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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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Kinetics of SAM transport in E. coli. (A) Time course of [14C]SAM uptake in E. coli at 37°C as a function of time. The time course assay was carried out in transport buffer supplemented with 10 µM [14C]SAM. Time point assays were done in triplicate. (B) Effect of substrate concentration on [14C]SAM uptake at 37°C. The data represent six independent experiments done in triplicate. The background transport seen with MG1655 (○) was subtracted from the transport seen with ATM915 (MG1655 expressing ctl843 [Δ]) with GraphPad Prism software, and then nonlinear regression analysis was performed to obtain the Km and Vmax values.
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f2: Kinetics of SAM transport in E. coli. (A) Time course of [14C]SAM uptake in E. coli at 37°C as a function of time. The time course assay was carried out in transport buffer supplemented with 10 µM [14C]SAM. Time point assays were done in triplicate. (B) Effect of substrate concentration on [14C]SAM uptake at 37°C. The data represent six independent experiments done in triplicate. The background transport seen with MG1655 (○) was subtracted from the transport seen with ATM915 (MG1655 expressing ctl843 [Δ]) with GraphPad Prism software, and then nonlinear regression analysis was performed to obtain the Km and Vmax values.

Mentions: To investigate CTL843 function, uptake of [14C]SAM into intact cells of E. coli was analyzed by a rapid filtration assay. Control cells showed a slow, low linear uptake of SAM (Fig. 2A). In contrast, expression of CTL843 in ATM915 showed a marked increase in the amount of intracellular label over time in a process that was linear during the first minute and then reached saturation indicative of carrier-mediated transport (Fig. 2A). Cell suspensions were also incubated with 5 to 100 µM [14C]SAM, and uptake was measured after 30 s to estimate the kinetic constants of recombinant CTL843 (Fig. 2B). From these data, the calculated apparent Km values (reflecting SAM affinity for CTL843) and Vmax values (reflecting CTL843 activity) were 5.88 ± 0.11 µM and 31.57 ± 0.09 pmol min−1 mg−1, respectively. To assess the role of the proton gradient in CTL843 function, ATM915 was pretreated for 5 minutes with 20 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), which acts as a channel through the inner membrane to dissipate the H+ gradient. CCCP treatment reduced [14C]SAM intracellular uptake by 50%.


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

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

Kinetics of SAM transport in E. coli. (A) Time course of [14C]SAM uptake in E. coli at 37°C as a function of time. The time course assay was carried out in transport buffer supplemented with 10 µM [14C]SAM. Time point assays were done in triplicate. (B) Effect of substrate concentration on [14C]SAM uptake at 37°C. The data represent six independent experiments done in triplicate. The background transport seen with MG1655 (○) was subtracted from the transport seen with ATM915 (MG1655 expressing ctl843 [Δ]) with GraphPad Prism software, and then nonlinear regression analysis was performed to obtain the Km and Vmax values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Kinetics of SAM transport in E. coli. (A) Time course of [14C]SAM uptake in E. coli at 37°C as a function of time. The time course assay was carried out in transport buffer supplemented with 10 µM [14C]SAM. Time point assays were done in triplicate. (B) Effect of substrate concentration on [14C]SAM uptake at 37°C. The data represent six independent experiments done in triplicate. The background transport seen with MG1655 (○) was subtracted from the transport seen with ATM915 (MG1655 expressing ctl843 [Δ]) with GraphPad Prism software, and then nonlinear regression analysis was performed to obtain the Km and Vmax values.
Mentions: To investigate CTL843 function, uptake of [14C]SAM into intact cells of E. coli was analyzed by a rapid filtration assay. Control cells showed a slow, low linear uptake of SAM (Fig. 2A). In contrast, expression of CTL843 in ATM915 showed a marked increase in the amount of intracellular label over time in a process that was linear during the first minute and then reached saturation indicative of carrier-mediated transport (Fig. 2A). Cell suspensions were also incubated with 5 to 100 µM [14C]SAM, and uptake was measured after 30 s to estimate the kinetic constants of recombinant CTL843 (Fig. 2B). From these data, the calculated apparent Km values (reflecting SAM affinity for CTL843) and Vmax values (reflecting CTL843 activity) were 5.88 ± 0.11 µM and 31.57 ± 0.09 pmol min−1 mg−1, respectively. To assess the role of the proton gradient in CTL843 function, ATM915 was pretreated for 5 minutes with 20 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), which acts as a channel through the inner membrane to dissipate the H+ gradient. CCCP treatment reduced [14C]SAM intracellular uptake by 50%.

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