Publications (22)173.28 Total impact
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Article: Rapid detection of fluoroquinolone-resistant and heteroresistant Mycobacterium tuberculosis by use of sloppy molecular beacons and dual melting-temperature codes in a real-time PCR assay.
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ABSTRACT: Fluoroquinolones (FQ) are important second-line drugs to treat tuberculosis; however, FQ resistance is an emerging problem. Resistance has been mainly attributed to mutations in a 21-bp region of the Mycobacterium tuberculosis gyrA gene, often called the quinolone resistance-determining region (QRDR). We have developed a simple, rapid, and specific assay to detect FQ resistance-determining QRDR mutations. The assay amplifies the M. tuberculosis gyrA QRDR in an asymmetrical PCR followed by probing with two sloppy molecular beacons (SMBs) spanning the entire QRDR. Mutations are detected by melting temperature (T(m)) shifts that occur when the SMBs bind to mismatched sequences. By testing DNA targets corresponding to all known QRDR mutations, we found that one or both of the SMBs produced a T(m) shift of at least 3.6°C for each mutation, making mutation detection very robust. The assay was also able to identify mixtures of wild-type and mutant DNA, with QRDR mutants identified in samples containing as little as 5 to 10% mutant DNA. The assay was blindly validated for its ability to identify the QRDR mutations on DNA extracted from clinical M. tuberculosis strains. Fifty QRDR wild-type samples, 34 QRDR mutant samples, and 8 heteroresistant samples containing mixtures of wild-type and mutant DNA were analyzed. The results showed 100% concordance to conventional DNA sequencing, including a complete identification of all of the mixtures. This SMB T(m) shift assay will be a valuable molecular tool to rapidly detect FQ resistance and to detect the emergence of FQ heteroresistance in clinical samples from tuberculosis patients.Journal of clinical microbiology 12/2010; 49(3):932-40. · 4.16 Impact Factor -
Article: A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy.
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ABSTRACT: Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.Nature Communications 08/2010; 1:57. · 7.40 Impact Factor -
Article: Descriptive proteomic analysis shows protein variability between closely related clinical isolates of Mycobacterium tuberculosis.
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ABSTRACT: The use of isobaric tags such as iTRAQ allows the relative and absolute quantification of hundreds of proteins in a single experiment for up to eight different samples. More classical techniques such as 2-DE can offer a complimentary approach for the analysis of complex protein samples. In this study, the proteomes of secreted and cytosolic proteins of genetically closely related strains of Mycobacterium tuberculosis were analyzed. Analysis of 2-D gels afforded 28 spots with variations in protein abundance between strains. These were identified by MS/MS. Meanwhile, a rigorous statistical analysis of iTRAQ data allowed the identification and quantification of 101 and 137 proteins in the secreted and cytosolic fractions, respectively. Interestingly, several differences in protein levels were observed between the closely related strains BE, C28 and H6. Seven proteins related to cell wall and cell processes were more abundant in BE, while enzymes related to metabolic pathways (GltA2, SucC, Gnd1, Eno) presented lower levels in the BE strain. Proteins involved in iron and sulfur acquisition (BfrB, ViuB, TB15.3 and SseC2) were more abundant in C28 and H6. In general, iTRAQ afforded rapid identification of fine differences between protein levels such as those presented between closely related strains. This provides a platform from which the relevance of these differences can be assessed further using complimentary proteomic and biological modeling methods.Proteomics 03/2010; 10(10):1966-84. · 4.43 Impact Factor -
Article: Molecular epidemiology of Mycobacterium tuberculosis in a South African community with high HIV prevalence.
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ABSTRACT: To explore the relationship between human immunodeficiency virus (HIV) and Mycobacterium tuberculosis genotypes, we performed IS6110-based restriction fragment-length polymorphism analysis on M. tuberculosis culture specimens from patients with smear-positive tuberculosis in a periurban community in South Africa from 2001 through 2005. Among 151 isolates, 95 strains were identified within 26 families, with 54% clustering. HIV status was associated with W-Beijing strains (P = .009) but not with clustering per se. The high frequency of clustering suggests ongoing transmission in both HIV-negative and HIV-positive individuals in this community. The strong association between W-Beijing and HIV infection may have important implications for tuberculosis control.The Journal of Infectious Diseases 10/2009; 200(8):1207-11. · 6.41 Impact Factor -
Article: Comparative in vitro activity of oritavancin against recent, genetically diverse, community-associated meticillin-resistant Staphylococcus aureus (MRSA) isolates.
International journal of antimicrobial agents 10/2009; 35(1):93-4. · 3.03 Impact Factor -
Article: Genotyping of Mycobacterium tuberculosis clinical isolates using IS6110-based restriction fragment length polymorphism analysis.
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ABSTRACT: A number of phylogenetic studies of Mycobacterium tuberculosis have suggested a highly clonal population structure. Despite the extreme homogeneity of M. tuberculosis strains, the genome is punctuated by a number of polymorphic regions that give rise to sufficient diversity, thus forming the basis for molecular epidemiologic studies of tuberculosis. As such, insertion sequence (IS) 6110, which is unique to members of the M. tuberculosis complex and is present in variable numbers and in discrete genomic locales among strains, has been extensively used in molecular epidemiologic studies. Genotyping, using IS6110-based restriction fragment length polymorphism (RFLP), was standardized by the international community, and this has facilitated inter- and intralaboratory comparison, thereby serving as a model system for subspeciation of M. tuberculosis. When IS6110-based RFLP was used in conjunction with conventional epidemiologic data, its utility was realized. In this chapter, we discuss the basic methodology for conducting IS6110-based RFLP and analyzing the resulting hybridization profiles.Methods in molecular biology (Clifton, N.J.) 02/2009; 551:173-88. -
Article: Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis.
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ABSTRACT: Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%--and by 23% in combination with spoligotyping--among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold--by threefold in combination with spoligotyping--under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.Journal of Clinical Microbiology 01/2007; 44(12):4498-510. · 4.15 Impact Factor -
Article: Identifying Mycobacterium tuberculosis complex strain families using spoligotypes.
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ABSTRACT: We present a novel approach for analysis of Mycobacterium tuberculosis complex (MTC) strain genotyping data. Our work presents a first step in an ongoing project dedicated to the development of decision support tools for tuberculosis (TB) epidemiologists exploiting both genotyping and epidemiological data. We focus on spacer oligonucleotide typing (spoligotyping), a genotyping method based on analysis of a direct repeat (DR) locus. We use mixture models to identify strain families of MTC based on their spoligotyping patterns. Our algorithm, SPOTCLUST, incorporates biological information on spoligotype evolution, without attempting to derive the full phylogeny of MTC. We applied our algorithm to 535 different spoligotype patterns identified among 7166 MTC strains isolated between 1996 and 2004 from New York State TB patients. Two models were employed and validated: a 36-component model based on global spoligotype database SpolDB3, and a randomly initialized model (RIM) containing 48 components. Our analysis both confirmed previously expert-defined families of MTC strains and suggested certain new families. SPOTCLUST, which is available online, can be further improved by incorporating data obtained using additional strain genetic markers and epidemiological information. We demonstrate on New York City (NYC) patient data how the resulting models can potentially form the basis of TB control tools using genotyping.Infection Genetics and Evolution 12/2006; 6(6):491-504. · 3.13 Impact Factor -
Article: Strain-specific differences in two large Mycobacterium tuberculosis genotype clusters in isolates collected from homeless patients in New York City from 2001 to 2004.
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ABSTRACT: We studied two large Mycobacterium tuberculosis genotype clusters associated with recent outbreaks in homeless persons to determine factors associated with these tuberculosis (TB) strains. Isolates from all culture-positive TB cases diagnosed from 1 January 2001 to 31 December 2004 were genotyped. Patients whose isolates had identical restriction fragment length polymorphism patterns and spoligotypes were considered clustered. Health department records were reviewed and reinterviews attempted for clustered cases. Patients with the Cs30 and BEs75 strains were compared to other genotypically clustered cases and to each other. The two largest genotype clusters among homeless persons were the Cs30 strain (n = 105) and the BEs75 strain (n = 47). Fifty-one (49%) patients with the Cs30 strain and 28 (60%) with the BEs75 strain were homeless. Compared to patients with the BEs75 strain, patients with the Cs30 strain were less likely to be respiratory acid-fast bacillus smear positive (51% versus 72%). Furthermore, patients with the BEs75 strain were more likely to be HIV infected (74% versus 42%), which suggests that most patients with this strain advanced to disease after recent infection. Cases in clusters of strains that have been circulating in the community over a long time period, such as the Cs30 strain, require additional investigation to determine whether clustering is a result of recent transmission or reactivation of remote infection.Journal of Clinical Microbiology 09/2006; 44(8):2890-6. · 4.15 Impact Factor -
Article: Which patients' factors predict the rate of growth of Mycobacterium tuberculosis clusters in an urban community?
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ABSTRACT: Factors influencing tuberculosis cluster growth are poorly understood. The authors examined clusters of two or more culture-confirmed Mycobacterium tuberculosis cases between January 1, 2001, and December 31, 2003, that had insertion sequence 6110 (IS6110) restriction fragment length polymorphism and spoligotype patterns identical to those of another study case. Genotypes first seen in New York, New York, before or during 1993 were considered historical; recent strains were those first seen after 1993. The authors examined the effect of the combined characteristics of infectiousness of the first two cases in a cluster on the rate of cluster growth. Genotyping was performed for 2,408 (91.8%) of the 2,623 tuberculosis cases diagnosed; 873 cases were in 212 clusters. Thirty-one clusters had historical strains, 153 were recent, and 28 were of unknown period. Patients' infectiousness was not associated with the rate of cluster growth among historical strain clusters. Among recent strain clusters, infectiousness of both of the initial cases was associated with a higher rate of cluster growth compared with clusters in which neither initial case was infectious, upon adjustment for male sex (rate ratio = 2.62, 95% confidence interval: 1.19, 5.78). The rate of genotype cluster growth should be monitored regardless of how long the strain has been present in the community. However, infectiousness in the first two cases may be useful to prioritize genotype cluster investigations.American Journal of Epidemiology 08/2006; 164(1):21-31. · 5.22 Impact Factor -
Article: Insights into the evolutionary history of tubercle bacilli as disclosed by genetic rearrangements within a PE_PGRS duplicated gene pair.
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ABSTRACT: The highly homologous PE_PGRS (Proline-glutamic acid_polymorphic GC-rich repetitive sequence) genes are members of the PE multigene family which is found only in mycobacteria. PE genes are particularly abundant within the genomes of pathogenic mycobacteria where they seem to have expanded as a result of gene duplication events. PE_PGRS genes are characterized by their high GC content and extensive repetitive sequences, making them prone to recombination events and genetic variability. Comparative sequence analysis of Mycobacterium tuberculosis genes PE_PGRS17 (Rv0978c) and PE_PGRS18 (Rv0980c) revealed a striking genetic variation associated with this typical tandem duplicate. In comparison to the M. tuberculosis reference strain H37Rv, the variation (named the 12/40 polymorphism) consists of an in-frame 12-bp insertion invariably accompanied by a set of 40 single nucleotide polymorphisms (SNPs) that occurs either in PE_PGRS17 or in both genes. Sequence analysis of the paralogous genes in a representative set of worldwide distributed tubercle bacilli isolates revealed data which supported previously proposed evolutionary scenarios for the M. tuberculosis complex (MTBC) and confirmed the very ancient origin of "M. canettii" and other smooth tubercle bacilli. Strikingly, the identified polymorphism appears to be coincident with the emergence of the post-bottleneck successful clone from which the MTBC expanded. Furthermore, the findings provide direct and clear evidence for the natural occurrence of gene conversion in mycobacteria, which appears to be restricted to modern M. tuberculosis strains. This study provides a new perspective to explore the molecular events that accompanied the evolution, clonal expansion, and recent diversification of tubercle bacilli.BMC Evolutionary Biology 02/2006; 6:107. · 3.52 Impact Factor -
Article: Insights into the evolutionary history of tubercle bacilli as disclosed by genetic rearrangements within a PE_PGRS duplicated gene pair
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ABSTRACT: Abstract Background The highly homologous PE_PGRS (Proline-glutamic acid_polymorphic GC-rich repetitive sequence) genes are members of the PE multigene family which is found only in mycobacteria. PE genes are particularly abundant within the genomes of pathogenic mycobacteria where they seem to have expanded as a result of gene duplication events. PE_PGRS genes are characterized by their high GC content and extensive repetitive sequences, making them prone to recombination events and genetic variability. Results Comparative sequence analysis of Mycobacterium tuberculosis genes PE_PGRS17 (Rv0978c) and PE_PGRS18 (Rv0980c) revealed a striking genetic variation associated with this typical tandem duplicate. In comparison to the M. tuberculosis reference strain H37Rv, the variation (named the 12/40 polymorphism) consists of an in-frame 12-bp insertion invariably accompanied by a set of 40 single nucleotide polymorphisms (SNPs) that occurs either in PE_PGRS17 or in both genes. Sequence analysis of the paralogous genes in a representative set of worldwide distributed tubercle bacilli isolates revealed data which supported previously proposed evolutionary scenarios for the M. tuberculosis complex (MTBC) and confirmed the very ancient origin of " M. canettii " and other smooth tubercle bacilli. Strikingly, the identified polymorphism appears to be coincident with the emergence of the post-bottleneck successful clone from which the MTBC expanded. Furthermore, the findings provide direct and clear evidence for the natural occurrence of gene conversion in mycobacteria, which appears to be restricted to modern M. tuberculosis strains. Conclusion This study provides a new perspective to explore the molecular events that accompanied the evolution, clonal expansion, and recent diversification of tubercle bacilli.BMC Evolutionary Biology. 01/2006; -
Article: Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology
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ABSTRACT: Abstract Background The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database. Results The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network. Conclusion Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.BMC Microbiology. 01/2006; -
Article: Clinical and radiographic correlates of primary and reactivation tuberculosis: a molecular epidemiology study.
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ABSTRACT: The traditional teaching that pulmonary tuberculosis characterized by lymphadenopathy, effusions, and lower or mid lung zone infiltrates on chest radiography represents "primary" disease from recently acquired infection, whereas upper lobe infiltrates and cavities represent secondary or reactivation disease acquired in the more distant past, is not based on well-established clinical evidence. Furthermore, it is not known whether the atypical radiograph common in human immunodeficiency virus (HIV)-associated tuberculosis is due to a preponderance of primary progressive disease or altered immunity. To analyze the relationship between recently acquired and remotely acquired pulmonary tuberculosis, clinical and demographic variables, and radiographic features by using molecular fingerprinting and conventional epidemiology. A retrospective, hospital-based series of 456 patients treated at a New York City medical center between 1990 and 1999. Eligible patients had to have had at least 1 positive respiratory culture for Mycobacterium tuberculosis and available radiographic data. Radiographic appearance as measured by the presence or absence of 6 features: upper lobe infiltrate, cavitary lesion, adenopathy, effusions, lower or mid lung zone infiltrate, and miliary pattern. Radiographs were considered typical if they had an upper lobe infiltrate or cavity whether or not other features were present. Atypical radiographs were those that had adenopathy, effusion, or mid lower lung zone infiltrates or had none of the above features. Human immunodeficiency virus infection was most commonly associated with an atypical radiographic appearance on chest radiograph with an odds ratio of 0.20 (95% confidence interval, 0.13-0.31). Although a clustered fingerprint, representing recently acquired disease, was associated with typical radiograph in univariate analysis (odds ratio, 0.68; 95% confidence interval, 0.47-0.99), the association was lost when adjusted for HIV status. Time from acquisition of infection to development of clinical disease does not reliably predict the radiographic appearance of tuberculosis. Human immunodeficiency virus status, a probable surrogate for the integrity of the host immune response, is the only independent predictor of radiographic appearance. The altered radiographic appearance of pulmonary tuberculosis in HIV is due to altered immunity rather than recent acquisition of infection and progression to active disease.JAMA The Journal of the American Medical Association 07/2005; 293(22):2740-5. · 30.03 Impact Factor -
Article: Differential monocyte activation underlies strain-specific Mycobacterium tuberculosis pathogenesis.
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ABSTRACT: In vitro infection of monocytes with Mycobacterium tuberculosis HN878 and related W/Beijing isolates preferentially induced interleukin-4 (IL-4) and IL-13, which characterize Th2 polarized immunity. In contrast, CDC1551 induced more IL-12 and other molecules associated with phagocyte activation and Th1 protective immunity. The differential cytokine-chemokine response was mediated by extracted lipids, suggesting that these molecules regulate host responses to infection.Infection and Immunity 10/2004; 72(9):5511-4. · 4.16 Impact Factor -
Article: Origins of community strains of methicillin-resistant Staphylococcus aureus.
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ABSTRACT: To characterize methicillin-resistant Staphylococcus aureus (MRSA) strains circulating in the community, we identified predictors of isolating community MRSA and genotyped a sample of MRSA collected from a community-based, high-risk population. Computerized databases of the Community Health Network of San Francisco and the Clinical Microbiology Laboratory were searched electronically for the years 1992-1999 to identify community-onset infections caused by MRSA. Sequential analyses were performed to identify predictors of MRSA strains. The majority (58%) of infections were caused by strains traceable to the hospital or to long-term care facilities. Injection drug use was associated with infections that were not associated with health care settings. Genotypes for 20 of 35 MRSA isolates recovered from injection drug users did not match any of >600 genotypes of clinical isolates. In a nonoutbreak setting, the hospital was the main source of community MRSA; however, the presence of genetically distinct and diverse MRSA strains indicates MRSA strains now also originate from the community.Clinical Infectious Diseases 07/2004; 39(1):47-54. · 9.15 Impact Factor -
Article: Hospital transmission of community-acquired methicillin-resistant Staphylococcus aureus among postpartum women.
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ABSTRACT: Infections caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are being increasingly observed in patients who lack traditional risk factors. We described 8 postpartum women who developed skin and soft-tissue infections caused by MRSA at a mean time of 23 days (range, 4-73 days) after delivery. Infections included 4 cases of mastitis (3 of which progressed to breast abscess), a postoperative wound infection, cellulitis, and pustulosis. The outbreak strains were compared with the prototype CA-MRSA strain MW2 and found to be indistinguishable by pulsed-field gel electrophoresis. All were spa type 131, all contained the staphylococcal chromosomal cassette mec type IV, and all expressed Panton-Valentine leukocidin and staphylococcal enterotoxins C and H. The route of transmission was not discovered: the results of surveillance cultures of samples obtained from employees of the hospital, the hospital environment, and newborns were negative for the outbreak strain. We report that MW2, which was previously limited to the midwestern United States, has spread to the northeastern United States and has become a health care-associated pathogen.Clinical Infectious Diseases 12/2003; 37(10):1313-9. · 9.15 Impact Factor -
Article: Mutations in putative mutator genes of Mycobacterium tuberculosis strains of the W-Beijing family.
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ABSTRACT: Alterations in genes involved in the repair of DNA mutations (mut genes) result in an increased mutation frequency and better adaptability of the bacterium to stressful conditions. W-Beijing genotype strains displayed unique missense alterations in three putative mut genes, including two of the mutT type (Rv3908 and mutT2) and ogt. These polymorphisms were found to be characteristic and unique to W-Beijing phylogenetic lineage. Analysis of the mut genes in 55 representative W-Beijing isolates suggests a sequential acquisition of the mutations, elucidating a plausible pathway of the molecular evolution of this clonal family. The acquisition of mut genes may explain in part the ability of the isolates of W-Beijing type to rapidly adapt to their environment.Emerging infectious diseases 08/2003; 9(7):838-45. · 6.17 Impact Factor -
Article: Targeting tuberculosis and malaria through inhibition of Enoyl reductase: compound activity and structural data.
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ABSTRACT: Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD+ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.Journal of Biological Chemistry 07/2003; 278(23):20851-9. · 4.77 Impact Factor -
Article: Targeting Tuberculosis and Malaria through Inhibition of Enoyl Reductase
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ABSTRACT: Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD+ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.Journal of Biological Chemistry 06/2003; 278(23):20851-20859. · 4.77 Impact Factor
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2007
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Institut Pasteur Paris
Paris, Ile-de-France, France
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