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

Abstract

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.

FIG 1

Growth of E. coli ∆metK mutant in the presence or absence of extracellular SAM. ATM778 (∆metK) transformed with the indicated plasmids was grown at 37°C in LB supplemented with Ap, Cm, 0.2% glucose (solid lines), or 0.2% arabinose (dotted lines), in the absence (A) or presence (B) of 1 mM SAM. Absorbance (OD₆₀₀) was measured in a Bioscreen growth curve analyzer and plotted against time (hours). Error bars represent the standard deviations from four replicates. Symbols: ○, pUC-empty vector; □, pREF77-ctl843; ▵, pRAK368-ctl843; ▿, pRAK368-ctl843 with IPTG.

FIG 2

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 K_m and V_max values.

FIG 3

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.

FIG 4

Sequence alignments of C. trachomatis L2 CTL843 and R. prowazekii RP076 SAM transporter. Amino acid sequences were first aligned on a web interface of MAFFT (53) and were subsequently aligned via Clustal W2 to illustrate the similarity. Identical residues are denoted with an asterisk and are highlighted in black boxes. Strongly conserved residues and weakly conserved residues are marked with a colon and a period, respectively, and are displayed in gray boxes.