Relevance of advance in the genetic engineering in leprosy

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CONTINUING MEDICAL EDUCATION

Year : 1999 | Volume : 65 | Issue : 1 | Page : 9-13

Relevance of advance in the genetic engineering in leprosy

MV Katoch
,

Correspondence Address:
M V Katoch

How to cite this article:
Katoch M V. Relevance of advance in the genetic engineering in leprosy. Indian J Dermatol Venereol Leprol 1999;65:9-13

How to cite this URL:
Katoch M V. Relevance of advance in the genetic engineering in leprosy. Indian J Dermatol Venereol Leprol [serial online] 1999 [cited 2013 May 24];65:9-13. Available from: http://www.ijdvl.com/text.asp?1999/65/1/9/4741

Introduction

Advances in molecular biology have influenced every discipline of life sciences including modern medicine. Among these advances the application of recombinant DNA technology or genetic engineering is the most important. Broadly it encompasses the ability to recombine the genes with appropriate vectors (plasmids, phages and others), make them enter suitable hosts and manipulate them for variety of functions specially the production of their gene products. Today we have several such genetically engineered products available for use. These include insulin, several chemokines/cytokines etc. As M.leprae is still not cultivable in any acceptable invitro medium system one has to depend upon alternative approaches to develop methods of diagnosis and management. Molecular biological technologies have enhanced the capacity to investigate these aspects. What began as a dream 15 years ago[1] has been translated into reality to a great extent.[2] Cloning and sequencing of genes of M.leprae have provided significant newer information about specific target sequences and has led to the development of several gene probes/amplification assays for diagnosis of leprosy.[2] Besides this the cloning and expression of metabolically or immunologically relevant components has several applications which range from preparation and production of immuno-diagnostic or immunoprophylactic reagents to identification of drug sensitivity sites.

Advances in cloning ofM.leprae genes: Last decade (1980s) wintessed a major progress in the development of techniques for cloning of genes of leprosy bacillus. Construction of lambda gt 11[3], cosmid libraries of M, leprae[4] and partial library of M. leprae using plasmid vectors[5] were important landmarks. Since then various immunologically/biologically relevant genes of M. leprae have been expressed in various vector-host systems.[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] Improtant achivements are:

(i) Production of recombinant proteins: A large number of M.leprae protein antigens have been expressed in lambdagt/ E.coli as well as other systems and have been immunologically characterized.[6][7][8][9][10][11][12][13][14][15][16][17][18][19] Some of these antigens have also been explored for their role in immunological responses [6][7][8][9][10][11][12][13],[15][16][17][18][19][20][21] and have been found to be highly immunodominant for both T and B cell responses.[6][7] Some have species and genus specific epitopes. One protein named as LS Rantigen has been shown to have epitopes which are preferentially recognized by ENL cases.[17] Most of these gene products are thought to be potentially useful for generation of immunodiagnostic and or immunoprophylactic reagents but none except for LSR protein have been adequately explored for clinical application. As most of these artificially expressed proteins belong to heat shock families, these may have limited scope as immunoprophylactic reagents because of fear of autoimmunity. Unlike many other pathogens, these recombinant proteins have not become established as alternate reagents for serology or substitute to lepromin/leprosin.

(ii) Biologically improtant genes: Recombinant DNA techniques have helped in identification of several new genes related to physiology of the M.leprae in terms of its multiplication, interaction with the host, drug action and intracellular survival. These include genes encoding for many important cell surface proteins, drug action related sites (ahpC, katG etc) genes associated with growth (thioredoxin reductase, ori C etc) and other regulatory proteins.[22-24] Practical application of these advances need to be investigated in future.

(iii) Mycobacterium as a host for gene cloning-genetically engineered vaccines:

Experience with cloning of genes in E.coli has been a major success but only a few mycobacterial genes have been expressed in E.coli. This led to the interest on other expression systems including streptomyces and mycobacteria. Introduction of foreign DNA using a shuttle phagemid has been reported in mycobacteria and replication of E coli plasmids in mycobacteria has been observed.[25] Vaccinia as well as mycobacterial vectors have also been described to be useful for cloning and expression of M.leprae genes.[26] It is believed that the ability to infect BCG vaccine strain with the shuttle vectors would be important for expression of genes of slow growing mycobacterial recombinant multivaccine vehicle.[26] These are encouraging reports and the impact of these advances on devising newer immunodiagnostic agents or immunoprophylactic agents is being ascertained.

Impact of genetic engineering methods:

While several genetic engineering methods to manipulate and investigate the genes of M.lepre have been standardized and applied, we have hardly any new generation of reagent(s) produced by this technology which is available for use. At present the reasons appear to belong to the following categories.

(a) Felt needs: From the success of public health campaigns of mass usage of multi-drug therapy (MDT) during the last decade it was felt that there is no problem about the diagnosis and treatment of leprosy. This led to thinking that there is no need for the application of these new technologies. As a result there was major decline in the interest in this area. Despite good initial success with the production of a few M.lepre proteins, very slow progress has been recently reported. Continued trasmission of leprosy despite mass MDT as indicated by high incidence rates has some sobering effect and it is hoped that the interest may be revived. In the emerging scenario, leprosy cases with low bacillary load are being detcted which are not suitable for production of lepromin of human origin. Further the number of colonies of animals (armadillos/nude mice) suitable for mass growth of M.leprae is dwindling. Thus we will have to depend upon genetic engineering methods for production of M.leprae specific components and this makes the technology even more relevant in future.

(b) Technical limitation: Besides the above factors, there are several technical limitations which need to be overcome for optimum use of this technology:

(i) Immunologically/therapeutically relevant antigens: One of the biggest bottlenecks in the exploitation of recombinant DNA technology has been the inadequate knowledge about the antigens of M.leprae which are important for protective immunity. The antigens which were readily expressed have been dissected off. [6, 7] As most of the genetically engineered proteins belonged to heat shock/stress families, these have not been found attractive either as immunodiagnostic or immunoprophylactic reagents.[27] With definitive idea of antigens of interest one is almost hunting for the unkown target(s). This technology can help in mass production but cannot help in identification of targets. These are to be identified by clinical and immunological investigations. It needs to be emphasized that the search for antigens which are of immunological interest should be vigorously pursued. Only then one can aim exploiting the recombinant technology to maximum.

Most of the currently available sero-assays in leprosy are handicapped as antibodies continued to persist for a long time after disease subsidence due to persistence of several of these antigens. This limits their application for monitoring the disease process. It would be better if sero assays are directed toward some component associated with live bacilli. Mycobacterial anzymes could be attractive, antigens for such purposes. For example superoxide dismutases from different mycobacteria including M.leprae have been immunologically characterized and divergences have been found.[28] Besides the mycobacterial enzymes, secretory proteins have been considered as important potential targets for immunological purposes.[29][30] Some such secretory proteins which are expected to be better indicators of presence of viable organisms have been identified. These are, however, all in the preliminary stages. The strategies for identification of unique specific fragments/specific amino acid sequences, their production by recombinant DNA technique and finally their application in measuring the immunological responses need to be further pursued.

(ii) Resistance/persistence: With the widespread application of MDT, the problem of drug resistance in leprosy appears to be under control. With the knowledge about the genes responsible for drug resistance studies to detect resistance mutants to rifampicin and may be other drugs from the biopsies can be planned using molecular methods.[31] Expression of target enzymes or molecules would help in understanding the mechanism(s) of drug sensitivity/resistance,[31][32] search of new drugs as well as development of new generation drug screening system(s).[33] Similarly molecular approaches may help in understanding the phenomenon of dormancy and persister stage.[34] Differential expression of genes in a active/dormant state and studying the effect of knocking off particular gene can help in understanding its functional role. The recombinant DNA technology has not been adequately exploited for this purpose.

(III) Sequencing of the genome of M.leprae: In the initial phase genetic engineering methods helped in generation of information about several important gene sequences of leprosy bacillus. Development of PCR based sequencing and other related methods subsequently have provided newer capabilities to sequence major part or eventually total genome of M.leprae. This started as an ambitious project even before M.tuberculosis but then the priority shifted to tuberculosis. During 1998, total genome sequence of M.tuberculosis has been published but work on M.leprae genome has become apparently slow. Neverthless this has been progressing and is perhaps nearing completion. Already significant newer information from this project and other related studies in this direction has become available.35 The information generated from the genome sequencing and the resultant improved undersrtanding of gene functions will hopefully stimulate the work in this area and we have genetically engineered products of M.leprae in the next millenium as replacement to native antigens such as lepromin/leprosin etc of animal/ human origin[35].

References

1.	Katoch VM. Recombinant DNA technology and its application to leprosy research. Indian J Lepr 1987;59:231-237.
2.	Katoch VM. New investigative techniques in leprosy. In: RG Valia, AR Valia Eds., Dermatology Update, Mumbai:Bhalani Publishing House, 1998:165-174.
3.	Young RA, Mehra V, Sweetser O, et al. Genes for major protein antigens of leprosy parasite Mycobacterium leprae. Nature 1985;316:450-452.
4.	Clark-Curtis JE, Jacobs WR, Docherty MA, Ritchie LR, Curtis R. Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. J Bacteriol 1985;161:1093-1102.
5.	Khandekar PS, Munshi A, Sinha S, et al. Construction of genomic libraries of mycobacterial origin; Identification of recombinants encoding mycobacterial specific protein. Int J Lepr 1986;54:416-421. [PUBMED] [FULLTEXT]
6.	Young DB, Gaebe T, Lathigra R, et al. Protein antigens: structure, function and regulation. In: McFadden JJ Ed, Molecular Biology of Mycobacteria. Surrey: Surrey University Press, 1990:1-35.
7.	Katoch VM, Bharadwaj VP, Katoch K. Towards production of immunodiagnostic reagents for leprosy by recombinant DNA techniques. CJIL Quarter Bull 1993;XII (1):1-5.
8.	Booth RJ, Harris DP, Love JM, et al. Antigenic proteins of M. leprae. Complete sequence of gene for 18 kDa protein. J Immunol 1988;140:597-601. [PUBMED] [FULLTEXT]
9.	Dockrell HM, Stocker NG, Lee SP, et al. T cell recognition of the 18 kilodalton Mycobacterial antigen. Infect Immun 1989;57:1979-1983.
10.	Lamb FI, Kingston AE, Iris-Estrada G, et al. Heterologous expression of the 65 kDa antigen of Mycobacterium leprae and murine T-cell responses to the gene product. Infect Immun 1988;56:1237-1241.
11.	Mustafa AS, Gill HK, Nerland A, et al. Human T cells recognise a major M. leprae protein antigen expressed in E coli. Nature 1986;319:63-66.
12.	Praputpittaya K, Ivanyi J. Study of idiotypes expressed by monoclonal antibodies to the 35 kD and 12 kD antigens of Mycobacterium leprae. Clin Exp Immun 1987;70:298-306.
13.	Thole JER, Stabel Fem, Suykerbuyk MEG, et al. A major immunogenic 36,000 molecular weight antigen from Mycobacterium leprae contains an immunoreactive region of proline-rich repeats. Infect Immun 1990;58:80-87.
14.	Thangaraj HS, lamb FL, Davis EO, et al. Identification, sequencing and expression of Mycobacterium leprae superoxide dismutase, a major antigen. Infect Immun 1990;58:1937-1942.
15.	Vega-Lopez F, Stoker NG, Lackniskar MF, et al. Recognition of mycobacterial antigen by sera from patients with leprosy. J Clin Microbiol 1988;26:2474-2479.
16.	Mehra V, Bloom BR, Bjardi AC, et al. A major T-cell antigen of Mycobacterium leprae is a 10 kDa heat shock cognate protein. J Exp Med 1992;175:275-284.
17.	Singh S, Shankarnarayan NP, Jenner PJ, et al. Sera of leprosy patients with type 2 reactions recognize sequences in Mycobacterium leprae recombinant LSR protein. Infect Immun 1994;62:86-90.
18.	Ilangumaran S, Shankaranarayan NP, Ramu G, et al. Antibody responses to recombinant 65 kDa, 70 kDa and 18 kDa mycobacterial antigens in leprosy patients and healthy contacts. Int J Lepr 1994;62:245-255.
19.	Pessolani MCV, Brennan PJ. Molecular definition and identification of new proteins of Mycobacterium leprae. Infect Immun 1996;64:5425-5427.
20.	Oskam L, Hartskeeri RA, Hermans CJ, et al. A 46 kDa integral membrane protein for Mycobacterium leprae resembles a number of bacterial and mammalian membrane target proteins. Microbiology 1995;141:1963-1968.
21.	Wiels B, Spierings E, Vannoort J, et al. Molecular characterization and T-cell stimulatory capacity of Mycobacterium leprae antigen T5. Infect Immun 1995;63:4682-4685.
22.	Nakata N, Matsuoka M, Kashiwabara Y, et al. Nucleotide sequence of the Mycobacterium leprae kat G region. J Bacteriol 1997;179:3053-3057.
23.	Wieles B, Ottenhoff THM, Steenwijk TM, et al. Increased intracellular survival of Mycobacterium smegmatis containing Mycobacterium leprae thioredoxin reductase gene. Infect Immun 1997;65:2537-2541.
24.	Fsihi H, De Rossi E, Salazar L, et al. Gene arrangement and organisation in a 76 kb gragment encompassing the ori C region of chromosome of Mycobacterium leprae. Microbiology 1996;142:3147-3161.
25.	Husson RN, James BE, Young RA. Gene replacement and expression of foreign DNA in mycobacteria. J Bacteriol 1990;172:519-524.
26.	Baumgart KW, Mckenzie KR, Radford AJ, et al. Immunogenecity and protection studies with recombinant mycobacteria and vaccinia vectors coexpressing 18 kDa protein of Mycobacterium leprae. Infect immun 1996;64:2274-2281.
27.	Sengupta U. Recombinant antigens: current situation and future. Indian J Lepr 1999;71:111-120.
28.	Shivannavar CT, Katoch VM, Sharma VD, et al. Determination of mycobacterial phylogeny by immunological relatedness of superoxide dismutases. Int J Syst Bacteriol 1996;46:1164-1169.
29.	Oflung F, Wiker HG, Deggerdal A, et al. A novel mycobacterial antigen relevant to cellular immunity belongs to a family of secreted lipoproteins. Scand J Immunol 1997;46:445-452.
30.	Harboe M, Wiker HG. Searching for secretory proteins of Mycobacterium leprae. Indian J lepr 1999;71:19-36.
31.	Honoroe N, Cole ST. Molecular basis of rifampicin resistance in Mycobacterium leprae. Antimicrob Agents Chemother 1993;37:414-417.
32.	Gillis TP, Williams DL. Dapsone resistance does not appear to be associated with a mutation in dihydropteroate synthase-2 gene in Mycobacterium leprae. Indian J lepr 1999;71:11-18.
33.	Katoch VM. Mechanisms of drug resistance in mycobacteria. In: RL Singhal and OP Sood Eds, Drug Resistance: Mehanisms and Management, Proc IV Annual Ranbaxy Symposium, New Delhi, Communicore, 1997:41-47.
34.	Gupta UD, Katoch VM. Understanding the phenomenon of persistence in mycobacterial infections. Indian J Lepr 1997;69:385-393.
35.	Smith DR, Richterich P, Rubenfield M, et al. Multiplex sequencing of 1.5 MB of the Mycobacterium leprae genome. Genome Res 1997;7:802-819.

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