Molecular epidemiology of leprosy in Cebu, Philippines and essentiality and characterization of an arabinosyltransferase involved in the cell wall synthesis of mycobacteria
Date
2009
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Abstract
Leprosy is a chronic granulomatous infectious disease caused by Mycobacterium leprae, which affects the skin, peripheral nerves and mucous membranes. Approximately a quarter million new cases are detected annually throughout the world. Since M. leprae is an obligate intracellular bacterium, not cultivable under in vitro conditions, and with a long incubation time, the causes for continued prevalence in endemic countries are still unknown. In this regard, molecular tools for differentiation of isolates of M. leprae are needed to track and control transmission of leprosy. Genome sequencing of M. leprae facilitated the identification of genetic markers such as variable number of tandem repeat (VNTR) and single nucleotide polymorphic (SNP) loci. We standardized methods for DNA extraction from the clinical samples and also developed rapid, high throughput and inexpensive methods like multiplex-PCR and fragment length analysis (FLA) for VNTR analysis; polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for SNPs to facilitate molecular strain typing of clinical samples. We have applied these methods on the DNA extracts of stored and new samples obtained from leprosy patients from Leonard Wood memorial (LWM), Cebu, Philippines. We compiled a database containing epidemiological information from over 200 leprosy patients. VNTR alleles were found to be highly concordant when obtained from biopsies and slit skin smears (SSS) from the same patients indicating that either can be used for strain typing. Based on parsimony cluster analysis of 207 clinical isolates, five major groups were observed. VNTR markers alone were able to differentiate the SNP type 3 isolates from the majority of the SNP type 1 isolates. VNTR profiles of the M. leprae isolates within the multicase families (MCFs) were similar indicating common source of transmission. In a continuation study, VNTR data was obtained for another seventy samples from newly diagnosed leprosy patients from LWM, Cebu, Philippines. Multiplex PCR and FLA methods were found to be more efficient and accurate compared to short tandem repeat (STR) amplification and DNA sequencing. Intra-patient VNTR variability using different SSS was found to be minimal. Population structure of M. leprae isolates from Cebu remained similar with stable clusters in the phylogentic tree, even with the inclusion of 70 additional samples. Drug resistance is a cause for concern in the treatment and control of any infectious disease. Drug resistance in leprosy was known to be caused during the dapsone monotherapy control program and patients' irregular treatment. WHO MDT which includes anti-leprosy drugs such as dapsone, rifampicin and clofazimine was introduced in 1982. However drug resistance was reported even in the areas where the MDT was implemented successfully. So to evaluate the primary drug resistance in newly diagnosed leprosy patients who are being treated with the standardized MDT regimen, we developed a multiplex PCR for amplifying the drug resistance determining regions (DRDR) in genes rpoB, folP1, gyrA and gyrB, which are targets of the drugs rifampicin, dapsone and oflaxacin, respectively. No mutations were detected in rpoB and folP1 DRDRs for 77 samples and in the gyrA DRDR for 15 samples tested thus far. A separate chapter describes the studies on the cell wall biosynthesis. The cell envelope of mycobacteria are comprised of a core macromolecule known as mycolyl arabinogalactan (mAG) which is covalently attached to the peptidoglycan layer forming the mAGP complex and other macromolecules such as lipomannan and lipoarabinomannan. The mAGP complex is essential for survival of the bacteria and is a permeability barrier. The synthesis and assembly of this mAGP complex is still not fully understood. In this regard, through comparative genome approaches we identified Rv3805c as a putative glycosyltransferase located in the cell wall synthesis gene cluster. Rv3805c predicted to have ten transmembrane domains, contains a conserved glycosyltransferase and proline rich motif also present in Emb proteins which are involved in cell wall biosynthesis. In this study we identified that, Rv3805c encodes an arabinosyltransferase through an in vitro arabinosyltransferase assay. Furthermore, a knock out mutant of an ortholog gene NCgl2780 was generated in C. glutamicum. The structural analysis of AG of NCgl2780 mutant in C. glutamicum revealed the loss of β(1→2) Araf residues at the non-reducing ends. We also indentified that the ortholog MSMEG_6400 is essential for M. smegmatis, as we were unable to obtain a genetic knock out mutant in the absence of an additional functional copy of the gene.
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Subject
arabinosyltransferase
corynebacteria
leprosy
Mycobacterium leprae
molecular biology
microbiology
biochemistry