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ABSTRACT: Cell mediated immunity (CMI) based in vitro interferon-γ released assay (IGRA) to Mycobacterium leprae specific antigens has potential as a promising diagnostic means to detect those individuals in the early stages of M. leprae infection. Diagnosis of leprosy is a major obstacle toward ultimate disease control, and has been compromised in the past by the lack of specific markers. Comparative bioinformatic analysis among mycobacterial genomes identified potential M. leprae unique proteins called "hypothetical unknowns". Due to massive gene decay and the prevalence of pseudogenes, it is unclear whether any of these proteins are expressed or are immunologically relevant. Here, we performed cDNA based quantitative real time PCR to investigate the expression status of 131 putative open reading frames (ORFs) encoding hypothetical unknowns. Twenty six of the M. leprae specific antigen candidates showed significant levels of gene expression compared to that of ESAT-6 (ML0049) which is an important T cell antigen of low abundance in M. leprae. Fifteen out of 26 selected antigen candidates were expressed and purified in Escherichia coli. The seroreactivity to these proteins using the pooled sera of lepromatous leprosy patients and cavitary tuberculosis patients revealed that 9 out of 15 recombinant hypothetical unknowns elicited M. leprae specific immune responses. These nine proteins may be good diagnostic reagents to improve both sensitivity and specificity in detection of individuals with asymptomatic leprosy.
Clinical and vaccine immunology: CVI 12/2012; · 2.37 Impact Factor
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Gérald Larrouy-Maumus,
Henrieta Skovierová,
Rabeb Dhouib,
Shiva Kumar Angala,
Sophie Zuberogoitia,
Ha Pham,
Anne Drumond Villela,
Katarina Mikusová,
Audrey Noguera,
Martine Gilleron,
Lucia Valentínová,
Jana Korduláková, Patrick J Brennan,
Germain Puzo,
Jérôme Nigou,
Mary Jackson
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ABSTRACT: The biosynthesis of Mycobacterium tuberculosis major cell envelope glycoconjugates is topologically split across the plasma membrane. Yet, nothing is known of the transporters required for the translocation of lipid-linked sugar donors and oligosaccharide intermediates from the cytoplasmic to the periplasmic side of the membrane in mycobacteria. One of the mechanisms used by prokaryotes to translocate lipid-linked phosphate sugars across the plasma membrane relies on translocases that share resemblance with small multidrug resistance (SMR) transporters. The presence of an SMR-like gene, Rv3789, located immediately upstream from dprE1/dprE2 responsible for the formation of decaprenyl-monophosphoryl-beta-D-arabinose (DPA) in the genome of M. tuberculosis led us to investigate its potential involvement in the formation of the major arabinosylated glycopolymers, lipoarabinomannan (LAM) and arabinogalactan (AG). Disruption of the ortholog of Rv3789 in Mycobacterium smegmatis resulted in a reduction of the arabinose content of both AG and LAM that accompanied the accumulation of DPA in the mutant cells. Interestingly, AG and LAM synthesis was restored in the mutant not only upon expression of Rv3789 but also upon that of the undecaprenyl phosphate aminoarabinose flippase arnE/F genes from E. coli. A bacterial two-hybrid system further indicated that Rv3789 interacts in vivo with the galactosyltransferase that initiates the elongation of the galactan domain of AG. Biochemical and genetic evidence is thus consistent with Rv3789 belonging to an AG biosynthetic complex where its role is to re-orient DPA to the periplasm, allowing this arabinose donor to then be used in the build-up of the arabinan domains of AG and LAM.
Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
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Anna E Grzegorzewicz,
Jana Korduláková,
Victoria Jones,
Sarah E M Born,
Juan M Belardinelli,
Adrien Vaquié,
Vijay A K B Gundi,
Jan Madacki,
Nawel Slama,
Françoise Laval,
Julien Vaubourgeix,
Rebecca M Crew,
Brigitte Gicquel,
Mamadou Daffé,
Hector R Morbidoni, Patrick J Brennan,
Annaik Quémard,
Michael R Mcneil,
Mary Jackson
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ABSTRACT: Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases
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Saioa Urresti,
David Albesa-Jové,
Francis Schaeffer,
Ha T Pham,
Devinder Kaur,
Petra Gest,
Mark J van der Woerd,
Ana Carreras-González,
Sonia López-Fernández,
Pedro M Alzari, Patrick J Brennan,
Mary Jackson,
Marcelo E Guerin
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ABSTRACT: Considerable progress has been made in recent years in our understanding of the structural basis of glycosyl transfer. Yet the nature and relevance of the conformational changes associated with substrate recognition and catalysis remain poorly understood. We have focused on the glucosyl-3-phosphoglycerate synthase (GpgS), a "retaining" enzyme, that initiates the biosynthetic pathway of methylglucose lipopolysaccharides in mycobacteria. Evidence is provided that GpgS displays an unusually broad metal ion specificity for a GT-A enzyme, with Mg(2+), Mn(2+), Ca(2+), Co(2+), and Fe(2+) assisting catalysis. In the crystal structure of the apo-form of GpgS, we have observed that a flexible loop adopts a double conformation L(A) and L(I) in the active site of both monomers of the protein dimer. Notably, the L(A) loop geometry corresponds to an active conformation and is conserved in two other relevant states of the enzyme, namely the GpgS·metal·nucleotide sugar donor and the GpgS·metal·nucleotide·acceptor-bound complexes, indicating that GpgS is intrinsically in a catalytically active conformation. The crystal structure of GpgS in the presence of Mn(2+)·UDP·phosphoglyceric acid revealed an alternate conformation for the nucleotide sugar β-phosphate, which likely occurs upon sugar transfer. Structural, biochemical, and biophysical data point to a crucial role of the β-phosphate in donor and acceptor substrate binding and catalysis. Altogether, our experimental data suggest a model wherein the catalytic site is essentially preformed, with a few conformational changes of lateral chain residues as the protein proceeds along the catalytic cycle. This model of action may be applicable to a broad range of GT-A glycosyltransferases.
Journal of Biological Chemistry 05/2012; 287(29):24649-61. · 4.77 Impact Factor
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ABSTRACT: Mannose-capped lipoarabinomannan (ManLAM) is a complex lipoglycan abundantly present in the Mycobacterium tuberculosis cell envelope. Many biological properties have been ascribed to ManLAM, from directly interacting with the host and participating in the intracellular survival of M. tuberculosis, to triggering innate and adaptive immune responses, including the activation of CD1b-restricted T cells. Due to its structural complexity, ManLAM is considered a heterogeneous population of molecules which may explain its different biological properties. The presence of various modifications such as fatty acids, succinates, lactates, phosphoinositides and methylthioxylose in ManLAM have proven to correlate directly with its biological activity and may potentially be involved in the interactions between CD1b and the T cell population. To further delineate the specific ManLAM epitopes involved in CD1b-restricted T cell recognition, and their potential roles in mediating immune responses in M. tuberculosis infection, we established a method to resolve ManLAM into eight different isoforms based on their different isoelectric values. Our results show that a ManLAM isoform with an isoelectric value of 5.8 was the most potent in stimulating the production of interferon-γ in different CD1b-restricted T-cell lines. Compositional analyses of these isoforms of ManLAM revealed a direct relationship between the overall charge of the ManLAM molecule and its capacity to be presented to T cells via the CD1 compartment.
Glycobiology 04/2012; 22(8):1118-27. · 3.58 Impact Factor
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Marcia V S B Martins,
Marjorie M da S Guimarães,
John S Spencer,
Mariana A V B Hacker,
Luciana S Costa,
Fernanda M Carvalho,
Annemieke Geluk,
Jolien J van der Ploeg-van Schip,
Maria A A Pontes,
Heitor S Gonçalves,
Janvier P de Morais,
Tereza J P G Bandeira,
Maria C V Pessolani, Patrick J Brennan,
Geraldo M B Pereira
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ABSTRACT: During recent years, comparative genomic analysis has allowed the identification of Mycobacterium leprae-specific genes with potential application for the diagnosis of leprosy. In a previous study, 58 synthetic peptides derived from these sequences were tested for their ability to induce production of IFN-γ in PBMC from endemic controls (EC) with unknown exposure to M. leprae, household contacts of leprosy patients and patients, indicating the potential of these synthetic peptides for the diagnosis of sub- or preclinical forms of leprosy. In the present study, the patterns of IFN-γ release of the individuals exposed or non-exposed to M. leprae were compared using an Artificial Neural Network algorithm, and the most promising M. leprae peptides for the identification of exposed people were selected. This subset of M. leprae-specific peptides allowed the differentiation of groups of individuals from sites hyperendemic for leprosy versus those from areas with lower level detection rates. A progressive reduction in the IFN-γ levels in response to the peptides was seen when contacts of multibacillary (MB) patients were compared to other less exposed groups, suggesting a down modulation of IFN-γ production with an increase in bacillary load or exposure to M. leprae. The data generated indicate that an IFN-γ assay based on these peptides applied individually or as a pool can be used as a new tool for predicting the magnitude of M. leprae transmission in a given population.
PLoS Neglected Tropical Diseases 04/2012; 6(4):e1616. · 4.69 Impact Factor
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ABSTRACT: Based on the discovery of three single nucleotide polymorphisms (SNPs) in Mycobacterium leprae, it has been previously reported that there are four major SNP types associated with different geographic regions around the world. Another typing system for global differentiation of M. leprae is the analysis of the variable number of short tandem repeats within the rpoT gene. To expand the analysis of geographic distribution of M. leprae, classified by SNP and rpoT gene polymorphisms, we studied 85 clinical isolates from Thai patients and compared the findings with those reported from Asian isolates. SNP genotyping by PCR amplification and sequencing revealed that all strains like those in Myanmar were SNP type 1 and 3, with the former being predominant, while in Japan, Korea, and Indonesia, the SNP type 3 was found to be more frequent. The pattern of M. leprae distribution in Thailand and Myanmar is quite similar, except that SNP type 2 was not found in Thailand. In addition, the 3-copy hexamer genotype in the rpoT gene is shared among the isolates from these two neighboring countries. On the basis of these two markers, we postulate that M. leprae in leprosy patients from Myanmar and Thailand has a common historical origin. Further differentiation among Thai isolates was possible by assessing copy numbers of the TTC sequence, a more polymorphic microsatellite locus.
Japanese journal of infectious diseases. 01/2012; 65(1):52-6.
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ABSTRACT: Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR-high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations.
Journal of clinical microbiology 12/2011; 50(3):742-53. · 4.16 Impact Factor
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ABSTRACT: PGL-I (phenolic glycolipid I) emerged in the early 1980s on the one hand as part of intensive efforts to define the typing antigens of a host of Mycobacterium spp. and also from characterisation of the lipids of skin biopsies from highly bacillary positive lepromatous leprosy patients. PGL-I, despite its extreme lipophilicity due to its inherent phthiocerol dimycocerosyl component, is highly antigenic evoking high titre IgM antibodies in lepromatous leprosy patients, attributable largely to the unique 3,6-di-O-methyl-beta-D-glucosyl entity at the non-reducing terminus of its trisaccharide. PGL-I itself or in the form of semisynthetic neoglycoproteins containing the synthetic terminal disaccharide or the whole trisaccharide chemically conjugated to such as bovine or human serum albumin, has found its greatest utility in the serological diagnosis, confirmation and management of lepromatous leprosy. PGL-I has also been implicated in the tropism of M. leprae for Schwann cells, through specific binding to laminin, and to play an important role in downregulation of the inflammatory immune response and inhibition of dendritic cell maturation and activation, thereby facilitating the persistence of M. leprae/leprosy.
Leprosy review 12/2011; 82(4):344-57. · 1.04 Impact Factor
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ABSTRACT: Our aim was to compare the performance of three serological assays in leprosy patients and their household contacts utilising two quantitative ELISA tests using native PGL-I (PGL-1 ELISA), synthetic ND-O-HSA (ND-O-HSA ELISA), and the semi-quantitative lateral flow test (ML Flow).
Comparisons among three immunological assays, PGL-I ELISA, ND-O-HSA ELISA, and ML Flow were performed in 154 leprosy patients, 191 household contacts and 52 health subjects.
The sensitivity results of the PGL-1, ND-O-HSA, and ML Flow were 68.83%, 63.84%, and 60.65%, respectively, with specificity of 98% for both ELISA assays. The native and synthetic PGL-I ELISA assays detected antibodies in 22.73% and 31.82% of the paucibacillary (PB) patients, respectively and the ML Flow test did not detect antibodies in this group. The ML Flow test was able to discriminate patients into PB or multibacillary (MB) forms, while the native PGL-I and ND-O-HSA was correlated with the bacillary load and the Ridley-Jopling clinical forms. In household contacts, the native PGL-I, ND-O-HSA, and ML Flow assays detected seropositivity of 25%, 17%, and 10%, respectively.
The use of ELISA and ML Flow tests are thus recommended as additional tools in the diagnosis and classification of the clinical forms, aiding in prescribing the correct treatment regimen to prevent subsequent nerve damage and disability, and besides, the PGL-I ELISA may be used to detect subclinical infection in leprosy.
Leprosy review 12/2011; 82(4):389-401. · 1.04 Impact Factor
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ABSTRACT: Two genes from the "mycobacterial arabinogalactan biosynthetic cluster" spanning the region from Rv3779 to Rv3809c in the genome of Mycobacterium tuberculosis H37Rv were annotated as possible components of the ATP-binding cassette transporter. Rv3781 encodes a nucleotide-binding domain and Rv3783 determines production of a membrane-spanning domain. We have examined possible roles of these genes in mycobacterial cell wall biosynthesis through inactivation of their respective orthologs in Mycobacterium smegmatis mc(2)155, phenotypic characterization of the mutant strains via metabolic labeling with [U-(14)C]-glucose, cell-free reactions with UDP-[U-(14)C]-galactose monitoring galactan build-up and transcriptional analysis. Several lines of evidence suggest that this ABC transporter is involved in biosynthesis of arabinogalactan, although more investigation is needed to establish its precise role or the transported substrate.
General Physiology and Biophysics 09/2011; 30(3):239-50. · 1.19 Impact Factor
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ABSTRACT: Leprosy is a disease of the skin and peripheral nervous system caused by the obligate intracellular bacterium Mycobacterium leprae. The clinical presentations of leprosy are spectral, with the severity of disease determined by the balance between the cellular and humoral immune response of the host. The exact mechanisms that facilitate disease susceptibility, onset and progression to certain clinical phenotypes are presently unclear. Various studies have examined lipid metabolism in leprosy, but there has been limited work using whole metabolite profiles to distinguish the clinical forms of leprosy.
In this study we adopted a metabolomics approach using high mass accuracy ultrahigh pressure liquid chromatography mass spectrometry (UPLC-MS) to investigate the circulatory biomarkers in newly diagnosed untreated leprosy patients. Sera from patients having bacterial indices (BI) below 1 or above 4 were selected, subjected to UPLC-MS, and then analyzed for biomarkers which distinguish the polar presentations of leprosy. We found significant increases in the abundance of certain polyunsaturated fatty acids (PUFAs) and phospholipids in the high-BI patients, when contrasted with the levels in the low-BI patients. In particular, the median values of arachidonic acid (2-fold increase), eicosapentaenoic acid (2.6-fold increase) and docosahexaenoic acid (1.6-fold increase) were found to be greater in the high-BI patients.
Eicosapentaenoic acid and docosahexaenoic acid are known to exert anti-inflammatory properties, while arachidonic acid has been reported to have both pro- and anti-inflammatory activities. The observed increase in the levels of several lipids in high-BI patients may provide novel clues regarding the biological pathways involved in the immunomodulation of leprosy. Furthermore, these results may lead to the discovery of biomarkers that can be used to investigate susceptibility to infection, facilitate early diagnosis and monitor the progression of disease.
PLoS Neglected Tropical Diseases 09/2011; 5(9):e1303. · 4.69 Impact Factor
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Wei Li,
Rama M Sakamuri,
Danielle E Lyons,
Florenda M Orcullo,
Vidyagouri Shinde,
Edred Lao Dela Pena,
Armi A Maghanoy,
Irene B Mallari,
Esterlina V Tan,
Indira Nath, Patrick J Brennan,
Marivic Balagon,
Varalakshmi Vissa
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ABSTRACT: Drug resistance surveillance identified six untreated leprosy patients in the Philippines with Mycobacterium leprae folP1 mutations which confer dapsone resistance. Five patients share a village of residence; four who carried the mutation, Thr53Val, were also linked by M. leprae variable-number tandem repeat (VNTR) strain types. In India, folP1 mutations were detected in two relapse patients with a history of dapsone treatment. Mutations were not found in the rifampin target gene rpoB. These findings indicate that dapsone resistance is being transmitted.
Antimicrobial Agents and Chemotherapy 08/2011; 55(11):5384-7. · 4.84 Impact Factor
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ABSTRACT: The cell wall of mycobacteria consists of an outer membrane, analogous to that of gram-negative bacteria, attached to the peptidoglycan (PG) via a connecting polysaccharide arabinogalactan (AG). Although the primary structure of these components is fairly well deciphered, issues such as the coverage of the PG layer by covalently attached mycolates in the outer membrane and the spatial details of the mycolic acid attachment to the arabinan have remained unknown. It is also not understood how these components work together to lead to the classical acid-fast staining of mycobacteria. Because the majority of Mycobacterium tuberculosis bacteria in established experimental animal infections are acid-fast negative, clearly cell wall changes are occurring. To address both the spatial properties of mycobacterial cell walls and to begin to study the differences between bacteria grown in animals and cultures, the cell walls of Mycobacterium leprae grown in armadillos was characterized and compared with that of M. tuberculosis grown in culture. Most fundamentally, it was determined that the cell wall of M. leprae contained significantly more mycolic acids attached to PG than that of in vitro grown M. tuberculosis (mycolate:PG ratios of 21:10 versus 16:10, respectively). In keeping with this difference, more arabinogalactan (AG) molecules, linking the mycolic acids to PG, were found. Differences in the structures of the AG were also found; the AG of M. leprae is smaller than that of M. tuberculosis, although the same basic structural motifs are retained.
Journal of Biological Chemistry 05/2011; 286(26):23168-77. · 4.77 Impact Factor
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John S Spencer,
Hee Jin Kim,
William H Wheat,
Delphi Chatterjee,
Marivic V Balagon,
Roland V Cellona,
Esterlina V Tan,
Robert Gelber,
Paul Saunderson,
Malcolm S Duthie,
Stephen T Reece,
William Burman,
Robert Belknap,
William R Mac Kenzie,
Annemieke Geluk,
Linda Oskam,
Hazel M Dockrell, Patrick J Brennan
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ABSTRACT: A simple serodiagnostic test based on the Mycobacterium leprae-specific phenolic glycolipid I(PGL-I), for individuals with leprosy is nearly universally positive in leprosy patients with high bacillary loads but cannot be used as a stand-alone diagnostic test for the entire spectrum of the disease process. For patients with early infection with no detectable acid-fast bacilli in lesions or with low or no antibody titer to PGL-I, as in those at the tuberculoid end of the disease spectrum, this diagnostic approach has limited usefulness. To identify additional M. leprae antigens that might enhance the serological detection of these individuals, we have examined the reactivity patterns of patient sera to PGL-I, lipoarabinomannan (LAM), and six recombinant M. leprae proteins (ML1877, ML0841, ML2028, ML2038, ML0380, and ML0050) by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Overall, the responses to ML2028 (Ag85B) and ML2038 (bacterioferritin) were consistently high in both multibacillary and paucibacillary groups and weak or absent in endemic controls, while responses to other antigens showed considerable variability, from strongly positive to completely negative. This analysis has given a clearer understanding of some of the differences in the antibody responses between individuals at opposite ends of the disease spectrum, as well as illustrating the heterogeneity of antibody responses toward protein, carbohydrate, and glycolipid antigens within a clinical group. Correlating these response patterns with a particular disease state could allow for a more critical assessment of the form of disease within the leprosy spectrum and could lead to better patient management.
Clinical and vaccine immunology: CVI 02/2011; 18(2):260-7. · 2.37 Impact Factor
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Pauline Peltier,
Martina Beláňová,
Petronela Dianišková,
Ruokun Zhou,
Ruixiang Blake Zheng,
Jean A Pearcey,
Maju Joe, Patrick J Brennan,
Caroline Nugier-Chauvin,
Vincent Ferrières,
Todd L Lowary,
Richard Daniellou,
Katarína Mikušová
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ABSTRACT: UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short "dead-end" intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis.
Chemistry & biology 12/2010; 17(12):1356-66. · 6.52 Impact Factor
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ABSTRACT: Phosphatidyl-myo-inositol mannosides (PIMs) are unique glycolipids found in abundant quantities in the inner and outer membranes of the cell envelope of all Mycobacterium species. They are based on a phosphatidyl-myo-inositol lipid anchor carrying one to six mannose residues and up to four acyl chains. PIMs are considered not only essential structural components of the cell envelope but also the structural basis of the lipoglycans (lipomannan and lipoarabinomannan), all important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy. Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis and regulation is still incomplete. Recent advances in the identification of key proteins involved in PIM biogenesis and the determination of the three-dimensional structures of the essential phosphatidyl-myo-inositol mannosyltransferase PimA and the lipoprotein LpqW have led to important insights into the molecular basis of this pathway.
Journal of Biological Chemistry 10/2010; 285(44):33577-83. · 4.77 Impact Factor
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Henrieta Skovierová,
Gérald Larrouy-Maumus,
Ha Pham,
Martina Belanová,
Nathalie Barilone,
Arunava Dasgupta,
Katarina Mikusová,
Brigitte Gicquel,
Martine Gilleron, Patrick J Brennan,
Germain Puzo,
Jérôme Nigou,
Mary Jackson
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ABSTRACT: The arabinogalactan (AG) of slow growing pathogenic Mycobacterium spp. is characterized by the presence of galactosamine (GalN) modifying some of the interior branched arabinosyl residues. The biosynthetic origin of this substituent and its role(s) in the physiology and/or pathogenicity of mycobacteria are not known. We report on the discovery of a polyprenyl-phospho-N-acetylgalactosaminyl synthase (PpgS) and the glycosyltransferase Rv3779 from Mycobacterium tuberculosis required, respectively, for providing and transferring the GalN substrate for the modification of AG. Disruption of either ppgS (Rv3631) or Rv3779 totally abolished the synthesis of the GalN substituent of AG in M. tuberculosis H37Rv. Conversely, expression of ppgS in Mycobacterium smegmatis conferred upon this species otherwise devoid of ppgS ortholog and any detectable polyprenyl-phospho-N-acetylgalactosaminyl synthase activity the ability to synthesize polyprenyl-phospho-N-acetylgalactosamine (polyprenyl-P-GalNAc) from polyprenyl-P and UDP-GalNAc. Interestingly, this catalytic activity was increased 40-50-fold by co-expressing Rv3632, the encoding gene of a small membrane protein apparently co-transcribed with ppgS in M. tuberculosis H37Rv. The discovery of this novel lipid-linked sugar donor and the involvement of a the glycosyltransferase C-type glycosyltransferase in its transfer onto its final acceptor suggest that pathogenic mycobacteria modify AG on the periplasmic side of the plasma membrane. The availability of a ppgS knock-out mutant of M. tuberculosis provides unique opportunities to investigate the physiological function of the GalN substituent and the potential impact it may have on host-pathogen interactions.
Journal of Biological Chemistry 10/2010; 285(53):41348-55. · 4.77 Impact Factor
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ABSTRACT: In order to generate substantial amounts of neoglycoconjugate needed for commercialization of diagnostic kits and high-throughput detection of leprosy, we developed a facile and high-yield synthesis of the corresponding disaccharide. Herein, the non-reducing disaccharide segment of phenolic glycolipid I from Mycobacterium leprae, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1-->4)-O-2,3-di-O-methyl-alpha-L-rhamnopyranose was synthesized by an improved procedure. The disaccharide was efficiently conjugated to bovine/human serum albumin, via acyl-azide intermediate, to form natural disaccharide-BSA/HSA neoglycoproteins that showed a high activity in serodiagnosis of leprosy. The disaccharide incorporated into the proteins was accurately measured by MALDI-TOF mass spectrometry. The serological activities of the neoglycoproteins against pooled human lepromatous leprosy sera were measured by ELISA and they were detectable at picogram amounts.
Bioorganic & medicinal chemistry letters 06/2010; 20(11):3250-3. · 2.65 Impact Factor
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Marc Monot,
Nadine Honoré,
Thierry Garnier,
Nora Zidane,
Diana Sherafi,
Alberto Paniz-Mondolfi,
Masanori Matsuoka,
G Michael Taylor,
Helen D Donoghue,
Abi Bouwman, [......],
Bishwa Raj Sapkota,
John S Spencer,
Jérôme Thomas,
Keith Harshman,
Pushpendra Singh,
Philippe Busso,
Alexandre Gattiker,
Jacques Rougemont, Patrick J Brennan,
Stewart T Cole
Nature Genetics 04/2010; 42(4):361. · 35.53 Impact Factor
