Mechanisms of drug resistance in Leishmania

The emergence of drug resistance in protozoan parasites is a major obstacle to their control. Since vaccines are not yet in sight for several of these parasites, there is on urgent need to develop new and better drugs. These antimicrobial agents will possibly be more expensive, and will therefore impose on additional burden in health-care costs and in the planning of public health policies of the developing countries. A better understanding of drug resistance, to try to circumvent or overcome it, and the search for new specific cellular targets of parasites are warranted. The development, in vitro, of drug-resistant parasite cell lines has been instrumental in our understanding of the mechanisms of drug resistance in parasitic protozoans. Marc Ouellette and Barbara Popodopoulou here present on overview of the recent progress on the elucidation of mechanisms of drug resistance in the protozoan parasite Leishmania, selected under laboratory conditions.     

Molecular mechanisms of drug resistance and its reversal in cancer

Chemotherapy is the main strategy for the treatment of cancer. However, the main problem limiting the success of chemotherapy is the development of multidrug resistance. The resistance can be intrinsic or acquired. The resistance phenotype is associated with the tumor cells that gain a cross-resistance to a large range of drugs that are structurally and functionally different. Multidrug resistance arises via many unrelated mechanisms, such as overexpression of energy-dependent efflux proteins, decrease in uptake of the agents, increase or alteration in drug targets, modification of cell cycle checkpoints, inactivation of the agents, compartmentalization of the agents, inhibition of apoptosis and aberrant bioactive sphingolipid metabolism. Exact elucidation of resistance mechanisms and molecular and biochemical approaches to overcome multidrug resistance have been a major goal in cancer research. This review comprises the mechanisms guiding multidrug resistance in cancer chemotherapy and also touches on approaches for reversing the resistance.     

Molecular mechanisms of drug resistance in tyrosine kinases cAbl and cKit

The inhibition of protein kinases has gained general acceptance as an effective approach to treat a wide range of cancers. However, in many cases, prolonged administration of kinase inhibitors often leads to acquired resistance, and the therapeutic effect is subsequently diminished. The wealth of recent studies using biochemical, kinetic, and structural approaches have revealed the molecular basis for the clinically observed resistance. In this review, we highlight several of the most common molecular mechanisms that lead to acquired resistance to kinase inhibitors observed with the cAbl (cellular form of the Abelson leukemia virus tyrosine kinase) and the type III receptor tyrosine kinase cKit, including a newly identified mechanism resulting from accelerated kinase activation caused by mutations in the activation loop. Strategies to overcome the loss of drug sensitivity that represents a challenge currently facing the field and the emerging approaches to circumvent resistance are discussed.     

Molecular mechanisms of drug resistance in tyrosine kinases cAbl and cKit

The inhibition of protein kinases has gained general acceptance as an effective approach to treat a wide range of cancers. However, in many cases, prolonged administration of kinase inhibitors often leads to acquired resistance, and the therapeutic effect is subsequently diminished. The wealth of recent studies using biochemical, kinetic, and structural approaches have revealed the molecular basis for the clinically observed resistance. In this review, we highlight several of the most common molecular mechanisms that lead to acquired resistance to kinase inhibitors observed with the cAbl (cellular form of the Abelson leukemia virus tyrosine kinase) and the type III receptor tyrosine kinase cKit, including a newly identified mechanism resulting from accelerated kinase activation caused by mutations in the activation loop. Strategies to overcome the loss of drug sensitivity that represents a challenge currently facing the field and the emerging approaches to circumvent resistance are discussed.     

Unusual genetic architecture of natural variation affecting drug resistance in Drosophila melanogaster

Naturally occurring genetic variation was quantified for survival time of adult Drosophila melanogaster exposed to chronic ingestion of the drugs nicotine, caffeine, dopamine, tyramine and octopamine. Responses to nicotine, tyramine and octopamine were genetically correlated in both sexes, whereas caffeine response correlated with starvation resistance. However, there is also genetic variation that is specific for each of the drugs. Females tended to be more resistant than males to nicotine and caffeine but sex-by-genotype interactions were also seen for these drugs and for the response to dopamine. An unusual and complex genetic architecture was observed in crosses between lines with different responses to caffeine ingestion. Additive and dominance components were clearly seen from the analysis of F1 individuals, but increased female resistance to caffeine in backcross generations and increased male sensitivity in F2 generations confused the interpretation of possible epistatic contributions.     

Estimating genetic correlations in natural populations

Information on the genetic correlation between traits provides fundamental insight into the constraints on the evolutionary process. Estimates of such correlations are conventionally obtained by raising individuals of known relatedness in artificial environments. However, many species are not readily amenable to controlled breeding programmes, and considerable uncertainty exists over the extent to which estimates derived under benign laboratory conditions reflect the properties of populations in natural settings. Here, non-invasive methods that allow the estimation of genetic correlations from phenotypic measurements derived from individuals of unknown relatedness are introduced. Like the conventional approach, these methods demand large sample sizes in order to yield reasonably precise estimates, and special precautions need to be taken to eliminate bias from shared environmental effects. Provided the sample consists of at least 20% or so relatives, informative estimates of the genetic correlation are obtainable with sample sizes of several hundred individuals, particularly if supplemental information on relatedness is available from polymorphic molecular markers.     

Quantitative genetic analysis of natural populations

Quantitative genetic studies in natural populations have been rare because they require large breeding programmes or known pedigrees. The relatedness that has been estimated from molecular markers can now be used to substitute for breeding, allowing studies of previously inaccessible species. Many behavioural ecologists have a sufficient number of markers and study species with characteristics that are amenable to this approach. It is now time to combine studies of selection with studies of genetic variation for a more complete understanding of behavioural evolution.     

The genetic consequences of selection in natural populations

The selection coefficient, s, quantifies the strength of selection acting on a genetic variant. Despite this parameter's central importance to population genetic models, until recently we have known relatively little about the value of s in natural populations. With the development of molecular genetic techniques in the late 20th century and the sequencing technologies that followed, biologists are now able to identify genetic variants and directly relate them to organismal fitness. We reviewed the literature for published estimates of natural selection acting at the genetic level and found over 3000 estimates of selection coefficients from 79 studies. Selection coefficients were roughly exponentially distributed, suggesting that the impact of selection at the genetic level is generally weak but can occasionally be quite strong. We used both nonparametric statistics and formal random‐effects meta‐analysis to determine how selection varies across biological and methodological categories. Selection was stronger when measured over shorter timescales, with the mean magnitude of s greatest for studies that measured selection within a single generation. Our analyses found conflicting trends when considering how selection varies with the genetic scale (e.g., SNPs or haplotypes) at which it is measured, suggesting a need for further research. Besides these quantitative conclusions, we highlight key issues in the calculation, interpretation, and reporting of selection coefficients and provide recommendations for future research.     

Molecular mechanisms of vancomycin resistance

Vancomycin and related glycopeptides are drugs of last resort for the treatment of severe infections caused by Gram‐positive bacteria such as Enterococcus species, Staphylococcus aureus, and Clostridium difficile. Vancomycin was long considered immune to resistance due to its bactericidal activity based on binding to the bacterial cell envelope rather than to a protein target as is the case for most antibiotics. However, two types of complex resistance mechanisms, each comprised of a multi‐enzyme pathway, emerged and are now widely disseminated in pathogenic species, thus threatening the clinical efficiency of vancomycin. Vancomycin forms an intricate network of hydrogen bonds with the d ‐Ala‐d ‐Ala region of Lipid II, interfering with the peptidoglycan layer maturation process. Resistance to vancomycin involves degradation of this natural precursor and its replacement with d ‐Ala‐d ‐lac or d ‐Ala‐d ‐Ser alternatives to which vancomycin has low affinity. Through extensive research over 30 years after the initial discovery of vancomycin resistance, remarkable progress has been made in molecular understanding of the enzymatic cascades responsible. Progress has been driven by structural studies of the key components of the resistance mechanisms which provided important molecular understanding such as, for example, the ability of this cascade to discriminate between vancomycin sensitive and resistant peptidoglycan precursors. Important structural insights have been also made into the molecular evolution of vancomycin resistance enzymes. Altogether this molecular data can accelerate inhibitor discovery and optimization efforts to reverse vancomycin resistance. Here, we overview our current understanding of this complex resistance mechanism with a focus on the structural and molecular aspects.     

Molecular genetic analysis of purine nucleobase transport in Leishmania major

Leishmania major and all other parasitic protozoa are unable to synthesize purines de novo and are therefore reliant upon uptake of preformed purines from their hosts via nucleobase and nucleoside transporters. L. major expresses two nucleobase permeases, NT3 that is a high affinity transporter for purine nucleobases and NT4 that is a low affinity transporter for adenine. nt3((-/-)) null mutant promastigotes were unable to replicate in medium containing 10 microM hypoxanthine, guanine, or xanthine and replicated slowly in 10 microM adenine due to residual low affinity uptake of that purine. The NT3 transporter mediated the uptake of the anti-leishmanial drug allopurinol, and the nt3((-/-)) mutants were resistant to killing by this drug. Expression of the NT3 permease was profoundly downregulated at the protein but not the mRNA level in stationary phase compared with logarithmic phase promastigotes. The nt4((-/-)) null mutant was quantitatively impaired in survival within murine bone marrow-derived macrophages. Extensive efforts to generate an nt3((-/-))/nt4((-/-)) dual null mutant were not successful, suggesting that one of the two nucleobase permeases must be retained for robust growth of the parasite. The phenotypes of these null mutants underscore the importance of purine nucleobase transporters in the Leishmania life cycle and pharmacology.     

Measuring genetic relatedness in natural populations: Methodology

The estimation of relatedness within social groups, such as the colonies of a population of social insects, is an important field for evaluating hypotheses concerning the evolution and maintenance of social behaviour. The methodology of this estimation from genetic data in the absence of pedigree information has been poorly understood; we develop this methodology for b, the regression coefficient of relatedness, and discuss its applications. Both b and G (the pedigree coefficient of relatedness) are potentially asymmetric coefficients, whereas φ, r, and F_ST are necessarily symmetric. We develop an estimator for b suitable for small samples, and also one for standard deviation, and examine the properties of both using sampling simulations. The b estimator returns values slightly below E(b), and the standard deviation estimator yields conservative confidence intervals. A comparative study of b and F_ST shows that, given the same set of data, b is estimated with greater reliability than is F_ST. As is the case for F_ST, b can be used to examine population structure at various levels, and b possesses the advantage of an estimator for its standard error, which can also be used to test for heterogeneity among the loci surveyed. The actual numbers of identical genes held in common by interacting individuals, and not simply their proportions, need to be considered in using coefficients of relatedness in inclusive fitness calculations. This necessity is handled by the weighted coefficients of relatedness, G′ and b′, which have been referred to in the literature as r (as have most relatedness measures).     

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

Leishmania spp., protozoan parasites with a digenetic life cycle, cause a spectrum of diseases in humans. Recently several Leishmania spp. have been sequenced which significantly boosted the number and quality of proteomic studies conducted. Here a historic review will summarize work of the pre-genomic era and then focus on studies after genome information became available. Firstly works comparing the different life cycle stages, in order to identify stage specific proteins, will be discussed. Identifying post-translational modifications by proteomics especially phosphorylation events will be discussed. Further the contribution of proteomics to the understanding of the molecular mechanism of drug resistance and the investigation of immunogenic proteins for the identification of vaccine candidates will be summarized. Approaches of how potentially secreted proteins were identified are discussed. So far 30-35% of the total predicted proteome of Leishmania spp. have been identified. This comprises mainly the abundant proteins, therefore the last section will look into technological approaches on how this coverage may be increased and what the gel-free and gel-based proteomics have to offer will be compared.     

达托霉素耐药分子机制研究进展

环脂肽抗生素达托霉素抗菌活性强,致病菌不容易产生耐药性,已成为治疗革兰氏阳性菌特别是耐药菌感染的一线药物。但由于广泛使用,仍然出现了达托霉素耐药菌。细胞膜磷脂代谢和细胞壁结构动态与致病菌达托霉素耐药密切相关。文中综述了达托霉素作用机制和耐药机制,以期对药物研发和临床用药有所裨益。