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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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Related in: MedlinePlus

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.
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f4: 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.

Mentions: An ORF similar to CTL843 was found in all genome sequences available for the order Chlamydiales, with amino acid identity of >90% for Chlamydia homologs, 60 to 67% for Chlamydophila homologs, and 34 to 38% for the Chlamydia-like homologs. All CTL843 homologs contain a duplication of the evolutionarily conserved domain EamA, also known as DUF6 (PF00892; http://pfam.sanger.ac.uk/family/PF00892). While EamA is a signature for transporters belonging to the drug and metabolite transporter (DMT) superfamily, the presence of two EamA domains further subclassifies these chlamydial ORFs in the drug/metabolite exporter (DME) family exhibiting 10 alpha-helical transmembrane spanners (TMSs) (http://www.tcdb.org). Interestingly, this family of transporters also contains the SAM transporters belonging to Rickettsieae, another group of obligate intracellular bacteria. However, Rickettsia prowazekii RP076 (15) shares only about 20% identity with C. trachomatis L2 CTL843 (Fig. 4). Additional phylogenetic analysis failed to support any evolutionary relationship between the rickettsial and the chlamydial SAM transporters (Fig. S5).


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

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

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: 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.
Mentions: An ORF similar to CTL843 was found in all genome sequences available for the order Chlamydiales, with amino acid identity of >90% for Chlamydia homologs, 60 to 67% for Chlamydophila homologs, and 34 to 38% for the Chlamydia-like homologs. All CTL843 homologs contain a duplication of the evolutionarily conserved domain EamA, also known as DUF6 (PF00892; http://pfam.sanger.ac.uk/family/PF00892). While EamA is a signature for transporters belonging to the drug and metabolite transporter (DMT) superfamily, the presence of two EamA domains further subclassifies these chlamydial ORFs in the drug/metabolite exporter (DME) family exhibiting 10 alpha-helical transmembrane spanners (TMSs) (http://www.tcdb.org). Interestingly, this family of transporters also contains the SAM transporters belonging to Rickettsieae, another group of obligate intracellular bacteria. However, Rickettsia prowazekii RP076 (15) shares only about 20% identity with C. trachomatis L2 CTL843 (Fig. 4). Additional phylogenetic analysis failed to support any evolutionary relationship between the rickettsial and the chlamydial SAM transporters (Fig. S5).

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