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Identification and characterization of the Chlamydia trachomatis L2 S-adenosylmethionine transporter.

Binet R, Fernandez RE, Fisher DJ, Maurelli AT - MBio (2011)

Bottom Line: Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843.The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target.The transporter, CTL843, allows Chlamydia trachomatis L2 to steal S-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxic S-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT).

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA. rachel.binet@fda.hhs.gov

ABSTRACT

Unlabelled: Methylation is essential to the physiology of all cells, including the obligate intracellular bacterium Chlamydia. Nevertheless, the methylation cycle is under strong reductive evolutionary pressure in Chlamydia. Only Parachlamydia acanthamoebae and Waddlia chondrophila genome sequences harbor homologs to metK, encoding the S-adenosylmethionine (SAM) synthetase required for synthesis of SAM, and to sahH, which encodes the S-adenosylhomocysteine (SAH) hydrolase required for detoxification of SAH formed after the transfer of the methyl group from SAM to the methylation substrate. Transformation of a conditional-lethal ΔmetK mutant of Escherichia coli with a genomic library of Chlamydia trachomatis L2 identified CTL843 as a putative SAM transporter based on its ability to allow the mutant to survive metK deficiency only in the presence of extracellular SAM. CTL843 belongs to the drug/metabolite superfamily of transporters and allowed E. coli to transport S-adenosyl-L-[methyl-(14)C]methionine with an apparent K(m) of 5.9 µM and a V(max) of 32 pmol min(-1) mg(-1). Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843. We propose that CTL843 acts as a SAM/SAH transporter (SAMHT) serving a dual function by allowing Chlamydia to acquire SAM from the host cell and excrete the toxic by-product SAH. The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target.

Importance: Obligate intracellular parasites like Chlamydia have followed a reductive evolutionary path that has made them almost totally dependent on their host cell for nutrients. In this work, we identify a unique transporter of a metabolite essential for all methylation reactions that potentially bypasses the need for two enzymatic reactions in Chlamydia. The transporter, CTL843, allows Chlamydia trachomatis L2 to steal S-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxic S-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT). In addition to reflecting the adaptation of Chlamydia to an obligate intracellular lifestyle, the specific and central roles of SAMHT in Chlamydia metabolism provide a target for the development of therapeutic agents for the treatment of chlamydial infections.

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Efflux of [14C]SAM from E. coli cells expressing CTL843. Preloading of labeled SAM (10 µM) in ATM915 was performed initially for 1 min at 37°C and then stopped by dilution and washes at 4°C. Samples were resuspended in duplicate in M9 minimal salts with or without 100 µM cold SAM or cold SAH and incubated at 37°C in the presence or absence of 20 µM CCCP. After 10 min, the partition of labeled SAM was determined by counting radioactivity in the supernatants and in the pellets after centrifugation at 13,000 rpm for 90 s.
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f3: Efflux of [14C]SAM from E. coli cells expressing CTL843. Preloading of labeled SAM (10 µM) in ATM915 was performed initially for 1 min at 37°C and then stopped by dilution and washes at 4°C. Samples were resuspended in duplicate in M9 minimal salts with or without 100 µM cold SAM or cold SAH and incubated at 37°C in the presence or absence of 20 µM CCCP. After 10 min, the partition of labeled SAM was determined by counting radioactivity in the supernatants and in the pellets after centrifugation at 13,000 rpm for 90 s.

Mentions: In order to further characterize CTL843 as an SAM/SAH antiporter, back-exchange studies were performed. Cultures of ATM915 expressing CTL843 were preloaded with labeled SAM, washed to remove external radioactivity, and resuspended in M9 minimal salts supplemented with putative counterexchange substrates at a 10-fold excess over labeled SAM and incubated at 37°C. After 10 min, the cells were centrifuged and radioactivity was counted in the cells and in the supernatant. Quantification of exported radioactivity allows differentiation between counterexchange and unidirectional transport. E. coli expressing CTL843 and preloaded with [14C]SAM released significant amounts of internal label (~80% of the initial amount) after resuspension in buffer medium supplemented with unlabeled SAM (thus, against the SAM concentration gradient) or SAH. CCCP treatment did not affect the counterexchange of labeled SAM with SAM or SAH (Fig. 3), indicating that exchange of internal SAM with external SAM or SAH in excess was energy independent. On the other hand, maintenance of the cellular SAM gradient observed in the absence of “competitors” required an intact proton motive force, as SAM concentration reached equilibrium across the membranes in the presence of CCCP (Fig. 3). Thus, CTL843 is active in both directions and can function as an active symporter or energy-independent antiporter (SAM uptake coupled to labeled SAM exit). The similitude between SAM and SAH in the ability to promote the efflux of labeled SAM from the cells strongly suggests that SAH is also being transported into the cells in exchange for SAM. Consequently, we propose that CTL843 be renamed C. trachomatis L2 SAMHT for SAM/SAH transporter.


Identification and characterization of the Chlamydia trachomatis L2 S-adenosylmethionine transporter.

Binet R, Fernandez RE, Fisher DJ, Maurelli AT - MBio (2011)

Efflux of [14C]SAM from E. coli cells expressing CTL843. Preloading of labeled SAM (10 µM) in ATM915 was performed initially for 1 min at 37°C and then stopped by dilution and washes at 4°C. Samples were resuspended in duplicate in M9 minimal salts with or without 100 µM cold SAM or cold SAH and incubated at 37°C in the presence or absence of 20 µM CCCP. After 10 min, the partition of labeled SAM was determined by counting radioactivity in the supernatants and in the pellets after centrifugation at 13,000 rpm for 90 s.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104491&req=5

f3: Efflux of [14C]SAM from E. coli cells expressing CTL843. Preloading of labeled SAM (10 µM) in ATM915 was performed initially for 1 min at 37°C and then stopped by dilution and washes at 4°C. Samples were resuspended in duplicate in M9 minimal salts with or without 100 µM cold SAM or cold SAH and incubated at 37°C in the presence or absence of 20 µM CCCP. After 10 min, the partition of labeled SAM was determined by counting radioactivity in the supernatants and in the pellets after centrifugation at 13,000 rpm for 90 s.
Mentions: In order to further characterize CTL843 as an SAM/SAH antiporter, back-exchange studies were performed. Cultures of ATM915 expressing CTL843 were preloaded with labeled SAM, washed to remove external radioactivity, and resuspended in M9 minimal salts supplemented with putative counterexchange substrates at a 10-fold excess over labeled SAM and incubated at 37°C. After 10 min, the cells were centrifuged and radioactivity was counted in the cells and in the supernatant. Quantification of exported radioactivity allows differentiation between counterexchange and unidirectional transport. E. coli expressing CTL843 and preloaded with [14C]SAM released significant amounts of internal label (~80% of the initial amount) after resuspension in buffer medium supplemented with unlabeled SAM (thus, against the SAM concentration gradient) or SAH. CCCP treatment did not affect the counterexchange of labeled SAM with SAM or SAH (Fig. 3), indicating that exchange of internal SAM with external SAM or SAH in excess was energy independent. On the other hand, maintenance of the cellular SAM gradient observed in the absence of “competitors” required an intact proton motive force, as SAM concentration reached equilibrium across the membranes in the presence of CCCP (Fig. 3). Thus, CTL843 is active in both directions and can function as an active symporter or energy-independent antiporter (SAM uptake coupled to labeled SAM exit). The similitude between SAM and SAH in the ability to promote the efflux of labeled SAM from the cells strongly suggests that SAH is also being transported into the cells in exchange for SAM. Consequently, we propose that CTL843 be renamed C. trachomatis L2 SAMHT for SAM/SAH transporter.

Bottom Line: Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843.The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target.The transporter, CTL843, allows Chlamydia trachomatis L2 to steal S-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxic S-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT).

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA. rachel.binet@fda.hhs.gov

ABSTRACT

Unlabelled: Methylation is essential to the physiology of all cells, including the obligate intracellular bacterium Chlamydia. Nevertheless, the methylation cycle is under strong reductive evolutionary pressure in Chlamydia. Only Parachlamydia acanthamoebae and Waddlia chondrophila genome sequences harbor homologs to metK, encoding the S-adenosylmethionine (SAM) synthetase required for synthesis of SAM, and to sahH, which encodes the S-adenosylhomocysteine (SAH) hydrolase required for detoxification of SAH formed after the transfer of the methyl group from SAM to the methylation substrate. Transformation of a conditional-lethal ΔmetK mutant of Escherichia coli with a genomic library of Chlamydia trachomatis L2 identified CTL843 as a putative SAM transporter based on its ability to allow the mutant to survive metK deficiency only in the presence of extracellular SAM. CTL843 belongs to the drug/metabolite superfamily of transporters and allowed E. coli to transport S-adenosyl-L-[methyl-(14)C]methionine with an apparent K(m) of 5.9 µM and a V(max) of 32 pmol min(-1) mg(-1). Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843. We propose that CTL843 acts as a SAM/SAH transporter (SAMHT) serving a dual function by allowing Chlamydia to acquire SAM from the host cell and excrete the toxic by-product SAH. The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target.

Importance: Obligate intracellular parasites like Chlamydia have followed a reductive evolutionary path that has made them almost totally dependent on their host cell for nutrients. In this work, we identify a unique transporter of a metabolite essential for all methylation reactions that potentially bypasses the need for two enzymatic reactions in Chlamydia. The transporter, CTL843, allows Chlamydia trachomatis L2 to steal S-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxic S-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT). In addition to reflecting the adaptation of Chlamydia to an obligate intracellular lifestyle, the specific and central roles of SAMHT in Chlamydia metabolism provide a target for the development of therapeutic agents for the treatment of chlamydial infections.

Show MeSH
Related in: MedlinePlus