1. Fluorescent Amplified Fragment Length Polymorphism (FAFLP) analysis of Mycobacterium tuberculosis: Molecular Epidemiological approach for typing multi-drug resistant (MDR) isolates 

Principal Investigator: Seyed E Hasnain*
Co-Principal Investigator: Niyaz Ahmed
Project Associate: Noman Siddiqi
Collaborator: V M Katoch
Central JALMA Institute for Leprosy, Agra

*Jointly with the National Institute of Immunology

Background:

Although tuberculosis is a curable disease, recent emergence of multidrug resistant strains has been of great concern. The scenario is alarming particularly in developing countries because of rapid spread of HIV. An understanding of the molecular mechanism of pathogenesis is imperative for effective control of this pathogen. Resistance to several drugs have been attributed to SNPs in different mycobaterial loci. However, a whole genome approach is likely to give a better understanding of drug resistance. Using FAFLP it is possible identify single base substitutions in a relatively less time. Using this powerful tool, the basic goals of this project are to develop high resolution fingerprints of M. tuberculosis and other pathogenic isolates as well as predictive modeling of single base substitutions associated with molecular pathogenesis, drug resistance and virulence. This project also aims to develop a national epidemiological database of available M. tuberculosis isolates based on their genotypic signatures.

High resolution genotyping methods based on predictive modeling of restricted fragments for pathogen identification has been developed. This method is being applied to M. tuberculosis, H. pylori, P. aeruginosa and E. coli. Fluorescent amplified fragment length polymorphism (FAFLP) analysis was performed with EcoRI/MseI microbial adapters to generate molecular signatures for individual isolates on a genome wide scale. This will help in monitoring micro-heterogeneity among the pathogenic isolates. FAFLP analysis was investigated for its ability to identify and subtype patient isolates of Mycobacterium tuberculosis in India. We assessed this technique for studying strain divergence and relatedness among pathogenic mycobacteria with 103 isolates of M. tuberculosis from geographically distributed patients from North India.

The FAFLP profiles derived from EcoRI/MseI fragments differentiated drug sensitive isolates of M. tuberculosis from MDR types and were found to be extremely reproducible. Multiplex FAFLP was used to exploit the resolving power of expert softwares in a multi-color detection mode. Scoring of large number of markers, generated in the process of FAFLP, was carried out by automating the procedure by Genescan3.1TM (Perkin Elmer, USA) and Genotyper2.5TM (Perkin Elmer, USA) software. A total of 23 different FAFLP profiles were observed suggesting strain divergence. We were successful in differentiating avirulent strains (H37Ra) of tubercle bacilli from highly virulent laboratory strain H37Rv. Differentially visualised green colored bands could be the possible virulence associated markers.

2. Rapid detection of M. tuberculosis in clinical samples by PCR

Principal Investigator: Seyed E Hasnain*
Co-Principal Investigator:  Niyaz Ahmed
Project Associate: Noman Siddiqi
Technical Officer: G R Savithri
Collaborator: V M Katoch
Central JALMA Institute for Leprosy, Agra

*Jointly with the National Institute of Immunology

Background:

Mycobacterium tuberculosis is a slow growing microorganism and confirmation of the diagnosis by conventional culture is a lengthy process. DNA based identification of pathogens has been a recent alternative to the microscopic and bacteriological techniques. We tested the efficacy of DNA amplification techniques to recognize the insertion sequence-1081 (IS1081) of the M. tuberculosis complex in clinical samples of diverse origin including sputum sediments, cerebrospinal fluid (CSF) and peripheral blood. 

Current work:

In our lab, two methods, namely the nested polymerase chain reaction (nested PCR) and polymerase chain reaction<96>restriction endonuclease analysis (PCR-REA) were developed to recognize a 639 bp segment of the IS1081 of the M tuberculosis complex. Nested PCR with NZ3/NZ4 primers could successfully amplify a 217 bp region nested within the 306 bp sequence amplified by the NZ1/NZ2 primers. An alternative system involving HindIII restricted digestion of the NZ1/NZ6 amplified 639 bp sequence was also developed and standardized. This amplicon on digestion with HindIII gave fragments of 371 bp and 268 bp. When tested on laboratory isolates and clinical samples such as peripheral blood and sputum samples, the assays were found to be 100% target specific with a concordance of 100% with pure cultures (n = 200), 94.33% (100 of 103 samples) with acid fast smear positive sputum samples, 100% with CSF (15 of 15) and 62.5% (10 of the 16 samples) with peripheral blood from immunocompetent patients with pulmonary tuberculosis. 

3. Genomic alterations in human meningioma

Principal Investigator: Seyed E Hasnain*
Other Members: Niyaz Ahmed
Collaborator: Subrata K Sinha
AIIMS, New Delhi

*Jointly with the National Institute of Immunology

Background:

Genetic alterations in tumor tissue can be detected by RAPD method and the advantage of this method is easy identification of loci involved in DNA rearrangements without prior knowledge of oncogenic loci. Earlier using RAPD we established 4 tumor specific markers termed as meng1 through meng4 altered in human meningioma.. The 1.2 kb amplicon corresponding to meng-1, was cloned and sequenced. The meng-1 SCAR primers failed to generate any amplifications from other tumor types implying that alterations in meng1 is meningioma specific. Zoo blot analysis using this probe revealed that meng-1 was conserved across evolution.

4. Baculovirus p35 gene expression and cell death

Principal Investigator: Seyed E Hasnain*
Project Associate: Sudhir, Sahdev*, Manjari Mohan

*Jointly with the National Institute of Immunology


Background:

The main objective is to unravel the mechanism of inhibition of apoptosis by the baculovirus p35 gene. P35 was known to inhibit apoptosis by interfering with caspase-1 activation as a suicide inhibitor resulting in arrest of apoptosis. We earlier discovered that p35 can also inhibit oxidant dependant apoptosis by acting as a free radical quencher. p35 was also shown to inhibit UV-B induced apoptosis in insect cells and release of cytochrome-c from mitochondria was correlated with UV-B induced apoptosis.

p35 mediated inhibition of Caspase-3 activation

H2O2-induced apoptosis in murine macrophages was established as a manifestation of time dependent activation of caspase-3. CMVp35 transfection 4 hours prior to H2O2 treatment however, resulted in inhibition of caspase activation. Cytochrome c release from mitochondria as a consequence of H2O2 treatment of macrophages as well as in Sf9 cells. Release of cytochrome c was observed to be time dependent and reached maximum levels at very early time periods. CMVp35 transfection 4 hours prior to H2O2 treatment showed significant reduction in the levels of cytochrome c release. 

Cloning, expression and purification of Sf-caspase-1

In order to understand the target of action of P35, the gene encoding the Sf9 caspase was isolated by PCR amplification and cloned in pET-23a(+) vector. The plasmid pRN-Cas has been transformed into BL21 cells and further work is underway. Since P35 acts as a substrate as well as an inhibitor of caspase, it would be worthwhile to study their interactions.

5. Gene expression in baculovirus-infected insect cells

Principal Investigator: Seyed E Hasnain*
Co-Principal Investigator: Murali Dharan Bashyam
Project Associates: Sudip Ghosh, Aruna Ramachandran
Ph.D Students: Priya Viswanathan*

*Jointly with the National Institute of Immunology

Background: The baculovirus expression vector system is most popular and most widely used because of its several advantages over other expression systems. The baculovirus system uses two very late gene promoters namely polh and p10 for driving expression of foreign genes under their control. The simple architecture but a very high level of transcription from these promoters has eluded most baculovirologists. We discovered a host protein PPBP from insect cells which binds specifically around the transcriptional start site of the polh and p10 promoter. PPBP was further found to be required for initiation of transcription from these promoters. We also found that activated transcription from these promoters also depends on the presence of cis-elements upstream to the transcriptional start site. Recently, we discovered the presence of novel Sp-like proteins in Sf9 cells that could regulate transcription from the polh promoter by binding to Sp-like cognate motif present upstream to the polh basal promoter.

Experiments are underway to further elucidate the role of Sp-family factor(s) present in Sf9 insect (Spodoptera frugiperda) cells in transcription from the polyhedrin (polh) gene promoter. Mutational analyses of the AcSp DNA motif were carried out to identify the exact binding sequence(s) responsible for the binding of the Sp-family protein(s). Electrophoretic mobility shift assays (EMSAs) using pure recombinant human Sp1 revealed that the host factor(s) binding to AcSp was not Sp1, but probably a new member of the larger Sp-superfamily. The insect Sp-like factor(s) also demonstrated a requirement for zinc as evidenced by their inability to bind the AcSp sequence in EMSAs in the presence of o-phenanthroline, a zinc chelator. In-vivo mopping experiments indicated that the transcriptional enhancement by the AcSp sequence motif was due to the binding of the host factor(s) and not a function of the cis-sequence per se. Recombinant viruses were constructed with varying lengths of sequences upstream to the polh promoter driving a luciferase reporter gene. Infection with these viruses revealed the involvement of the AcSp sequence in the viral context as well, pointing to the critical role played by this motif in transcription from the polh promoter.

6. Molecular Epidemiology, Microevolution and Functional Genomics of M. tuberculosis in India

Principal Investigator(s): N. Ahmed, Seyed E Hasnain*
Collaborators: V M Katoch, CJIL, Agra, SK Sharma, AIIMS, New Delhi, KJR Murthy, Bhagwan Mahavir Hospital, Hyderabad

The M. tuberculosis genome is a circular chromosome of 4,403,765 base pairs with an average G + C content of 65.6%. There are a total of 4,033 predicted open reading frames (ORFs). Predicted biological roles were assigned to 1,734 ORFs (43%); 605 ORFs (15%) match hypothetical proteins from other species, and 1,694 ORFs (42%) have no database match and presumably represent novel genes. For analysis of patient isolates from across the country, we make use of Fluorescent Amplified Fragment Length Polymorphism (FAFLP) analysis. Linkage analysis and genotyping of FAFLP markers is an easy method for identification of possible virulence factors, the genetic elements involved in molecular pathogenesis, multi-drug resistance (MDR) and intra-cellular survival. It provides a means of examining DNA segments distributed over the entire genome of an organism, which is advantageous over methods that examine restriction site changes in selected genes, for example, PCR-RFLP techniques.

Apart from tracing the origin of outbreaks, automation of scoring of the large number of markers further increases the potential of the technique for mapping of the loci which are linked to the molecular pathogenicity of tuberculosis such as resistance to one or more anti-tubercular drugs. Also, the amplitypes could serve as molecular ID cards for the isolates and possible laboratory contaminations with other slow growing mycobacteria and the possibilities of duplicate strain accessions in the repositories could be ruled out. FAFLP profiles are suitable for rapid electronic transmission for interlaboratory comparison and are well suited for storage in epidemiological databases for future comparison. Differential display PCR and cDNA-FAFLP are the two powerful tools that are being adopted to identify novel genes expressed under varied drug pressure or as a result of host-pathogen interactions. We are also exploring to harness microarray hybridisation as a powerful approach in inverse genetics, which will result in identification of differentially-expressed genes important in pathogenesis. This will provide useful targets for rational design of new drugs and vaccine candidates for important bacterial pathogens. Analysis of gene expression for all predicted ORF<92>s (~6000) in the yeast genome and the M. tuberculosis genome has recently been described. Together, these studies confirm the reality of "transcriptomics" as a tool for mycobacterial pathogenesis, vaccine and drug design. Technical developments will continuously emerge in this cutting edge technology and we are ready to exploit them. Recently some markers on chromosomes 15q and Xq showed evidence of linkage to Tuberulosis in Africans. Our FAFLP approach could be used for identifying host factors and genetic loci associated with susceptibility to infection and pathogenesis of M. tuberculosis complex in Indian populations.

7. National Epidemiological Database of M. tuberculosis (NED-MT): an object oriented database

Principal Investigator(s): N. Ahmed, Seyed E Hasnain*
Project Associate: Ahmed A. Majeed

Essentially a DNA Fingerprint resource database of FAFLP profiles, NED-MT aims at examining DNA segments distributed over the entire genome of M. tuberculosis. The amplitypes of clinical isolates developed in our laboratory will serve as reference material in the form of 'molecular ID cards' for the individual strains. Inter -laboratory exchange of data will allow faster comparisons to rule out possible laboratory contaminations with other slow growing mycobacteria and the possibilities of duplicate strain accessions. Database of mapped routes of infections and molecular fingerprints for all the isolates in India will be generated in an object oriented database management system (OODBMS) (http://www.sysra.com/eyedb) like ACEDB (A Caenorhabditis elegans Database) or MycDB. Searchable profiles, electropherograms, amplitypes, genotyper tables, allele frequencies, clinical data and linkage maps etc. corresponding to known isolates in our country, will be deposited to this database and will be posted on to the web server for inter-laboratory comparison purposes.

8. Molecular epidemiology and pathogenesis of Helicobacter pylori infection in Hyderabad

Principal Investigator(s): N. Ahmed, Seyed E Hasnain*
Collaborator: C M Habibullah
H. pylori isolates (n=65) from patient populations with duodenal ulcers have been fingerprinted by MseI/EcoRI FAFLP mathod. Markers representing pathogenicity island sequences and those corresponding to Metronidazole resistance are being looked into. The next step is to perform cDNA AFLP with organisms grown under different drug pressures and those from different pathogenic spectrum (invasive and non-invasive strains).

9. Biology of hepatocyte growth and regeneration

Principal Investigator(s): N. Ahmed, Seyed E Hasnain*
Collaborator: C M Habibullah (DMC, Hyderabad)

Major objectives of this project include

• Studies on liver cell proliferation and liver regeneration by development of hepatocyte growth factor - green fluorescent protein (HGF-GFP) gene therapy tools
• Development of asialoglycoprotein receptor mediated gene delivery system for hepatocytes
• Studies on HGF expression in-vivo and liver cell proliferation marker assays using immunohistochemistry tools

10. Genome-sequence-based analysis of germ line mutations in patients with ocular disorders

Principal Investigator(s): N. Ahmed, Seyed E Hasnain*
Collaborator(s): Chitra K, Shirley G P (LVPEI, Hyderabad)

In most patients with isolated unilateral retinoblastoma, tumor development is initiated by somatic inactivation of both the alleles of the Rb1 gene. However, some of these patients can transmit retinoblastoma predisposition to their offspring. To determine the frequency and nature of constitutional Rb1 mutations in retinoblastoma patients in India, this program was taken up. It aims at sequencing of more than 1400 samples from classified patient populations and the healthy controls for known and novel mutations in all the 27 exons of Rb1 gene. We analyzed DNA from peripheral blood and from fresh and formalin-fixed tumor samples. Automated DNA sequencing of Rb1 gene was carried out for about 600 samples covering 20 of the 27 exons. Several hotspots have been short-listed for germline mutations after a careful analysis of these coding regions using Lasergene Navigator (DNAstar Inc. USA) package. A C -->T transition in codon 579 of exon 18 that changes arginine (CGA) to stop codon (TGA) is identified for the Indian populations for the first time. Another gene of interest under this program is the CYP1B1 gene associated with primary congenital glaucoma. All the 3 exons of the gene have been sequenced for mutational analysis and are being analysed for novel mutations. Sequence data under this program will be analysed from the surveillance point of view and pooled to the existing DNA sequence database at the automated genome analysis facility of the CDFD.