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 ~  Abstract
 ~ Introduction
 ~ Patients and Methods
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 34  |  Issue : 4  |  Page : 471-475
 

Molecular analysis of Rv0679c and Rv0180c genes of Mycobacterium tuberculosis from clinical isolates of pulmonary tuberculosis


1 Department of Biotechnology, Sreenidhi Institute of Science and Technology, Hyderabad, Telangana, India
2 Microbiology Division, LEPRA Society-Blue Peter Public Health Research Center, Hyderabad, Telangana, India

Date of Submission28-May-2015
Date of Acceptance17-Jul-2016
Date of Web Publication8-Dec-2016

Correspondence Address:
KSR Sivasai
Department of Biotechnology, Sreenidhi Institute of Science and Technology, Hyderabad, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.195357

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 ~ Abstract 

Context: Two novel proteins/genes Rv0679c and Rv0180c of Mycobacterium tuberculosis (MTB) H37Rv were classified as a hypothetical membrane and transmembrane proteins which might have a role in the invasion. Molecular analysis of these genes in human clinical isolates of pulmonary tuberculosis (PTB) patients was not well characterised. Aims: To assess the molecular diversity of Rv0679c and Rv0180c genes of MTB from clinical isolates of PTB patients. Settings and Design: DNA from 97 clinical isolates was extracted and subjected to amplification using selective primers by polymerase chain reaction (PCR). The PCR product obtained was sequenced commercially. Patients and Methods: Clinical isolates obtained from tuberculosis patients were investigated for polymorphisms in the Rv0679c and Rv0180c genes by PCR and DNA sequencing. Genomic DNA isolated by cetyltrimethylammonium bromide method was used for amplification of genes. Results: Rv0679c gene was highly conserved in 61 out of 65 clinical isolates assessed for sequence homology with wild-type H37Rv gene and was identical using ClustalW. Fifty-five out of 78 (70.5%) clinical isolates assessed for Rv0180c were positive for single nucleotide polymorphism (SNP) at 258th position where the nucleotide G was replaced with T (G to T). In clinical isolates of untreated cases, the frequency was 54.5% for SNP at 258th position which is low compared to cases undergoing treatment where the frequency was 73.1%. Conclusions: Molecular analysis of Rv0180c in clinical isolates of PTB assessed in this study was the first report, where an SNP at 258th position G to T was identified within the gene. Rv0679c gene was highly conserved (94%), within Indian clinical isolates as compared to reports from other nations.


Keywords: Polymerase chain reaction, sequencing, single nucleotide polymorphism


How to cite this article:
Rupa L, Srikantam A, Lakshmana Rao S S, Devi U, Sivasai K. Molecular analysis of Rv0679c and Rv0180c genes of Mycobacterium tuberculosis from clinical isolates of pulmonary tuberculosis. Indian J Med Microbiol 2016;34:471-5

How to cite this URL:
Rupa L, Srikantam A, Lakshmana Rao S S, Devi U, Sivasai K. Molecular analysis of Rv0679c and Rv0180c genes of Mycobacterium tuberculosis from clinical isolates of pulmonary tuberculosis. Indian J Med Microbiol [serial online] 2016 [cited 2017 Mar 28];34:471-5. Available from: http://www.ijmm.org/text.asp?2016/34/4/471/195357



 ~ Introduction Top


Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) attributes for leading cause of human mortality worldwide. In 2013, alone around nine million of new cases diagnosed and 1.5 million died of TB.[1] The World Health Organization reported that the multidrug-resistant-TB prevalence is increasing, and it was 3.4% in new and 20.5% in previously treated TB cases.[1]

The MTB, apart from high conservation,[2],[3],[4],[5],[6] a large number of long sequence polymorphisms, a variation in repetitive elements, and single nucleotide polymorphisms (SNPs) have been detected in the genome.[7],[8] Some of these mutations caused drug resistance.[9],[10],[11] Variation among MTB strains genotypically [12] and human genetic polymorphism linked to TB have resulted in the changing relationship between MTB and the human host and further complicated TB control efforts.[13]

SNPs can affect the bacterial phenotype in various ways, which may have an impact on the outcome of TB infection and disease. Assessment on clinical isolates from seven different MTB lineages/genotypes evolved leading to the influence of strain genetic background on transmissibility, clinical presentation/outcome and resistance development.[14]

High-resolution genotyping for characterisation of strains in larger studies is mandatory for understanding the mechanisms of host–pathogen interaction and to improve TB control. The present study aims to analyse the polymorphism status of two novel genes Rv0679c and Rv0180c in clinical isolates from pulmonary TB (PTB).

The Rv0679c gene of MTB was 498 bp, and its protein was classified as a hypothetical membrane protein of the cell envelope. Its protein homologue in Mycobacterium bovis Bacillus Calmette–Guérin was a putative lipoprotein that has been shown to be tightly associated to lipoarabinomannan.[15] Cifuentes et al.[16] demonstrated the presence of this gene in H37Rv and H37Ra genome and also reported the possibility of transcription, which were not observed in other mycobacterial spp. They reported that Rv0679c proteins might have a role in the invasion of pathogen because the truncated peptides of Rv0679c could inhibit the binding and uptake of MTB by epithelial and macrophage cell lines. Recently, Nakajima et al.[17] reported that a specific mutation in Rv0679c enabled them in identifying Beijing family lineage-specific MTB isolates and developed a simple multiplex polymerase chain reaction (PCR) assay for characterisation.

The Rv0180c gene of MTB was 1359 bp, and its protein was shown to be conserved transmembrane protein of unknown function that shares high sequence similarity with putative conserved membrane and transmembrane proteins, Mb0186c from M. bovis and ML2600c from M. leprae and other mycobacterial spp.[18] Cáceres et al.[18] demonstrated that the peptides of Rv0180c were capable of inhibiting mycobacterial entry into host cells indicating potential candidates for anti-TB vaccine. However, both studies were carried out using cell lines.

Apart from this as of date, there are not many reports on molecular characterisation status of Rv0679c and Rv0180c genes of MTB from clinical isolates. In light of above, we were interested in molecular characterisation of Rv0679c and Rv0180c genes of MTB from clinical isolates obtained from PTB patients for identification of SNPs.


 ~ Patients and Methods Top


Clinical isolates

Ninety-seven clinical isolates obtained from sputum of PTB patients, which were confirmed by culture were included in the study. The Rv0679c and Rv0180c genes were assessed in 65 and 78 isolates, respectively, where 46 are common for both the gene analysis. Clinical isolates were provided by Blue Peter Research Centre (LEPRA Society, India), and the study was approved by the Institutional Ethics Committee of Cherlapally, Hyderabad. Clinical isolates were obtained and grouped into untreated and cases under treatment for PTB.

Extraction of genomic DNA from Mycobacterium tuberculosis isolates

Genomic DNA was isolated from clinical isolates using cetyltrimethylammonium bromide method as described by Del Portillo et al.[19] The concentration was determined by measuring OD at 260 nm and purity by assessing the ratio between 260/280.

Rv0679c and Rv0180c gene amplification by polymerase chain reaction

We assessed for SNPs in partially amplified region of genes which are shown in [Table 1]. Amplification of selected regions of Rv0679c and Rv0180c gene was carried out using genomic DNA isolated from clinical isolates. The primers used for amplification of genes and methodologies followed were of Cifuentes et al.,[16] and Cáceres et al.,[18] respectively. Briefly, genes of Rv0679c and Rv0180c were subjected to amplification by primers [Table 1], and the expected amplified product is around 346 bp for both the genes. The amplified product was a partially amplified product of both the genes from the genome of MTB isolates [Table 1]. PCR was carried out in a thermocycler (Bio-Rad, California, USA) where reaction mixture contains 1 µL of 5 pmol of each primer, 22 µL of molecular grade water, 1 µL of genomic DNA and 25 µL of 2× master mix (Bangalore Genei, Bangalore, India) with Taq DNA Polymerase, dNTPs and an optimum reaction buffer, in a total volume of 50 µL. The amplification conditions used were same for both the genes, 5 min at 95°C followed by 30 cycles of 94°C for 1 min for dissociation; 72°C for 45 s for synthesis, followed by a 5 min final extension. The annealing temperatures for Rv0679c and Rv0180c gene were different and were 59°C for 1 min (Rv0679c) and 56°C for 45 s (Rv0180c), respectively. Genomic DNA of MTB H37Rv was used as control and PCR grade water was used as negative control. The 346 bp amplified PCR products of both the genes were confirmed by visualising on 2% agarose gel.
Table 1: Primers used for amplification of the Rv0679c and Rv0180c genes

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Gene sequencing for identification of single nucleotide polymorphisms

The 346 bp PCR products obtained from different clinical isolates for Rv0679c and Rv0180c genes were confirmed by agarose gel electrophoresis and then subjected to DNA sequencing. The DNA sequence was obtained by outsourcing to SciGenom Labs where ABI 3730XL sequencer was used (SciGenom Labs, Kerala, India). The obtained sequences were assessed for SNPs using ClustalW2 Programme, with the sequence of wild-type genes of MTB H37Rv as a control. ClustalW2 is a general purpose multiple sequence alignment programme for DNA, available online at http://www.ebi.ac.uk/Tools/clustalw2/index.html. It calculated the best match for the selected sequences and line them so that the identities, similarities and differences could be seen.


 ~ Results Top


Molecular analysis of a part of Rv0679c and Rv0180c genes

The expected 346 bp amplified region of Rv0679c gene was observed in all 65 clinical isolates assessed. [Figure 1]a is a representation of the PCR product obtained from clinical isolates for Rv0679c. The selectively amplified region of 346 bp was a part of 498 bp of whole Rv0679c gene which falls between 30 and 376 bp of the gene [Table 1].
Figure 1: Amplified region of Rv0679c gene (a) and Rv0180c gene (b) from seven clinical isolates of pulmonary tuberculosis patients

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Seventy-eight clinical isolates were assessed for Rv0180c gene and expected 346 bp product was observed in all. [Figure 1]b is a representation of the PCR product obtained for Rv0180c gene from isolates. The 346 bp was a part of 1359 bp of whole Rv0180c gene which falls between 28 and 374 bp of the gene [Table 1].

Forty-six clinical isolates of 65 assessed for Rv0679c gene and 78 assessed for Rv0180c gene are common for both the genes.

Identification of single nucleotide polymorphisms in amplified part of Rv0180 gene from clinical isolates

The Rv0180c gene was around 1359 bp, out of which we assessed for 346 bp region ranging from 28 to 374 sequence of the gene ([Table 1] location in H37Rv genome was 210920-211266). The amplified products of the Rv0180c gene were sequenced commercially, and homology assessment was done comparing with Rv0180c of H37Rv wild-type strain. Our homology analysis revealed an SNP at location 258th position of Rv0180c gene. Fifty-five out of 78 (70.5%) clinical isolates assessed were positive for SNP at 258th position where the nucleotide G was replaced with T (G258T) [Table 2]. Out of 55, 6 belong to untreated cases and 49 belong to undergoing treatment cases Another SNP was observed at 292th position where nucleotide A was replaced with C (A to C) in only one isolate from untreated cases. The SNPs observed in Rv0180c gene at 258th and 292nd position correspond to the 211150 and 211184 position of the H37Rv genome. Even though the frequency of SNPs observed in untreated cases was 63.6%, the frequency for SNP at 258th position was 54.5% compared to cases undergoing treatment where the frequency was 73.1% [Table 2].
Table 2: Single nucleotide polymorphism analysis in Rv0180c and Rv0679c genes of Mycobacterium tuberculosis from clinical isolates

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Rv0679c gene of clinical isolates is comparatively conserved

The Rv0679c gene was around 498 bp, out of which we assessed for 346 bp region ranging from 30 to 376 sequence of the gene ([Table 1] location in H37Rv genome was 779573-779919). The comparative analysis between H37Rv wild-type strain and 65 clinical isolates revealed that, in 61 isolates, the sequence was identical to wild type indicating that Rv0679c was highly conserved (93.8%). Out of 61 conserved isolates, 5 belong to untreated cases and 56 belong to cases undergoing treatment.

While in four isolates, we could observe SNPs at 183, 202, 337 and 343 positions where G to C, G to A, A to C and G to A, nucleotide change, respectively. These locations are equivalent to 779726, 779745, 779880 and 779886, of the genome of H37Rv wild-type strain [Table 2]. All these four clinical isolates belong to the people who are undergoing treatment as shown in [Table 2].


 ~ Discussion Top


With the objective of molecular analysis of Rv0679c and Rv0180c genes in clinical isolates, we observed that the Rv0679c was highly conserved (93.8%). In contrast, an SNP of G258T was observed in Rv0180c gene in 70.5% of the isolates. The Rv0679c and Rv0180c proteins of MTB have been predicted to be the membrane proteins and might play a role in host cell invasion. Thus, identified SNPs in the genes may have an impact on the expressed proteins leading to either alteration in the amount of protein or change of function in terms of host cell invasion.

Strain variation in MTB complex was shown to play a role in the outcome of TB infection and disease.[3] Genomic diversity in the strains of MTB complex was an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Earlier studies by Gao et al.[4] revealed that clinical MTB isolates have variable gene expression profiles and have different numbers of genes deleted from their chromosome.[6] Molecular epidemiological studies in human infections have indicated that certain MTB types, identified by DNA fingerprinting, could be especially prone to drug resistance acquisition.[20]

In our study, we amplified a part of the Rv0679c and Rv0180c genes for assessment but not complete genes [Table 1]. We got the expected 346 bp product from all the isolates assessed, which were observed by Cifuentes et al.,[16] and Cáceres et al.,[18] in their study in different mycobacterial spp. expect Mycobacterium canettii. The outcome of Cifuentes et al.,[16] Caceres et al.,[18] Nakajima et al.[17] and our study indicates that both these genes are present in the majority of mycobacterial spp. and MTB obtained from clinical isolates.

To assess the molecular diversity of Rv0679c and Rv0180c genes in MTB obtained from clinical isolates, we sequenced the PCR products and assessed for homology alignment with the wild type sequences of both the genes from H37Rv. Our study revealed that the Rv0679c was highly conserved in 93.8% of the isolates. Nakajima et al.[17] in their study using Rv0679c gene sequence analysis of clinical isolates collected from Japan, Bangladesh and Nepal reported that the gene was conserved in lineage-specific backgrounds except Beijing genotype family. They reported that an SNP C to G at position 426, of Rv0679c was lineage-specific mutation observed only in members of the Beijing genotype family. In our study, the part of Rv0679c gene amplified was between 30 and 376 bp; we could not comment whether this SNP was there in our isolates. Studies are ongoing to amplify the full gene and assess more number of isolates to establish further. However, a report by Thomas et al.,[21] on lineages of modern and ancestral genotypes of Andhra Pradesh suggested that the lineage was predominantly EAI and CAS with Beijing genotype was less. With this in background, we speculate that 426 SNP observed by Nakajima et al.[17] might not be possible in the majority of our clinical isolates assessed. Apart from high conservation of Rv0679c gene in isolates, Nakajima et al.[17] reported unique SNPs in three of the 66 MTC strains assessed. MTB strains C has an SNP C to T at position 185, T17 has an SNP cytosine was inserted at position 92 and M. canettii CIPT 140010059, two SNPs and a codon insertion, ACC at position 154. In our study, on Rv0679c gene analysis, we could not find any of the above SNPs in our isolates. Interestingly, we observed different SNPs in four clinical isolates at locations 183, 202, 337 and 343 where nucleotide G was replaced by C, G to A, A to C and G to A, respectively [Table 2].

Molecular analysis of a part of Rv0180c gene in our study revealed an SNP G to T at position 258 in 70.5% of clinical isolates. The Rv0180c protein expression was confirmed as cell membrane protein;[16] however, its function was not well characterised. Caceres et al.[18] demonstrated that Rv0180c protein could play a role in the host cell invasion. One would expect that the SNP observed at the gene level in our study may have a beneficial or detrimental effect. Studies are designed to assess this effect and are ongoing. We also observed another SNP at position 292 where A was replaced by C in one clinical isolate.

Another interesting observation of the study was that the frequency of SNP G to T at 258th position was high (73.1%) in isolates from cases undergoing treatment compared to untreated cases which was 54.5%. The difference in the frequency observed was interesting; however, statistical analysis was not carried out as the sample size was small. This observation suggests a possibility that it may be associated with how individuals respond to drugs either leading to susceptibility or resistance. Studies are ongoing to amplify the full gene and assess more number of isolates to establish the significance of identified SNPs and also in identifying new SNPs in the rest of the gene. This sought of study would help in drawing a relationship between SNP observed confers decrease or increase in the risk of contracting PTB and its significance in a diseased situation.


 ~ Conclusion Top


Ours is the first report on identifying an SNP G258T in novel Rv0180c gene in clinical isolates obtained from PTB patients globally, apart from Rv0679c gene which was highly conserved in Indian clinical isolates.

Financial support and sponsorship

We thank Indian Council of Medical Research, New Delhi, India, for Senior Research Fellowship assistance to Lavarti Rupa. We are grateful to the management and administration of Sreenidhi Institute of Science and Technology and TEQIP-II Programme, World Bank Scheme, for financial support of the study.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Coscolla M, Gagneux S. Does M. tuberculosis genomic diversity explain disease diversity? Drug Discov Today Dis Mech 2010;7:e43-59.  Back to cited text no. 3
    
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Hirsh AE, Tsolaki AG, DeRiemer K, Feldman MW, Small PM. Stable association between strains of Mycobacterium tuberculosis and their human host populations. Proc Natl Acad Sci U S A 2004;101:4871-6.  Back to cited text no. 5
    
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Cox HS, Kubica T, Doshetov D, Kebede Y, Rüsch-Gerdess S, Niemann S. The Beijing genotype and drug resistant tuberculosis in the Aral Sea region of Central Asia. Respir Res 2005;6:134.  Back to cited text no. 9
    
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Filliol I, Motiwala AS, Cavatore M, Qi W, Hazbón MH, Bobadilla del Valle M, et al. Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: Insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set. J Bacteriol 2006;188:759-72.  Back to cited text no. 12
    
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Dye C, Williams BG. The population dynamics and control of tuberculosis. Science 2010;328:856-61.  Back to cited text no. 13
    
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Coscolla M, Gagneux S. Consequences of genomic diversity in Mycobacterium tuberculosis. Semin Immunol 2014;26:431-44.  Back to cited text no. 14
    
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Matsuba T, Suzuki Y, Tanaka Y. Association of the Rv0679c protein with lipids and carbohydrates in Mycobacterium tuberculosis/Mycobacterium bovis BCG. Arch Microbiol 2007;187:297-311.  Back to cited text no. 15
    
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Cifuentes DP, Ocampo M, Curtidor H, Vanegas M, Forero M, Patarroyo ME, et al. Mycobacterium tuberculosis Rv0679c protein sequences involved in host-cell infection: Potential TB vaccine candidate antigen. BMC Microbiol 2010;10:109.  Back to cited text no. 16
    
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Nakajima C, Tamaru A, Rahim Z, Poudel A, Maharjan B, Khin Saw Aye, et al. Simple multiplex PCR assay for identification of Beijing family Mycobacterium tuberculosis isolates with a lineage-specific mutation in Rv0679c. J Clin Microbiol 2013;51:2025-32.  Back to cited text no. 17
    
18.
Cáceres SM, Ocampo M, Arévalo-Pinzón G, Jimenez RA, Patarroyo ME, Patarroyo MA. The Mycobacterium tuberculosis membrane protein Rv0180c: Evaluation of peptide sequences implicated in mycobacterial invasion of two human cell lines. Peptides 2011;32:1-10.  Back to cited text no. 18
    
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21.
Thomas SK, Iravatham CC, Moni BH, Kumar A, Archana BV, Majid M, et al. Modern and ancestral genotypes of Mycobacterium tuberculosis from Andhra Pradesh, India. PLoS One 2011;6:e27584.  Back to cited text no. 21
    


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