Dataset Information

Isolation in endothelial cell cultures of chlamydia trachomatis LGV (Serovar L2) from a lymph node of a patient with suspected cat scratch disease.

ABSTRACT: An inguinal lymph node, removed from a 21-year-old Romanian man suspected of having cat scratch disease, was sent to our laboratory for Bartonella culture. Lymph node specimens were inoculated on blood-enriched agar and in an endothelial cell culture system using the centrifugation shell vial technique. Bacteria were grown in cell monolayers and detected as positive with an anti-Bartonella henselae rabbit serum. However, such bacteria were identified as Chlamydia trachomatis biovar LGV serovar L2 by PCR sequencing techniques. Pathological examination of tissue biopsies was compatible with either lymphogranuloma venereum or cat scratch disease. The shell vial system is suitable for isolation of intracellular pathogens responsible for chronic lymphadenopathies, including C. trachomatis, Bartonella species, Francisella tularensis, and mycobacteria. However, care should be taken when identifying Chlamydia spp. and Bartonella spp. using polyclonal antibodies, since species of both genera have common antigens which are responsible for cross-reactions.

SUBMITTER: Maurin M

PROVIDER: S-EPMC86728 | biostudies-literature | 2000 Jun

REPOSITORIES: biostudies-literature

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Isolation in endothelial cell cultures of chlamydia trachomatis LGV (Serovar L2) from a lymph node of a patient with suspected cat scratch disease.

Maurin M M Raoult D D

Journal of clinical microbiology 20000601 6

An inguinal lymph node, removed from a 21-year-old Romanian man suspected of having cat scratch disease, was sent to our laboratory for Bartonella culture. Lymph node specimens were inoculated on blood-enriched agar and in an endothelial cell culture system using the centrifugation shell vial technique. Bacteria were grown in cell monolayers and detected as positive with an anti-Bartonella henselae rabbit serum. However, such bacteria were identified as Chlamydia trachomatis biovar LGV serovar L ...[more]

PMID: 10834954

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Project description:UnlabelledMethylation 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.ImportanceObligate 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.

Project description:Chlamydia have reduced genomes that reflect their obligately parasitic lifestyle. Despite their different tissue tropisms, chlamydial strains share a large number of common genes and have few recognized pseudogenes, indicating genomic stability. All of the Chlamydiaceae have homologs of the aaxABC gene cluster that encodes a functional arginine:agmatine exchange system in Chlamydia (Chlamydophila)pneumoniae. However, Chlamydia trachomatis serovar L2 strains have a nonsense mutation in their aaxB genes, and C. trachomatis serovar A and B strains have frameshift mutations in their aaxC homologs, suggesting that relaxed selection may have enabled the evolution of aax pseudogenes. Biochemical experiments were performed to determine whether the aaxABC genes from C. trachomatis strains were transcribed, and mutagenesis was used to identify nucleotide substitutions that prevent protein maturation and activity. Molecular evolution techniques were applied to determine the relaxation of selection and the scope of aax gene inactivation in the Chlamydiales.The aaxABC genes were co-transcribed in C. trachomatis L2/434, during the mid-late stage of cellular infection. However, a stop codon in the aaxB gene from this strain prevented the heterologous production of an active pyruvoyl-dependent arginine decarboxylase. Replacing that ochre codon with its ancestral tryptophan codon rescued the activity of this self-cleaving enzyme. The aaxB gene from C. trachomatis D/UW-3 was heterologously expressed as a proenzyme that failed to cleave and form the catalytic pyruvoyl cofactor. This inactive protein could be rescued by replacing the arginine-115 codon with an ancestral glycine codon. The aaxC gene from the D/UW-3 strain encoded an active arginine:agmatine antiporter protein, while the L2/434 homolog was unexpectedly inactive. Yet the frequencies of nonsynonymous versus synonymous nucleotide substitutions show no signs of relaxed selection, consistent with the recent inactivation of these genes.The ancestor of the Chlamydiaceae had a functional arginine:agmatine exchange system that is decaying through independent, parallel processes in the C. trachomatis lineage. Differences in arginine metabolism among Chlamydiaceae species may be partly associated with their tissue tropism, possibly due to the protection conferred by a functional arginine-agmatine exchange system against host nitric oxide production and innate immunity. The independent loss of AaxB activity in all sequenced C. trachomatis strains indicates continual gene inactivation and illustrates the difficulty of recognizing recent bacterial pseudogenes from sequence comparison, transcriptional profiling or the analysis of nucleotide substitution rates.

Dataset Information

Isolation in endothelial cell cultures of chlamydia trachomatis LGV (Serovar L2) from a lymph node of a patient with suspected cat scratch disease.

Publications

Isolation in endothelial cell cultures of chlamydia trachomatis LGV (Serovar L2) from a lymph node of a patient with suspected cat scratch disease.

Similar Datasets

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