Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Barrett KF

PROVIDER: S-EPMC7020975 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Barrett Kayleigh F KF Dranow David M DM Phan Isabelle Q IQ Michaels Samantha A SA Shaheen Shareef S Navaluna Edelmar D ED Craig Justin K JK Tillery Logan M LM Choi Ryan R Edwards Thomas E TE Conrady Deborah G DG Abendroth Jan J Horanyi Peter S PS Lorimer Donald D DD Van Voorhis Wesley C WC Zhang Zhongsheng Z Barrett Lynn K LK Subramanian Sandhya S Staker Bart B Fan Erkang E Myler Peter J PJ Soge Olusegun O OO Hybiske Kevin K Ojo Kayode K KK

Protein science : a publication of the Protein Society 20200128 3

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 t ...[more]

PMID: 31930578

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Project description:BackgroundLaboratory detection of Human papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae in liquid-based cervicovaginal cytology specimens is now based on identification of the DNA sequences unique to these infectious agents. However, current commercial test kits rely on nucleotide probe hybridization to determine DNA sequences, which may lead to diagnostic errors due to cross-reactivity. The aim of this study was to find a practical approach to perform automated Sanger DNA sequencing in clinical laboratories for validation of the DNA tests for these three infectious agents.MethodsA crude proteinase K digest of 5% of the cells collected in a liquid-based cervicovaginal cytology specimen was used for the detection of DNA molecules specific for HPV, C trachomatis and N gonorrhoeae, and for preparation of materials suitable for direct automated DNA sequencing. Several sets of commercially available polymerase chain reaction (PCR) primers were used to prepare nested PCR amplicons for direct DNA sequencing.ResultsSome variants of HPV-16 and HPV-31 were found to share an at least 34-base long sequence homology downstream of the GP5+ binding site, and all HPV-6 and HPV-11 variants shared an upstream 34-base sequence including part of the GP5+ primer. Accurate HPV genotyping frequently required more than 34-bases for sequence alignments to distinguish some of the HPV genotype variants with closely related sequences in this L1 gene hypervariable region. Using the automated Sanger DNA sequencing method for parallel comparative studies on split samples and to retest the residues of samples previously tested positive for C trachomatis and/or for N gonorrhoeae, we also found false-negative and false-positive results as reported by two commercial nucleic acid test kits.ConclusionIdentification of a signature DNA sequence by the automated Sanger method is useful for validation of HPV genotyping and for molecular testing of C trachomatis and N gonorrhoeae in liquid-based cervicovaginal Papanicolaou (Pap) cytology specimens for clinical laboratories with experience in molecular biology to increase the specificity of these DNA-based tests. However, even a highly specific test for high-risk HPV genotyping may have unacceptably low positive predictive values for precancer lesion in populations with a low cervical cancer prevalence rate.

Project description:Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) represent the most common agents of sexually transmitted rectal infections among men having sex with other men (MSM). In this study, we assessed the bacterial composition of the rectal microbiota associated with CT and/or NG infections in a cohort of men reporting unsafe rectal intercourse. A total of 125 rectal swabs were collected and four groups were compared: non-infected subjects (n = 53), patients with CT (n = 37), or NG rectal infection (n = 17) and patients with contemporary positivity for CT/NG (n = 18). CT and NG infections were detected by a real-time commercial test and the rectal microbiota composition was analyzed from rectal swabs through sequencing of the hypervariable V3-V4 regions of the 16S rRNA gene. The rectal microbiota of all subgroups was dominated by Prevotellaceae, Enterobacteriaceae, and Ruminococcaceae families. Irrespective of the analyzed subgroup, we found that the rectal environment of all the enrolled MSM was rich in Prevotella and Escherichia genera. Moreover, a shift in the bacterial composition between patients with sexually transmitted rectal infections and controls was noticed: infected patients were characterized by a depletion of Escherichia species, associated with an increase of anaerobic genera, including Peptoniphilus, Peptostreptococcus, and Parvimonas. Overall, the presence of rectal symptoms did not significantly modify the rectal microbiota profiles among the four groups of analyzed patients. We confirmed that HIV-positive patients are characterized by a lower bacterial richness than HIV-negative subjects. However, we found that the presence of HIV has a different impact on bacterial rectal communities compared to CT and NG infections, modifying the relative abundance of several genera, including Gardnerella, Lactobacillus, Corynebacterium, and Sutterella. Information about the rectal microbiota composition in CT and NG infections could shed light on the pathogenesis of these conditions and could contribute to the onset of new strategies for their control.

Project description:BackgroundThe global burden of sexually transmitted infections (STIs) is high and there have been reports of increasing chlamydial and gonorrheal infections. High-volume screening programs for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are an important component of STI control. This study evaluated the high-volume workflow and performance of the cobas® CT/NG assay for use on the automated Roche cobas® 6800 system, with the cobas p 480 instrument for pre-analytics, compared with the Aptima Combo 2 assay on the Hologic Panther system.MethodsHigh-volume workflow and performance were evaluated using paired female urine specimens. Workflow analysis (n?=?376) included hands-on time (HoT), number of manual interventions, and time to first and last results. For performance assessment, paired results from the cobas CT/NG and Aptima Combo 2 assays, for both CT and NG, were compared and two-sided 95% confidence intervals calculated to provide estimates of positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) between the tests. McNemar's test was used for significance testing.ResultsPre-analytical preparations and system start-up on the cobas 6800 system required 00:27:38 (hr:min:sec) HoT whilst the Panther system required 00:30:43. The cobas 6800 system required eight interactions and 00:43:59 HoT to process 376 samples. The Panther system required six interactions and 00:39:10 HoT. Time to first results was 02:53:00 on the cobas c6800 system for 96 samples and 03:28:29 on the Panther system for five samples. The cobas 6800 system delivered all 376 results 3?h faster than the Panther system (07:45:26 and 10:47:30, respectively). The performance correlation between both assays was high (PPA, NPA and OPA >?99% for both CT and NG). McNemar's test revealed no statistically significant difference between the assays.ConclusionFor high-volume automated CT/NG testing, both the cobas 6800 system and Panther system provided accurate results. Although less manual intervention steps were needed for the Panther system, improved turnaround time was obtained with the cobas 6800 system with less risk for contamination. The additional testing capacity on the cobas 6800 system would allow a growing service to deliver more results in a single shift.

Dataset Information

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

Publications

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

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