Indian Journal of Dermatology
ORIGINAL ARTICLE
Year
: 2015  |  Volume : 60  |  Issue : 4  |  Page : 351--355

Unique TTC repeat base pair loss mutation in cases of pure neural leprosy: A survival strategy of Mycobacterium leprae?


Abhishek De1, Abu Hena Hasanoor Reja2, Supratik Biswas2, Basudev Bhattacharya2, Gobinda Chatterjee1, Keya Basu3, Aarti Sarda4, Nidhi Chowdhury1,  
1 Department of Dermatology, Institute of Post-Graduate Medical Education and Research (IPGMER), Kolkata, West Bengal, India
2 Department of Biochemistry, Institute of Post-Graduate Medical Education and Research (IPGMER), Kolkata, West Bengal, India
3 Department of Pathology, Institute of Post-Graduate Medical Education and Research (IPGMER), Kolkata, West Bengal, India
4 Department of Dermatology, KPC Medical College, Kolkata, West Bengal, India

Correspondence Address:
Dr. Abhishek De
242 AJC Bose Road, Bhowanipur, Kolkata - 700 020, West Bengal
India

Abstract

Background: Genomic reduction helps obligate intracellular microbes to survive difficult host niches. Adaptation of Mycobacterium leprae in cases of pure neural leprosy (PNL) in the intracellular niche of peripheral nerves can be associated with some gene loss. Recently, a stable but variable number of tandem repefzats (TTC) have been reported in strains of M. leprae. FolP and rpoB genes are the two common mutation sites which deal with the susceptibility of the bacteria to drugs. Aim: We attempted to find if genomic reduction of M. leprae in context of these TTC repeats or mutations in folP1 and rpoB can be the reason for the restriction of M. leprae in the nerves in PNL. Materials and Methods: DNA extracts taken from fine needle aspiration of affected nerves of 24 PNL cases were studied for tandem repeats with 21TTC primer in multiplex-PCR. Mutations were also studied by PCR Amplification of SRDR (Sulphone Resistance Determining Region) of the folP1 and multiple primer PCR amplification refractory mutation system (MARS) of the rpoB. Results: Of the 24 PNL, only 1 patient showed mutation in the rpoB gene and none in the folp1 gene. Studying the mutation in TTC region of the M. leprae gene we found that all the cases have a loss of a few bases in the sequence. Conclusion: We can conclude that there is consistent loss in the bases in the TTC region in all cases of pure neural Hansen and we postulate that it may be an adaptive response of the bacteria to survive host niche resulting in its restriction to peripheral nerves.



How to cite this article:
De A, Hasanoor Reja AH, Biswas S, Bhattacharya B, Chatterjee G, Basu K, Sarda A, Chowdhury N. Unique TTC repeat base pair loss mutation in cases of pure neural leprosy: A survival strategy of Mycobacterium leprae?.Indian J Dermatol 2015;60:351-355


How to cite this URL:
De A, Hasanoor Reja AH, Biswas S, Bhattacharya B, Chatterjee G, Basu K, Sarda A, Chowdhury N. Unique TTC repeat base pair loss mutation in cases of pure neural leprosy: A survival strategy of Mycobacterium leprae?. Indian J Dermatol [serial online] 2015 [cited 2019 Sep 22 ];60:351-355
Available from: http://www.e-ijd.org/text.asp?2015/60/4/351/160478


Full Text

 Introduction



Unlike other mycobacteria, Mycobacterium leprae is unique in the fact that it is an obligate intracellular parasite and serves as excellent model to study how these bacteria exploit the functions of their host cells. Obligatory intracellular parasites possess a small genome and have a tendency for genomic reduction. Genome reduction is a common phenomenon of these obligatory intracellular parasites that target overlapping subsets of potentially dispensable genes while adapting to the selective pressure of different niches. [1] However, the deletion of genes in pathogens may also have a detrimental effect on their fitness and on their ability to cause disease. [2]

TTC repeat is a variable number of tandem repeat stable and specific for a strain of M. leprae. This has been found to be 21 in cases of non-mutant strains. [2] Also, two folP homologs folP1 and folP2 have been found to be important to the bacterial susceptibility to the treatment. [3]

In case of Pure Neural Hansen (PNL), M. leprae is restricted to the peripheral nerves. Recently, the authors have described a simple but objective method of diagnosis of PNL by using fine needle aspiration and multiplex PCR. [4] However, we perceive that there is a considerable lack of data regarding the genomic changes in M. leprae which confines itself to a low grade infection of peripheral nerves for years. We postulate that this adaptation to this intra-cellular niche may be accompanied by either gene loss or mutation in susceptibility genes.

In this study we attempted to find if there is a consistent pattern of gene loss at TTC tandem repeats or susceptibility mutation in either folP or rpoB genes which may be the reason for the restriction and survival of M. leprae in the nerves of the host.

 Materials and Methods



Patients' selection criteria

All clinically suspected cases of PNL were subjected to fine-needle aspiration (FNA) from the affected nerves after duly signing an informed consent form. Aspirates were subjected to DNA extraction and multiplex PCR for detection of M. leprae. PNL cases with a confirmed microbiological diagnosis by multiplex cases were only included for further study of genomic reduction and susceptibility mutation.

Fine-needle aspiration

FNA was done as described by Theuvenet et al. [5],[6] We used a 10 ml syringe with 23-G needle for FNA cytology (FNAC). Most commonly ulnar nerve was taken for FNAC due to its easy approachability. Small amount of normal saline was first injected into the nerve for easier tapping. Aspirates were then subjected to cytology (hematoxylin and eosin), special staining with Ziehl-Neelsen and DNA extraction.

Extraction of DNA from the FNAC samples

Genomic DNA was extracted from a portion of the fine-needle aspirates, collected aseptically with the standard precautions by the standard Phenol Chloroform method after proteinase-K digestion as described by Banerjee et al. [7]

PCR amplification of TTC repeats

The primer used for region flanking entire 21TTC repeat sequences was originally designed by Shin et al. [2] A non-mutant Thai 53 strain was used as a negative control, which was previously used by other workers. [8] Both the primers were designed based on the M. leprae genome sequences flanking the TTC repeats to make sure that only the M. leprae genomic DNA would anneal with the primers [Table 1].{Table 1}

Detection of mutations in the folp1 gene

The molecualar detection of mutation in the folp1 gene of M. leprae was done as explained by Williams and Gillis. [3] Briefly this assay required PCR Amplification of SRDR (Sulphone Resistance Determining Region) of the folP1 by the primers explained in [Table 1] and mixing of these PCR products with a universal heteroduplex generator (UHG - DDS - 441). The SRDR PCR products were further analyzed for single-stranded conformational polymorphism as explained by Bannerjee et al. [7]

Detection of mutation in the rpoB gene

Molecular detection in the rpoB gene of M. leprae was determined. Multiple primer PCR Amplification Refractory mutation System (MARS) assay was done as explained by Sapkota et al. [8] Briefly, eliquotes of FNA homogenate were subjected to five cold and heat shocks, achieved by snap freezing in liquid nitrogen for 1 min followed by boiling at 100 C in a dry heat block. The crude lysate was used as template in PCR amplification by using the primers explained in [Table 1].

PCR conditions

Approximately 100 ng genomic DNA was amplified with Ampli Taq Gold (Applied Biosystems, Inc. [ABI], Foster City, CA) in a PCR reaction mixtures, containing 1x PCR buffer (Applied Biosystems), 2 mM MgCl 2 , 0.25 mM each dNTP, 20 picomoles primers TTC-A and TTC-B, folP1F and folP1R, CFP, CRP and M425. The primer sequences, primer annealing temperature (Ta°), and PCR product sizes are given in [Table 1]. The PCR reactions were performed under the following conditions: 95°C for 4 minutes, followed by 35 cycles of 95°C for 1 min, Ta° for the internal control as in [Table 1] for 1 min, 72°C for 1 min, and then 72°C for 10 minutes for the final extension. The amplified products were separated by electrophoresis on 2% agarose gel stained with 0.5 mg/mL ethidium bromide and visualized and photographed under a UV transilluminator.

 Results



A total of 33 cases were subjected to fine needle aspiration of which 24 cases were found to be positive for M. leprae in multiplex PCR as in [Table 2]. These cases were included in this study and their aspirates were subjected to genome reduction analysis at TTC tandem repeats and susceptibility mutation analysis for the genes folP and rpoB.{Table 2}

Out of the 24 cases 23 cases were males and 1 patient was female as shown in [Table 2]. In multiplex PCR study, all 24 cases were found positive for the presence of M. leprae DNA. The mutation study was done by amplifying the concerned gene by PCR and [Figure 1] further subjecting to DNA sequencing as shown in [Figure 2] and [Figure 3]. The obtained data was analyzed by NCBI Basic Local Alignment Search Tool (NCBI BLAST). On analysis of mutation pattern 15 out of 24 positive patients showed a loss of 5bp in the nucleotide sequence of TTC repeats as in [Figure 4], while the other 9 patients showed 3bp loss in the nucleotide sequence as shown in [Figure 5] and [Figure 6]. Moreover, the mutation analysis for folP1 did not show any mutation in bacterium genome in any of the patients as in [Table 2] while 1 out of 24 patients showed to have a few point mutations in the rpoB gene of the bacterium as shown in [Figure 3].{Figure 1}{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Figure 6}

 Discussion



Although neural leprosy cases were not extensively studied for the genetic and biochemical changes in the bacilli, it provokes questions regarding the unusual ability and tendency of the bacilli to proliferate inside neural tissue surviving the host defense. Many obligate inter-cellular bacteria have been found to show the phenomenon of genomic reduction which supposedly help them to adapt their particular intercellular niche. [9] We tried to find out, in these unusual subset of leprosy, if there is any changes in the bacterial genome either in the form of genomic reduction or in the form susceptibility gene mutation.

We found the rate of mutation in the mycobacterium leprae genome obtained from the fine-needle aspirate of the pure neural Hansen cases is considerably higher in the variable number tandem repeats like TTC repeats. We observed a loss of 5 base pairs in 15 cases as in [Figure 4] and loss of 3 base pair in 9 cases as shown in [Figure 4]. TTC repeat is the number of tandem repeats in M. leprae, reported first by Shin et al. as shown in [Figure 3]. It had been reported that non-mutant strain of M. leprae has 21 repeats. [2] The stability of this TTC repeat was studied and reported by Masanori Matsuoaka et al. in 2004 and was found that the copy number of the TTC repeats in each laboratory maintained isolates remain stable even after several serial passages in the nude mice footpads. [10] Our findings thus show that there is definitely some amount of genomic reduction in the bacilli affecting and restricting itself to nerves.

Also, as earlier reports by Sapkota et al. and Williams et al. suggest, some point mutations in the folp1 gene and rpoB gene may cause the change in metabolic activities of the bacterium; hence, we studied the mutation pattern in these genes. [3],[11]

Genome sequencing of M. leprae revealed that it possesses two folP homologs (folP1 and folP2). Among these two homologues folP1 appears to be part of an operon containing three other genes involved in folate biosynthesis which is similar to that of M. tuberculosis. [3] Williams et al. showed that mutation in this gene contributes to change in susceptibility of the bacterium to drugs. [3] Sapkota et al. showed the mutation in other short, highly conserved region called rifampicin resistance determining region (RRDR) of the rpoB gene effecting codon 401-427 may also contribute to susceptibility of the bacterium to drugs. [12]

We found that none of these cases had any mutation in the folP1 gene of the bacterium ruling out any possibilities of resistance against Dapson. Also, the study for the mutation in the rpoB gene showed point mutations in the rpoB gene of only one patient as shown in [Figure 2]. Thus, of the 24 cases only 1 case may have primary resistance against Rifampicin.

Matsuoaka et al. suggested that changes in TTC may result in strain difference. [10] However, it remained unclear how this change affect bacterial susceptibility and pathogenecity. Since, 23 of our 24 cases did not have any susceptibility gene mutation but all of our cases showed definite sign of genomic reduction; it raises the question if genomic reduction at TTC repeat could be the cause of this unique restriction of the bacilli in the neural tissue in the cases of pure neural Hansen disease. However, further studies with larger number of cases and with studies of detailed biochemical changes may give us further clues to understand the survival strategies of the bacteria.

 Conclusion



All cases of pure neural leprosy showed consistent genomic reduction at the TTC base pair tandem repeats which could well be the cause of this unique clinical restriction to neural tissue of this subset of leprosy.

References

1Andersson JO, Andersson SG. Insights into the evolutionary process of genome degradation. Curr Opin Genet Dev 1999;9:664-71.
2Shin YC, Lee H, Lee H, Walsh GP, Kim JD, Cho SN. Variable numbers of TTC repeats in Mycobacterium leprae DNA from leprosy patients and use in strain differentiation. J Clin Microbiol 2000;38:4535-8.
3Williams DL, Gillis TP. Molecular detection of drug resistance in Mycobacterium leprae. Lepr Rev 2004;75:118-30.
4Reja AH, De A, Biswas S, Chattopadhyay A, Chatterjee G, Bhattacharya B, et al. Use of fine needle aspirate from peripheral nerves of pure-neural leprosy for cytology and PCR to confirm the diagnosis: A pilot study. Indian J Dermatol Venereol Leprol 2013;79:789-94.
5Theuvenet WJ, Miyazaki N, Roche P, Shrestha I. Cytological needle aspiration for the diagnosis of pure neural leprosy. Indian J Lepr 1996;68:109-12.
6Theuvenet WJ, Miyazaki N, Roche P, Shrestha I. Cytological needle aspiration of the nerve for the diagnosis of pure neural leprosy. Int J Lepr Other Mycobact Dis 1993;61:597-9.
7Banerjee S, Sarkar K, Gupta S, Mahapatra PS, Gupta S, Guha S, et al. Multiplex PCR technique could be an alternative approach for early detection of leprosy among close contacts--a pilot study from India. BMC Infect Dis 2010;10:252.
8Matsuoka M, Zhang L, Budiawan T, Saeki K, Izumi S. Genotyping of Mycobacterium leprae on the basis of the polymorphism of TTC repeats for analysis of leprosy transmission. J Clin Microbiol 2004;42:741-5.
9Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson NR, Wheeler PR et al. Massive gene decay in the leprosy bacillus. Nature 2001;409:1007-11.
10Matsuoka M, Kashiwabara Y, Namisato M. A Mycobacterium leprae isolate resistant to dapson, rifampicin, ofloxacin and sparfloxacin. Int J Lepr Other Mycobact Dis 2000;68:452-5.
11Sakharkar KR, Dhar PK, Chow VT. Genomic reduction in prokaryotic obligatory intracellular parasites of humans: A comparative analysis. Int J Syst Evol Microbiol 2004;54:1937-41.
12Sapkota BR, Ranjit C, Neupane KD, Macdonald M. Development and evaluation of a novel multiple-primer PCR amplification refractory mutation system for the rapid detection of mutations conferring rifampicin resistance in codon 425 of the rpoB gene of mycobacterium leprae. J Med Microbiol 2008;57:179-84.