Review ArticleIndian J Med Res 120, October 2004, pp 207-212

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Mycobacterium tuberculosis is reputed to have thehighest annual global mortality among all of thepathogens1. The rise in tuberculosis (TB) incidenceover the last two decades is partly due to TB deathsin HIV-infected patients and partly due to theemergence of multidrug resistant strains of thebacteria. This rapid increase in the disease has led topotential funding arrangements aimed at a large efforttowards stopping this disease before it becomes aglobal epidemic. Due to its slow growth and highvirulence it is extremely difficult to work with theTB bacterium. However, rapidly evolvingmycobacterial genomics with complete genomesequences known along with powerful bioinformaticsapproaches, one can realize better therapeutics andprophylactics in the near future2. A comparativegenomic analysis of these species has a potential toreveal the genetic basis of disease phenotypes, whichwill be invaluable for the development of muchneeded drugs and newer vaccines.

Problem of multi drug resistant tuberculosis(MDR-TB)

One of the classical threats of the tuberculosisepidemic has been the MDR-TB. Use, and often abuseor misuse, of antimicrobial agents has encouragedthe evolution of bacteria toward resistance, resultingoften in therapeutic failure. There are evidences thatbacteria have the ability to adapt to this deficit andrecover fitness on serial passage1. Resistance toantituberculosis drugs has been a problem since theera of chemotherapy began. After dramatic outbreaksof MDR-TB in the early 1990s, resistance becamerecognized as a global problem. MDR-TB nowthreatens the inhabitants of countries in Europe, Asia,Africa, and the Americas2. An understanding of themolecular basis of drug resistance may contribute tothe development of new drugs3. Management ofMDR-TB relies on prompt recognition and treatmentwith at least 3 drugs to which an isolate is susceptible.

Genomics of Mycobacterium tuberculosis: Old threats & new trends

Niyaz Ahmed & Seyed E. Hasnain

Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India

Received July 22, 2003

Tuberculosis (TB) has been declared as a global health emergency by the World Health Organization(WHO). This has been mainly due to the emergence of multiple drug resistant strains and the synergybetween tubercle bacilli and the human immunodeficiency virus (HIV). Genomic analysis of strainsfor outbreak investigations is in vogue for about a decade now. However, information available fromwhole genome sequencing efforts and comparative genomics of laboratory and field strains is likelyto revolutionize efforts towards understanding molecular pathogenesis and dissemination dynamicsof this dreaded disease. Genomic information is also going to fuel discovery projects where newtargets will be identified and explored towards a new drug for TB. Besides this, efforts of informationtechnologists, chemists, population biologists, freelance workers, media persons, non-governmentalorganizations and administrators to needed to handle the problem of tuberculosis to prevent it frombecoming a pandemic.

Key words Genomics - Mycobacterium tuberculosis - multidrug resistant strains

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The roles of drug containing environments, and theimmunological status of the host and bacterialmolecular mechanisms of development of drugresistance to M. tuberculosis have been examined andresults are helpful in implementation of modifieddrug regimens in tuberculosis control programmes.Multidrug resistant strains of M. tuberculosisseriously threaten tuberculosis control and preventionefforts. Molecular studies of the mechanism of actionof antitubercular drugs have elucidated the geneticbasis of drug resistance. Drug resistance in M.tuberculosis has been primarily attributed to themutations in the drug target genes, however, thepresence of efflux pumps in clinical MDR isolatescannot be ruled out4. These mutations lead either toan altered target or to a change in titration of the drug.A diverse array of strategies is already available toassist in rapid detection of drug resistance-associatedgene mutations. In spite of remarkable advances inthis area, much remains to be learned about themolecular genetic basis of drug resistance in M.tuberculosis. During the last decade, there has beena marked increase in the number and gravity oftuberculosis cases both in developing countries andin industrialized nations. One of the more insidiousconsequences of this resurgence has been the recentemergence of nosocomial transmission of multi drugresistant strains of M. tuberculosis, thus creatinguntreatable forms of the disease and these strains maybecome widespread. That the various clinical isolatesof M. tuberculosis are geographically partitioned atthe global level. Ahmed et al5,6 has provided evidenceto the concept of geographic genomics7.

The TB- HIV symphony: Supporting the idea oflow virulent clones

The emergence of the HIV/acquiredimmunodeficiency syndrome (AIDS) pandemic hasled to major shift in our approaches towardsepidemiological studies of TB both in resource poorand developed countries, with HIV infectionbecoming a risk factor for the development of activeTB infection. This might be a consequence ofincreased re-activation of previously acquired,dormant bacilli and an increase in susceptibility toboth re-infection and primary infection. Studiesinvolving DNA fingerprinting of M. tuberculosis

isolates obtained from AIDS patients by the techniqueof restriction fragment length polymorphism (RFLP)analysis showed that re-infection and new infectionboth occur in AIDS patients5. It has been speculatedthat the HIV/AIDS patients constituting an ecologicalniche for M. tuberculosis, where less virulent strainsmultiply freely without the selection pressureprovided by an immunocompetent patient.Consequently, it should be possible to differentiallyidentify pathogenic bacterial clones and differentiatethem on the basis of epidemiological parametersrelated to co-infection, relapse versus recent infectionand multiple drug resistance. Recently, onestudy6 has been conducted under the auspices of theM. tuberculosis evolutionary genomics interest group,where significantly different genotypes were observedfor HIV-associated tubercle bacilli as compared tobacilli recovered from non-HIV patients. This hasbeen the only study of its kind in the post genomicscenario where high resolution genomic patternanalysis was systematically applied to a wellcharacterized strain collection from a clinically testedpatient population. While statistically significantassociation of a clone to the disease outbreak in acommunity may reflect increased fitness of thegenome, it can be suggested that this phenomenonshould be observed in multiple settings before theycan be linked with certainty to a particular hostpopulation. Frequent travel and social links amongthe people of economically productive and sexuallyactive age groups as seen in some African and SouthAmerican countries, often provide for fastmultiplication of low virulent strains and thereforehigh clustering is seen in such settings.

The post genomic approaches

New developments in molecular biology andfunctional genomics have fostered major advancesin our understanding of genetic variability among M.tuberculosis. Complete genome sequenceinformation8 has been released for this organismopening new vistas in diagnosis, epidemiology andvaccine development. Automated DNA sequencingallows for the direct comparison of specific genesamong large populations of isolates, anddetermination of complete genomic sequence of twostrains together with new technologies like DNA

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microarrays and computational biology, has provideda whole genome perspective on genomic content,gene regulation, and metabolism.

The face of infection epidemiology has changedin the post genomic scenario. Bacterial genomesignatures permit whole genome comparisonsbetween different bacterial strains or isolates by usingdifferential amplification techniques and automatedgenotyping for the determination of genetic signaturesor barcodes. These signatures could be a group ofmarkers or DNA fragments of unique sizes. In thisway, gene deletions may be readily identified thatcorrelate with alteration in virulence or otherphenotypes.

The use of differential expression to monitorwhole genome expression is now a powerfulapproach, which will result in definit ion ofdifferentially expressed genes important inpathogenesis, and will provide useful targets forrational design of new drugs and vaccine candidatesfor M. tuberculosis. These studies are likely toconfirm the reality of “transcriptomics” as a technicaldeliverable for hypothesis driven research intomycobacterial pathogenesis, vaccine and drugresearch.

The emergence of protein-based analysis orproteomics is likely to facilitate determination ofchanges in the expression profile that leads topathogenicity, drug resistance and immune responses.This approach has a very high potentiality andaccuracy to identify proteins involved inpathogenicity and drug resistance leading todevelopment of medical intervention. Similarapproaches can be adopted to understand M.tuberculosis pathogenicity and multidrug resistanceamong different strains.

The recent accumulation of copious data about theextent and nature of genetic and phenotypic variationin tubercle bacill i provides an unprecedentedopportunity to determine the phenotypicconsequences of genetic polymorphism in thisorganism. However, a coherent synthesis of thisknowledge is lacking, reflecting the formidablechallenges of this endeavour. Because environmentaland host factors clearly contribute to the clinical and

epidemiologic behaviour of strains, these factors mustbe carefully integrated into the investigative process.

Post genomic databases for epidemiologists

A large number of molecular markers have beenmade available for epidemiological and evolutionarystudies as a result of comparative genomics studies(Fig.). These markers include Mycobacterialinterspersed repeat units-variable number tandemrepeats (MIRU-VNTRs)9, fluorescent amplifiedfragment length polymorphism (FAFLPs)6 and largegenomic deletions10. The present day challenge is tocompile standardized fingerprint patterns originatingfrom highly networked, multi-centric, genotypicanalyses in the databases for interlaboratory use andfuture references7. This requires an accuratemeasurement of fragment lengths. Analysis viamolecular weight markers in adjacent lanes isstraightforward and can be done automatically ondigitated images. A correction for the variation inmigration rates and gel distortion is achieved by co-electrophoresis in each lane of both sample andmarker fragment. One option is the use of invariantfragments with known lengths as internal markers.Digitated images in a standard graphical file may beobtained as scanned autoradiographs. These data canbe imported in gel analysis software, which carriesout the normalization and fragment sizing for analysisby the dedicated software. Many databases have beendeveloped recently for rapid genotypic searches andcomparisons on the world-wide web: SpolDB:Spoligotyping resource database at Institut Pasteurof Guadilope (http://www.cdc.gov/ncidod/EID/vol7no3/sola_data.htm) Tbase: RFLP resourcedatabase at RIVM, The Netherlands (https://hypoc ra tes . r i vm.n l /bnwww/ IS6110-RFLP-bands.htm) AmpliBASE: AFLP resource database atCDFD Hyderabad (http:www.cdfd.org.in/amplibase)

MiruDB:VNTR resource database at IBL Lille,France (http://www.ibl.fr/mirus/mirus.html)

Post genomic discovery efforts: TB structuralgenomics

It is quite unfortunate to realize that while theworld is faced with the horrors of TB epidemic, nonovel compound has been introduced for widespread

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210 INDIAN J MED RES, OCTOBER 2004

treatment of this disease after rifampicin wasdeveloped in 1963. At this point, however, it isbelieved that new chemotherapies for treatment ofTB should have the following major objectives: (i)development of faster acting drugs to shorten theduration of treatment; (ii ) development of novelantimicrobials to counter the emergence of bacteriaresistant to current therapies; and (iii ) developmentof chemotherapeutics that specifically target dormantbacilli and hence provide more effective treatmentof latent TB infections.

In the post genomic scenario, a strong emphasishas been paid on structural genomics based discoveryof new powerful drugs. Structural genomics is thelarge scale determination and analysis of proteinstructures. It is a new field that has been boosted bymajor technological advances in structuredetermination and by the availability of as many as95 to 200 completed genome sequences related tomany microbial and protozoan species. Severalcomputational methods can predict the biologicalroles of proteins by analyzing functional relationships

among proteins rather than sequence similarities.Three of these methods are phylogenetic profiling,domain fusion and gene clustering. These methodscan help in identifying potential drug targets such asproteins functionally linked to known targets of anti-TB drugs and to proteins known to be essential forbacterial survival. Inhibiting these functionallyrelated proteins should have a similar effect on theorganism as inhibiting the present drug targets, sincethe same processes or pathways would be disrupted.An international effort in the form of the TBStructural Genomics Consortium11 has been a mostsuccessful partnership of investigators working ondifferent targets throughout the world. Objectives ofthis partnership include determining the structuresof many of the mycobacterial proteins facilitatingdrug discovery efforts using high throughputscreening, genetic identifications of key TB genes,and structure-based approaches. Other proteins thatmight be good targets for anti-TB therapy includeextracellular proteins that are involved in virulence,and persistence determinants, since the M.tuberculosis cell envelope is impermeable to many

Fig. Screen dump of AmpliBASE MT®, a M. tuberculosis diversity knowledgebase, that is based on MIRU(A) and FAFLP(B)markers.

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antibacterial agents. Potential therapeutic targets alsoinclude secreted antigens and proteins involved iniron acquisition, which is a critical process for M.tuberculosis survival. These proteins include ironregulatory proteins and enzymes involved in theproduction of secreted iron siderophores such asexocholins and mycobactins. Proteins specific totubercle bacilli might constitute possible targets,since these may provide adaptations exclusive to thevirulence and pathogenicity of mycobacteria. Inparticular, 10 per cent of the M. tuberculosis genomeconsists of genes that encode M. tuberculosis-specificPE, PPE and PE-PGRS proteins. The PE family isnamed after the presence of the motif proline-gluamicacid (PE) at positions 8 and 9 in a highly conservedN-terminal domain of approximately 110 aminoacids, the C-terminal region is however, variablewithin the family. The PPE family resembles the PEfamily with a highly conserved N-terminal region(about 180 amino acids), containing the motifproline-prolineglutamic acid at positions 7-9. Lastly,the (PE-PGRS) (polymorphic GC-rich sequence)family represents an extension of the PE proteinfamily with multiple repeats of glycine-glycine-alanine (asparagine) motifs. The function or functionsof these proteins are unknown though they have beenimplicated in virulence and immune surveillance12,13.

Conclusion and expert opinion

In the post genomic scenario, future of TBelimination holds a lot of promise but it is yet to berealized. With the unravelling of the M. tuberculosisgenome, newer paradigms in the areas ofepidemiology, evolution and diagnostics are almostcertain. On the discovery front, scientists can nowselectively target enzymes of M. tuberculosis whichdisplay strong antigenic attributes14. Geneticproducts of mycobacteria can be modified to causetheir own death. The international initiative underthe auspices of the Structural Genomics Consortiumis all set to harvest the fruits of such a new biology.In the future, the distinction between drugs,immunomodulators and vaccines may disappearleading to amalgamation of all these as a superantimycobacterial to rid the world from the deadlyshadow of TB. With the unprecedented growth ofbioinformatics and comparative genomics, state of

the art technologies are essential to utilize the vastgene pools sitt ing in the databases. Genomicdiscovery alone will not be sufficient to tackle theproblem. Apart from basic scientists, the role of healthcare workers, clinicians and epidemiologists, public-private partnerships and the non- governmentorganizations will be highly needed on all the frontiers.

Acknowledgment

Authors thank all the collaborators of M. tuberculosisevolutionary genomics interest group and Centre for DNAFingerprinting and Diagnostics, Hyderabad for supporting thisinit iat ive. The generous support from Department ofBiotechnology (DBT) and Council for Scientific & IndustrialResearch (CSIR), New Delhi is gratefully acknowledged.

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Reprint requests : Dr S.E. Hasnain, Director, Centre for DNA Fingerprinting & Diagnostics, ECIL Road, Nacharam,Hyderabad 500076, Indiae-mail: hasnain@cdfd.org.in