PubMed Health⌕ Search

Biomedical subjects

David Alland

Publications and source records attributed to David Alland.

17 recordsLinked to original sources
PubMed2006

Role of large sequence polymorphisms (LSPs) in generating genomic diversity among clinical isolates of Mycobacterium tuberculosis and the utility of LSPs in phylogenetic analysis.

Mycobacterium tuberculosis strains contain different genomic insertions or deletions called large sequence polymorphisms (LSPs). Distinguishing between LSPs that occur one time versus ones that occur repeatedly in a genomic region may provide insights into the biological roles of LSPs and identify useful phylogenetic markers. We analyzed 163 clinical M. tuberculosis isolates for 17 LSPs identified in a genomic comparison of M. tuberculosis strains H37Rv and CDC1551. LSPs were mapped onto a single-nucleotide polymorphism (SNP)-based phylogenetic tree created using nine novel SNP markers that were found to reproduce a 212-SNP-based phylogeny. Four LSPs (group A) mapped to a single SNP tree segment. Two LSPs (group B) and 11 LSPs (group C) were inferred to have arisen independently in the same genomic region either two or more than two times, respectively. None of the group A LSPs but one group B LSP and five group C LSPs were flanked by IS6110 sequences in the references strains. Genes encoding members of the proline-glutamic acid or proline-proline-glutamic acid protein families were present only in group B or C LSPs. SNP- versus LSP-based phylogenies were also compared. We classified each isolate into 58 LSP types by using a separate LSP-based phylogenetic analysis and mapped the LSP types onto the SNP tree. LSPs often assigned isolates to the correct phylogenetic lineage; however, significant mistakes occurred for 6/58 (10%) of the LSP types. In conclusion, most LSPs occur in genomic regions that are prone to repeated insertion/deletion events and were responsible for an unexpectedly high degree of genomic variation in clinical M. tuberculosis. Group B and C LSPs may represent polymorphisms that occur due to selective pressure and affect the phenotype of the organism, while group A LSPs are preferable phylogenetic markers.

Gene Deletion↗
PubMed2006

Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid.

Isoniazid is one of the most effective antituberculosis drugs, yet its precise mechanism of action is still controversial. Using specialized linkage transduction, a single point mutation allele (S94A) within the putative target gene inhA was transferred in Mycobacterium tuberculosis. The inhA(S94A) allele was sufficient to confer clinically relevant levels of resistance to isoniazid killing and inhibition of mycolic acid biosynthesis. This resistance correlated with the decreased binding of the INH-NAD inhibitor to InhA, as shown by enzymatic and X-ray crystallographic analyses, and establishes InhA as the primary target of isoniazid action in M. tuberculosis.

Antitubercular Agents↗
PubMed2006

Probing genomic diversity and evolution of Escherichia coli O157 by single nucleotide polymorphisms.

Infections by Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) are the predominant cause of bloody diarrhea and hemolytic uremic syndrome in the United States. In silico comparison of the two complete STEC O157 genomes (Sakai and EDL933) revealed a strikingly high level of sequence identity in orthologous protein-coding genes, limiting the use of nucleotide sequences to study the evolution and epidemiology of this bacterial pathogen. To systematically examine single nucleotide polymorphisms (SNPs) at a genome scale, we designed comparative genome sequencing microarrays and analyzed 1199 chromosomal genes (a total of 1,167,948 bp) and 92,721 bp of the large virulence plasmid (pO157) of eleven outbreak-associated STEC O157 strains. We discovered 906 SNPs in 523 chromosomal genes and observed a high level of DNA polymorphisms among the pO157 plasmids. Based on a uniform rate of synonymous substitution for Escherichia coli and Salmonella enterica (4.7x10(-9) per site per year), we estimate that the most recent common ancestor of the contemporary beta-glucuronidase-negative, non-sorbitolfermenting STEC O157 strains existed ca. 40 thousand years ago. The phylogeny of the STEC O157 strains based on the informative synonymous SNPs was compared to the maximum parsimony trees inferred from pulsed-field gel electrophoresis and multilocus variable numbers of tandem repeats analysis. The topological discrepancies indicate that, in contrast to the synonymous mutations, parts of STEC O157 genomes have evolved through different mechanisms with highly variable divergence rates. The SNP loci reported here will provide useful genetic markers for developing high-throughput methods for fine-resolution genotyping of STEC O157. Functional characterization of nucleotide polymorphisms should shed new insights on the evolution, epidemiology, and pathogenesis of STEC O157 and related pathogens.

Chromosome Mapping↗
PubMed2006

Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis.

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.

Alleles↗
PubMed2006

Arginine homeostasis in J774.1 macrophages in the context of Mycobacterium bovis BCG infection.

The competition for L-arginine between the inducible nitric oxide synthase and arginase contributes to the outcome of several parasitic and bacterial infections. The acquisition of L-arginine, however, is important not only for the host cells but also for the intracellular pathogen. In this study we observe that strain AS-1, the Mycobacterium bovis BCG strain lacking the Rv0522 gene, which encodes an arginine permease, perturbs l-arginine metabolism in J774.1 murine macrophages. Infection with AS-1, but not with wild-type BCG, induced l-arginine uptake in J774.1 cells. This increase in L-arginine uptake was independent of activation with gamma interferon plus lipopolysaccharide and correlated with increased expression of the MCAT1 and MCAT2 cationic amino acid transport genes. AS-1 infection also enhanced arginase activity in resting J774.1 cells. Survival studies revealed that AS-1 survived better than BCG within resting J774.1 cells. Intracellular growth of AS-1 was further enhanced by inhibiting arginase and ornithine decarboxylase activities in J774.1 cells using L-norvaline and difluoromethylornithine treatment, respectively. These results suggest that the arginine-related activities of J774.1 macrophages are affected by the arginine transport capacity of the infecting BCG strain. The loss of Rv0522 gene-encoded arginine transport may have induced other cationic amino acid transport systems during intracellular growth of AS-1, allowing better survival within resting macrophages.

Amino Acid Transport Systems, Basic↗
PubMed2006

Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set.

We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of the M. tuberculosis samples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting that M. tuberculosis first arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction of M. tuberculosis into these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured by GST. Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences of M. tuberculosis.

Algorithms↗
PubMed2005

The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol.

Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR-pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two-dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.

Adaptation, Physiological↗
PubMed2005

Altered NADH/NAD+ ratio mediates coresistance to isoniazid and ethionamide in mycobacteria.

The front-line antituberculosis drug isoniazid (INH) and the related drug ethionamide (ETH) are prodrugs that upon activation inhibit the synthesis of mycolic acids, leading to bactericidal activity. Coresistance to INH and ETH can be mediated by dominant mutations in the target gene inhA, encoding an enoyl-ACP reductase, or by recessive mutations in ndh, encoding a type II NADH dehydrogenase (NdhII). To address the mechanism of resistance mediated by the latter, we have isolated novel ndh mutants of Mycobacterium smegmatis and Mycobacterium bovis BCG. The M. smegmatis ndh mutants were highly resistant to INH and ETH, while the M. bovis BCG mutants had low-level resistance to INH and ETH. All mutants had defects in NdhII activity resulting in an increase in intracellular NADH/NAD(+) ratios. Increasing NADH levels were shown to protect InhA against inhibition by the INH-NAD adduct formed upon INH activation. We conclude that ndh mutations mediate a novel mechanism of resistance by increasing the NADH cellular concentration, which competitively inhibits the binding of INH-NAD or ETH-NAD adduct to InhA.

Antitubercular Agents↗
PubMed2005

Role of embB codon 306 mutations in Mycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance.

Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P = 0.008) and katG315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.

Antitubercular Agents↗
PubMed2004

Novel hairpin-shaped primer assay to study the association of the -44 single-nucleotide polymorphism of the DEFB1 gene with early-onset periodontal disease.

A powerful, cost-effective new method for studying single-nucleotide polymorphisms (SNPs) is described. This method is based on the use of hairpin-shaped primers (HP), which give a sensitive and specific PCR amplification of each specific allele, without the use of costly fluorophore-labeled probes and any post-PCR manipulation. The amplification is monitored in real-time using SYBR Green I dye and takes only 2 h to yield results. The HP assay has a simple design and utilizes a conventional real-time PCR apparatus. The -44 C-->G transversion in the DEFB1 gene (which encodes human beta-defensin 1) has been previously associated with Candida carriage in oral epithelia. In this study, we analyzed the association between early-onset periodontal disease (EOP) and the -44 SNP. We used an HP assay to study the distribution of the -44 SNP in 264 human DNAs obtained from two cohorts of EOP patients and healthy controls from different ethnic backgrounds. The results indicate that the -44 SNP has a similar distribution between EOP and healthy patients, suggesting that it is not associated with the disease.

Alleles↗
PubMed2004

Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay.

We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis.

Antibiotics, Antitubercular↗
PubMed2004

Hairpin primers for simplified single-nucleotide polymorphism analysis of Mycobacterium tuberculosis and other organisms.

We describe a novel, simple, rapid, and highly sensitive method to detect single-nucleotide polymorphisms (SNPs) in Mycobacterium tuberculosis and other organisms. Amplification refractory mutation (ARMS) SNP assays were modified by converting the SNP-detecting linear primers in the ARMS assay to hairpin-shaped primers (HPs) through the addition of a 5' tail complementary to the 3' end of the linear primer. The improved ability of these primers to detect SNPs in M. tuberculosis was compared in a real-time PCR with SYBR-I green dye. Linear primers resulted in incorrect or indeterminate allele designation for 6 of the 13 SNP alleles tested in seven different SNP assays, while HPs determined the correct SNP in all cases. We compared the cycle threshold differences (DeltaC(t)) between the reactions containing primer-template matches and the reactions containing primer-template mismatches (where a larger DeltaC(t) indicates a more robust assay). The use of HPs dramatically improved the mean DeltaC(t) values for the SNP assays (7.6 for linear primers and 11.2 for HPs). We designed 98 different HP assays for SNPs previously associated with resistance to the antibiotic isoniazid to test the large-scale utility of the HP approach. Assay design was successful in 72.4%, 83.7%, 88.8%, and 92.9% of the assays after one to four rounds of assay design, respectively. HP SNP assays are simple, sensitive, robust, and inexpensive. These advantages favor the application of this technique for SNP assays of M. tuberculosis and other organisms.

Base Sequence↗
PubMed2003

Targeting tuberculosis and malaria through inhibition of Enoyl reductase: compound activity and structural data.

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.

Animals↗
PubMed2003

Modeling bacterial evolution with comparative-genome-based marker systems: application to Mycobacterium tuberculosis evolution and pathogenesis.

The comparative-genomic sequencing of two Mycobacterium tuberculosis strains enabled us to identify single nucleotide polymorphism (SNP) markers for studies of evolution, pathogenesis, and epidemiology in clinical M. tuberculosis. Phylogenetic analysis using these "comparative-genome markers" (CGMs) produced a highly unusual phylogeny with a complete absence of secondary branches. To investigate CGM-based phylogenies, we devised computer models to simulate sequence evolution and calculate new phylogenies based on an SNP format. We found that CGMs represent a distinct class of phylogenetic markers that depend critically on the genetic distances between compared "reference strains." Properly distanced reference strains generate CGMs that accurately depict evolutionary relationships, distorted only by branch collapse. Improperly distanced reference strains generate CGMs that distort and reroot outgroups. Applying this understanding to the CGM-based phylogeny of M. tuberculosis, we found evidence to suggest that this species is highly clonal without detectable lateral gene exchange. We noted indications of evolutionary bottlenecks, including one at the level of the PHRI "C" strain previously associated with particular virulence characteristics. Our evidence also suggests that loss of IS6110 to fewer than seven elements per genome is uncommon. Finally, we present population-based evidence that KasA, an important component of mycolic acid biosynthesis, develops G312S polymorphisms under selective pressure.

Bacterial Proteins↗
PubMed2002

Methodological problems in the molecular epidemiology of tuberculosis.

In systematic studies of the molecular epidemiology of tuberculosis, DNA fingerprinting is used to estimate the fraction of incident cases attributable to recent transmission of Mycobacterium tuberculosis rather than reactivation disease and to identify risk factors for recent transmission. This approach is based on the premise that tuberculosis cases that share a DNA fingerprint are epidemiologically related while cases in which fingerprints are unique are due to remote infection that has reactivated. In this paper, the authors review the objectives and design of molecular epidemiologic studies of tuberculosis, describe current analytical approaches, and consider the impact of these different approaches on study results. Using data from a previously published investigation of the epidemiology of tuberculosis conducted from 1990 to 1993 among tuberculosis patients in New York City, New York, the authors show how selecting different measures of disease frequency, comparison groups, and sampling strategies may impact the results and interpretability of the study. They demonstrate ways to conduct sensitivity analyses of estimated results and suggest strategies that may improve the usefulness of this approach to studying tuberculosis.

Bias↗
PubMed2002

Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis.

The inhA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed inhA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either inhA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy inhA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing inhA plasmids uniformly exhibited 20-fold or greater increased resistance to INH and 10-fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH-susceptible M. tuberculosis strains. Using molecular beacons, quantified inhA and kasA mRNA levels showed that increased inhA mRNA levels corre--lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring inhA or kasA plasmids yielded the same conclusion: overexpressed inhA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, InhA is the primary target of action of INH and ETH in all three species.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗