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Evaluation of the Ribosomal Protein S1 Gene (rpsA) as a Novel Biomarker for Mycobacterium Species Identification.

ABSTRACT: To evaluate the resolution and reliability of the rpsA gene, encoding ribosomal protein S1, as a novel biomarker for mycobacteria species identification.A segment of the rpsA gene (565?bp) was amplified by PCR from 42 mycobacterial reference strains, 172 nontuberculosis mycobacteria clinical isolates, and 16 M. tuberculosis complex clinical isolates. The PCR products were sequenced and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method.Comparative sequence analysis of the rpsA gene provided the basis for species differentiation within the genus Mycobacterium. Slow- and rapid-growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. The sequences discrepancy was obvious between M. kansasii and M. gastri, M. chelonae and M. abscessus, M. avium and M. intracellulare, and M. szulgai and M. malmoense, which cannot be achieved by 16S ribosomal DNA (rDNA) homologue genes comparison. 183 of the 188 (97.3%) clinical isolates, consisting of 8 mycobacterial species, were identified correctly by rpsA gene blast.Our study indicates that rpsA sequencing can be used effectively for mycobacteria species identification as a supplement to 16S rDNA sequence analysis.

SUBMITTER: Duan H

PROVIDER: S-EPMC4402190 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Evaluation of the Ribosomal Protein S1 Gene (rpsA) as a Novel Biomarker for Mycobacterium Species Identification.

Duan Hongfei H Liu Guan G Wang Xiaobo X Fu Yuhong Y Liang Qian Q Shang Yuanyuan Y Chu Naihui N Huang Hairong H

BioMed research international 20150405

<h4>Objectives</h4>To evaluate the resolution and reliability of the rpsA gene, encoding ribosomal protein S1, as a novel biomarker for mycobacteria species identification.<h4>Methods</h4>A segment of the rpsA gene (565 bp) was amplified by PCR from 42 mycobacterial reference strains, 172 nontuberculosis mycobacteria clinical isolates, and 16 M. tuberculosis complex clinical isolates. The PCR products were sequenced and aligned by using the multiple alignment algorithm in the MegAlign package (D ...[more]

PMID: 25945329

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Project description:Resistance to key first-line drugs is a major hurdle to achieve the global end tuberculosis (TB) targets. A prodrug, pyrazinamide (PZA) is the only drug, effective in latent TB, recommended in drug resistance and susceptible Mycobacterium tuberculosis (MTB) isolates. The prodrug conversion into active form, pyrazinoic acid (POA), required the activity of pncA gene encoded pyrazinamidase (PZase). Although pncA mutations have been commonly associated with PZA resistance but a small number of resistance cases have been associated with mutationss in RpsA protein. Here in this study a total of 69 PZA resistance isolates have been sequenced for pncA mutations. However, samples that were found PZA resistant but pncA wild type (pncAWT), have been sequenced for rpsA and panD genes mutation. We repeated a drug susceptibility testing according to the WHO guidelines on 18 pncAWT MTB isolates. The rpsA and panD genes were sequenced. Out of total 69 PZA resistant isolates, 51 harbored 36 mutations in pncA gene (GeneBank Accession No. MH46111) while, fifteen different mutations including seven novel, were detected in the fourth S1 domain of RpsA known as C-terminal (MtRpsACTD) end. We did not detect any mutations in panD gene. Among the rpsA mutations, we investigated the molecular mechanism of resistance behind mutations, D342N, D343N, A344P, and I351F, present in the MtRpsACTD through molecular dynamic simulations (MD). WT showed a good drug binding affinity as compared to mutants (MTs), D342N, D343N, A344P, and I351F. Binding pocket volume, stability, and fluctuations have been altered whereas the total energy, protein folding, and geometric shape analysis further explored a significant variation between WT and MTs. In conclusion, mutations in MtRpsACTD might be involved to alter the RpsA activity, resulting in drug resistance. Such molecular mechanism behind resistance may provide a better insight into the resistance mechanism to achieve the global TB control targets.

Dataset Information

Evaluation of the Ribosomal Protein S1 Gene (rpsA) as a Novel Biomarker for Mycobacterium Species Identification.

Publications

Evaluation of the Ribosomal Protein S1 Gene (rpsA) as a Novel Biomarker for Mycobacterium Species Identification.

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