Structures of glyceraldehyde 3-phosphate dehydrogenase in Neisseria gonorrhoeae and Chlamydia trachomatis.

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co-infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng-Ct co-infections. Development of a safe, effective, and inexpensive dual therapy for Ng-Ct co-infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X-ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high-throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.

High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases.

Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L(4)Mn(IV)(µO)(2)Fe(III)L(4) (metal-metal distance of ~2.75Å, L = ligand) prevailing in metal-grown R2, or L(4)Mn(IV)(µO)(µOH)Fe(III)L(4) (~2.90Å) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55Å) with a L(4)Fe(IV)(µO)(2)Fe(III)L(3) core structure. Several Mn,Fe(III)Fe(III) (~2.9-3.1Å) and Mn,Fe(III)Fe(II) species (~3.3-3.4Å) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the Mn(IV)(µO)(µOH)Fe(III) core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.

Eradication of Chlamydia trachomatis parallels symptom regression in chronic bacterial prostatitis patients treated with a fluoroquinolone-macrolide combination.

To investigate the association between eradication of Chlamydia trachomatis (CT) and symptom regression in chronic prostatitis, 55 symptomatic patients were subjected to segmented tests to localise CT in first voided urine (VB1), prostatic secretions (EPS), post-massage voided (VB3) or semen specimens. Patients were divided in three treatment groups: the 'urethral involvement' group ('U': VB1 positive, EPS/VB3/Semen negative) was treated with 500 mg day(-1) azithromycin for 3 days. The 'prostatitis' group ('P': VB1 negative, EPS/VB3/semen positive) with 4-week levofloxacin-azithromycin combination. A third group, 'U+P' (VB1, EPS/VB3/semen positive) received both treatments in sequence. In P patients, eradication of CT was paralleled by marked, sustained symptom improvement and by significant decrease of serum prostate-specific antigen (PSA) levels. Compared with U patients, undergoing rapid regression of symptoms related to painful micturition after short-term azithromycin, U+P patients showed symptom and pathogen persistence in VB3/EPS/semen and required additional treatment with 4-week levofloxacin-azithromycin to achieve pathogen eradication, symptom regression, and decrease of PSA. Our results support a causative role of CT in chronic bacterial prostatitis. In the presence of a positive urethral localisation of the pathogen, thorough microbiological investigation together with focused symptom analysis may reveal an underlying chlamydial prostatitis and direct effective therapy with appropriate antibacterial agents.

4th meeting of the EU research network EUROME: from the identification of genes and cellular networks in murine models of arthritis to novel therapeutic intervention strategies in rheumatoid arthritis, London, UK, 9 March 2004.

Rheumatoid arthritis (RA) is a common human disease with a prevalence of about 1% in most parts of the world. At the time of symptom onset it is difficult to predict the severity of subsequent disease course. After 2 years joint erosions are seen in most patients, and most patients become clinically disabled within 20 years. A recent meeting at the Kennedy Institute of Rheumatology (Imperial College, London) brought together representatives from several European centres of excellence, to discuss research funded by the EU Framework 5 Quality of Life Programme. This research network combines gene and protein expression profiling with different animal models of RA to identify cells, genes and pathways contributing to arthritis initiation, progression and chronicity. The studies discussed highlight the reality that collaboration between different research groups is the basis of groundbreaking research and, it is hoped, eventual new therapies for RA.

Chlamydia trachomatis genes whose products are related to energy metabolism are expressed differentially in active vs. persistent infection.

The Chlamydia trachomatis genome encodes glycolysis and pentose phosphate pathway enzymes, two ATP/ADP exchange proteins, and other energy transduction-related components. We asked if and when chlamydial genes specifying products related to energy transduction are expressed during active vs. persistent infection in in vitro models and in synovia from Chlamydia-associated arthritis patients. Hep-2 cells infected with K serovar were harvested from 0-48 h post-infection (active infection). Human monocytes identically infected were harvested at 1, 2, 3, 5 days post-infection (persistent). RNA from each preparation and from synovial samples PCR-positive/-negative for Chlamydia DNA was subjected to RT-PCR targeting (a) chlamydial primary rRNA transcripts and adt1 mRNA, (b) chlamydial mRNA encoding enzymes of the glycolysis (pyk, gap, pgk) and pentose phosphate (gnd, tal) pathways, the TCA cycle (mdhC, fumC), electron transport system (cydA, cydB), and sigma factors (rpoD, rpsD, rpoN). Primary rRNA transcripts and adt1 mRNA were present in each infected preparation and patient sample; controls were negative for chlamydial RNA. In infected Hep-2 cells, all energy transduction-related genes were expressed by approximately 11 h post-infection. In monocytes, pyk, gap, pgk, gnd, tal, cydA mRNA were present in 1-2-day-infected cells but absent at 3 days and after; cydB, mdhC, fumC were expressed through 5 days post-infection. RT-PCR targeting mRNA from sigma factor genes indicated that lack of these gene products cannot explain selective transcriptional down-regulation during persistence. Analyses of RNA from synovial tissues mirrored those from the monocyte system. These data suggest that in the first phase of active chlamydial infection, ADP/ATP exchange provides energy required for metabolism; in active growth, glycolysis supplements host ATP. In persistence host, rather than bacterially produced, ATP is the primary energy source. Metabolic rate in persistent C. trachomatis is lower than in actively growing cells, as judged from assays for relative chlamydial primary rRNA transcript levels in persistent vs. actively growing cells.