an ND-1000 spectrophotometer (Nanodrop, Wilmington, DE) and electrophoretically with an Agilent Bioanalyzer (Santa Clara, CA). Cy3- and Cy5-labeled cRNA was produced using an Agilent low-RNA-input fluorescent linear amplification kit. Labeled specimens were hybridized on Agilent whole-human-genome oligonucleotide microarrays for 17 h. The microarrays were washed according to the manufacturer's instructions and were scanned with an Agilent microarray scanner. The mean expression ratios for two technical replicates with reverse labeling were calculated for each gene, and the replicates were considered one experiment. Experiments were performed in duplicate.

Two-dimensional gel electrophoresis.HeLa cells were grown to 80% confluence in 75-cm² cell culture flasks and incubated in IM containing 10 mM Leu at 37°C in the presence of 5% CO₂ for 24 h. As a control, HeLa cells were incubated in IM without supplements. After incubation, the cells were washed once with 30 mM Tris-HCl (pH 7.3) containing 1 mM NaF and 1 mM NaVo₃ and detached using a cell scraper. Samples containing 60 μg total protein were prepared and separated on 15% polyacrylamide gels (30 by 40 cm) as previously described (19). Protein spots were visualized by silver staining. Biologically independent quadruplicate experiments were performed, and only spots showing the same regulation tendency in three of four experiments were considered significantly regulated.

Growth and purification of chlamydial stocks. C. trachomatis LGV serovar L2 (ATCC VR-902B) was routinely propagated in HeLa cells (ATCC CCL-2). Stock organisms were prepared as previously described (6, 7).

Chlamydial infections, amino acid treatment, and progeny infectivity titration.HeLa cells were seeded onto six-well plates in CGM and incubated overnight in the presence of 5% CO₂ at 37°C to allow adherence. C. trachomatis was suspended in IM, and cells were infected at a multiplicity of infection (MOI) of 0.5. Infected monolayers were rinsed once after 2 h in the presence of 5% CO₂ at 35°C, amino acid-supplemented IM was added, and incubation was continued under the same conditions. Infected cells were incubated in IM as a control. Infected cells were detached at 44 h postinfection (p.i.) using sterile glass beads, lysed by vortexing, and titrated onto fresh HeLa cells as previously described (4). Infectivity titers were determined, and the progeny infectivity was expressed as the percentage of inclusion-forming units compared to the untreated control.

Confocal immunofluorescence microscopy.HeLa cells seeded onto coverslips were infected as described above. Amino acid treatment was started 2 h p.i. Cells were washed with phosphate-buffered saline (PBS) at 44 h p.i., fixed with methanol, and subsequently stained with an Imagen Chlamydia detection kit. Coverslips were mounted on glass slides with Mowiol, and the preparations were examined with a Leica TCS-SP laser scanning confocal microscope with a krypton/argon laser. Images were processed using Adobe Photoshop 6.0.

Transmission electron microscopy.Infected cells were washed with PBS and fixed in 2.5% glutaraldehyde, and then they were postfixed with 1% osmium tetroxide, contrasted with uranyl acetate and tannic acid, dehydrated, and embedded in Polybed (Polysciences, Warrington, PA). After polymerization, 60-nm sections were cut and contrasted with lead citrate. Specimens were analyzed with a Leo 906E transmission electron microscope (Oberkochen, Germany) using a Morada digital camera (SIS, Münster, Germany). Images were processed using Adobe Photoshop 6.0.

High-performance liquid chromatography analysis of intracellular amino acids.HeLa cells were seeded onto six-well plates in CGM and incubated overnight in the presence of 5% CO₂ at 37°C to allow adherence. The cells were then rinsed once and incubated under the same conditions in IM containing 10 mM of the indicated amino acids. The medium was removed after 24 h, and the cells were rapidly chilled on ice and washed four times with 2 ml of ice-cold PBS. Free amino acids were extracted by incubating each monolayer in 5% TCA for 2 h at 4°C without detaching the cells from the cell culture dish, as described previously (15). The amino acid-containing TCA was carefully removed and centrifuged for 10 min at 20,000 × g and 4°C. Amino acids in the supernatant were identified by ion-exchange chromatography using a Biochrom 30 amino acid analyzer (Biochrom Ltd., Cambridge, United Kingdom) with a lithium hydroxide/lithium acetate system and a postcolumn ninhydrin reaction. All buffers and reagents were obtained ready to use from Laborservice Onken (Gründau/Breitenborn, Germany). The remaining intact cell monolayers were lysed in 0.1 M NaOH for protein determination with the BCA protein assay kit. Intracellular Val concentrations were normalized to the protein contents and expressed as percentages of the untreated control.

Quantitative RT-PCR.HeLa cells were infected with C. trachomatis at an MOI of 1 and incubated in IM for 2, 8, 16, 24, or 44 h. RNA was isolated with Trizol (Invitrogen) used according to the manufacturer's instructions, and quantitative real-time PCR was performed with the Qiagen SYBR green reverse transcription (RT)-PCR system using an ABI Prism 7000 sequence detection system. The primers used were CT554-RT-F (5′-CCT CGT TGG GAG ACA AAC AT-3′), CT554-RT-R (5′-AAG GCT TGT TGC TTC GAA AA-3′), 16S-RT-F (5′-GGA GAA AAG GGA ATT TCA CG-3′), and 16S-RT-R (5′-TCC ACA TCA AGT ATG CAT CG-3′) (BioTez, Berlin, Germany). Expression was normalized to chlamydial 16S rRNA, and mRNA levels relative to those at 24 h p.i. were calculated as previously described (32).

Bacterial strains, cloning, and functional complementation. Escherichia coli TOP10 cells (Invitrogen) were used for cloning and isolation of genomic DNA. The Ile auxotrophic E. coli K-12 derivative strain B7634 that is deficient for transport of BCAA (ileA hrbBCD brnQ) was kindly provided by Y. Anraku, Science University of Tokyo, Tokyo, Japan (42). All strains were grown in Luria-Bertani medium supplemented with 100 μg/ml ampicillin for selection and propagation of transformed clones. Isolation of genomic DNA and plasmids, PCR, restriction digestion, and electrotransformation were carried out as described by Sambrook et al. (34). Restriction enzymes were purchased from New England Biolabs (Ipswich, MA), and primers were purchased from BioTez. The E. coli brnQ gene and its homolog in C. trachomatis serovar L2, CT554, were both cloned into the low-copy-number plasmid pACYC177 (EMBL accession number X06402), because BCAA transporters were shown to be toxic at high expression levels (37). The natural E. coli promoter region of brnQ (promoter 191) was inserted upstream of brnQ and CT554 for comparable and constitutive expression. The primers used for gene isolation and cloning were Promo191-F (5′-ATG GAT CCT ACT GGT TGG TGA TTT CTT ATC-3′), Promo191-R (5′-ATC CCG GGT ACT GCC TGT GGA TGT GGT-3′), EcoQ-F (5′-ATC CCG GGA TGA CCC ATC AAT TAA GAT CG-3′), EcoQ-R (5′-ATC TCG AGT TAG TGA GCG CTG GAG GTC-3′), CT554-F (5′-ATC CCG GGA TGC ATA AGA AAA CAC AAT CAC-3′), and CT554-R (5′-TAC TCG AGT TAA ACG GAC AGC TTA TAG AG-3′). Plasmids were verified by DNA sequencing (SMB, Berlin, Germany). Functional expression of the cloned transporters was verified using E. coli strain B7634 cultured on M9 minimal medium agar plates supplemented with 50 μg/ml ampicillin and 5 μg/ml Ile. Transformants expressing the cloned transporters grew at this limiting Ile concentration, in contrast to B7634 carrying the empty vector.

Valine transport assays.The B7634 E. coli strain harboring pACYC177 derivatives was grown in 50 ml Luria-Bertani medium supplemented with 100 μg/ml ampicillin to an optical density at 600 nm (OD₆₀₀) of 0.5, harvested by centrifugation (10 min, 3,000 × g, 4°C), resuspended in 10 ml of ice-cold assay buffer (50 mM potassium phosphate [pH 6.5], 5 mM MgSO₄, 10 mM d-lactate), and centrifuged again. The pellet was then resuspended in fresh assay buffer to an OD₆₀₀ of 5, and [¹⁴C]valine uptake assays were performed at 30°C. For these assays, bacteria were diluted in prewarmed assay buffer to obtain an OD₆₀₀ of 1.7 and preincubated for 5 min. Then [¹⁴C]valine was added to produce the final concentrations indicated, and 180-μl aliquots were removed after 1 min, filtered through a 0.45-μm cellulose nitrate membrane, and immediately washed with 5 ml of ice-cold assay buffer. Filters were transferred to scintillation tubes containing 4.5 ml of scintillation fluid, and the radioactivity was measured with a Packard Tri-Carb 2900TR liquid scintillation analyzer. The initial uptake rates in the presence of 1 to 80 μM Val were used to determine the V _max and K_m values of Val transport using a Hanes-Wilkinson transformation and linear regression.

Statistical analysis.Experiments were conducted in triplicate unless otherwise stated. The data were expressed as means ± standard errors. Differences were considered significant if the P value was ≤0.01, as calculated using the Student t test.

The host cell is not affected by high amino acid concentrations.We tested whether high concentrations of amino acids resulted in stress or dramatic changes to the host cells. The host cell viability was not affected by high concentrations of antichlamydial amino acids, as measured using the WST-1 assay (see Fig. S1 in the supplemental material). Microarray analysis also revealed that there were no changes in the host transcriptome. None of the >41,000 human genes represented on this microarray were regulated more than 1.8-fold differently after the addition of 10 mM Leu. Analysis on two-dimensional gels also revealed no differences in the proteomes of Leu-treated and control HeLa cells (data not shown).

The mTOR signal transduction pathway is not involved in chlamydial growth arrest.BCAAs, particularly Leu, are known to activate the host signal transduction pathway of the mTOR (mammalian target of rapamycin) kinase, to upregulate host protein synthesis, and to inhibit autophagy (22). We detected mTOR pathway activation during chlamydial infection (see Fig. S2 in the supplemental material). The mTOR pathway-specific inhibitor rapamycin was employed to examine the influence of this pathway on amino acid-induced chlamydial growth inhibition. Rapamycin treatment changed neither the outcome of the productive infection nor the rate of chlamydial growth in the presence of excess Leu, Ile, Met, or Phe (Fig. 1).

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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.

Inhibition of host cell protein synthesis partially restores chlamydial growth.We investigated the influence of host cell protein synthesis during amino acid-induced chlamydial growth inhibition. Blocking protein synthesis in infected, amino acid-treated cells with cycloheximide resulted in inclusions that were larger than the inclusions observed without cycloheximide treatment (Fig. 2). Infected cell cultures treated with cycloheximide together with Leu, Ile, or Met contained inclusions that were roughly one-half the diameter of the inclusions in infected cells grown without additives. Unexpectedly, infectious EBs could not be recovered from these samples. Transmission electron micrographs revealed that these inclusions contained more chlamydiae than the inclusions in amino acid-treated specimens without cycloheximide but that the chlamydiae had a characteristic aberrant morphology. The bacteria were larger, and no EBs were detected. In brief, blocking host protein synthesis in infected cells treated with Leu, Ile, or Met led to resumption of inclusion growth and chlamydial proliferation but not to completion of the developmental cycle and production of infectious progeny. In Phe-treated cell cultures, cycloheximide treatment resulted in inclusions that were similar sizes but reduced the infectivity of progeny (54.6% ± 3.2% of the untreated control). Electron micrographs showed inclusions with few ABs and a high number of EBs (Fig. 2). These experiments showed that cycloheximide treatment led to partial reconstitution of chlamydial growth and development in infected, amino acid-treated cells. This rescue effect was more effective against the growth suppression caused by Phe than against the growth suppression caused by Leu, Ile, or Met.

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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.

Valine supplementation reverses chlamydial growth inhibition by leucine, isoleucine, methionine, or phenylalanine.The inability of cycloheximide to efficiently overcome the adverse effects of Leu, Ile, Met, or Phe indicates that a mechanism independent of the host cell is responsible for the inhibition of chlamydial growth. One attractive hypothesis is that the added amino acid may compete with one or more amino acids that are essential for chlamydial development and are transported by the same amino acid transporter. To test this hypothesis, cultures exposed to Leu, Ile, Met, or Phe were concomitantly supplemented with other single amino acids essential for C. trachomatis serovar L2 growth (2, 21). The growth arrest induced by elevated levels of Leu, Ile, Met, or Phe was completely reversed by simultaneous administration of Val (Fig. 3). Val supplementation fully restored both primary inclusion development and the production of infectious progeny. This ability to overcome amino acid-induced growth suppression was specific for Val, as other single amino acids did not have this effect. Even addition of a mixture containing elevated concentrations of all other essential amino acids except Val (Leu, Ile, Phe, Met, glutamine [Gln], histidine [His], and tryptophan [Trp]) did not restore chlamydial growth (Fig. 3). These results indicate that antagonism between amino acids is the principal mechanism underlying the inhibitory effects of elevated amino acid concentrations. Furthermore, Leu, Ile, Met, and Phe are all antagonists of the essential amino acid Val.

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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.

Valine supplementation does not reverse the chlamydial growth suppression caused by glycine, serine, or threonine.In our previous work, we found that addition of Gly, Ser, or Thr substantially impaired the development of infectious progeny but that the effects were not as strong as those of the hydrophobic amino acids discussed above (5). Here we investigated whether concomitant addition of Val could restore chlamydial development. Val did not reverse the antichlamydial effects of Gly, Ser, or Thr, since cell cultures treated with these amino acids with and without additional Val contained inclusions that were similar sizes (Fig. 4A). The yield of infectious chlamydial progeny in cultures treated with Gly, Ser, or Thr in the presence of Val was no different from the yield in the absence of Val (Fig. 4B). In contrast to valine's counteracting effect on Leu, Ile, Met, and Phe, no rescue was observed for the growth-inhibitory effects of Gly, Ser, or Thr, indicating that these hydrophilic amino acids do not act as Val antagonists.

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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.

The cytoplasmic valine concentration is not disturbed by the addition of other amino acids.Our results indicate that the growth-inhibitory effects of Leu, Ile, Met, or Phe on Chlamydia most likely result from competition with Val for a common membrane transporter, depriving Chlamydia of this essential amino acid. To localize this antagonism to a cellular compartment, we compared the intracellular Val concentrations in amino acid-treated and untreated cell cultures. Untreated HeLa cells had an average intracellular Val concentration of 10.0 ± 3.3 pmol per mg cellular protein. The intracellular Val concentration was not reduced upon treatment with Leu, Ile, Met, or Phe as potential competitors or with the noncompetitive control amino acid Thr (Fig. 5). To examine whether the antichlamydial amino acids were transported into the host cell, their intracellular concentrations were also assessed. The intracellular concentrations increased with increases in the extracellular concentrations (data not shown). These findings suggest that competitive inhibition of Val transport by either Leu, Ile, Met, or Phe does not occur at the host cell membrane.

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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.

The chlamydial homolog of brnQ, CT554, is expressed throughout chlamydial infection.Since the host cell membrane was ruled out as the site of competition, we speculated that amino acid antagonism might occur at the inclusion membrane, the chlamydial membrane, or both. We searched the chlamydial genome (http://stdgen.northwestern.edu/stdgen/bacteria/chlamy/index.html ) for genes encoding transporters for hydrophobic amino acids, which could serve as potential molecular sites of Val competition. The only candidate found was the CT554 gene, predicted to encode a BCAA transport system of the BrnQ (or LIV-II) family. This protein has amino acid sequence similarities of 47% to the BrnQ transporter of E. coli and up to 53% to the BrnQ transporters of other gram-negative bacteria (e.g., Clostridium tetani E88). These permeases are located in the inner bacterial membrane and rely on the proton motive force to transport Leu, Ile, and Val into the bacterium (37, 40). All sequenced members of the Chlamydiaceae and Parachlamydiaceae possess homologs of this gene, implying that it has an essential role. We examined the expression of the chlamydial brnQ homolog throughout the developmental cycle in C. trachomatis. Quantitative real-time RT-PCR of chlamydial RNA isolated at various time points revealed that this gene was expressed throughout the developmental cycle of C. trachomatis and that the expression was strongest during the early phase of infection (Fig. 6).

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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.

Characterization and competitive inhibition of the chlamydial BrnQ transporter.In order to functionally characterize the putative chlamydial BrnQ transporter, the CT554 gene was heterologously expressed in an E. coli strain deficient for BCAA transport, and the uptake of [¹⁴C]valine was monitored. C. trachomatis BrnQ facilitated transport of Val into E. coli with a V _max of 428 nmol·min⁻¹·mg total protein⁻¹ under standard conditions, compared to 1,046 nmol·min⁻¹·mg total protein⁻¹ for the E. coli transporter. Both transporters were inhibited in the presence of the ionophore CCCP, confirming their dependence on the proton gradient at the inner bacterial membrane. The K_m value for Val transport by the chlamydial BrnQ was 26.6 μM, compared to the 4.3 μM observed for Val transport by E. coli BrnQ (Fig. 7A).

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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. K_m and V _max values were calculated directly from regression lines. The regression lines intercept the horizontal axis at −K_m 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.

To verify our hypothesis that this transporter could be involved in the amino acid-induced growth inhibition, we examined which amino acids could competitively inhibit Val transport. We tested not only the hydrophobic amino acids Leu, Ile, Met, and Phe but also Gly, Ser, and Thr. Only Leu, Ile, Met, and Phe, which also cause valine-reversible growth inhibition in Chlamydia, completely blocked the chlamydial BrnQ transporter. In contrast, Met inhibited Val uptake by the E. coli BrnQ transporter much less. Gly, Ser, and Thr had little impact on Val transport by both transporters (Fig. 7B). Taken together, our results support the hypothesis that competitive inhibition of Val uptake by the chlamydial BrnQ transporter is a major mechanism underlying the antichlamydial effects of hydrophobic amino acids.