Caractérisation de la famille des protéines ABC et étude transcriptomique de la résistance à l'antimoine chez le parasite protozoaire Leishmania

Les parasites protozoaires du genre Leishmania sont responsables de différentes pathologies et représentent une cause importante de morbidité et de mortalité au niveau mondial. En absence de vaccins, le contrôle de la leishmaniose repose sur l’administration d’agents chimiothérapeutiques. Les médicaments disponibles sont peu nombreux et la plupart d’entre eux sont associés à des facteurs limitants, comme une toxicité relative à leur administration ou un coût trop élevé pour être utilisés de routine au niveau des zones endémiques les plus pauvres. Les composés à base d’antimoine pentavalent sont utilisés en thérapie depuis plusieurs décennies et demeurent encore aujourd’hui un traitement de première ligne contre les différentes formes de leishmanioses. Cependant, l’augmentation du nombre d’infections réfractaires au traitement, associée à la présence de foyers épidémiques de résistance, amenuisent le potentiel thérapeutique de ces molécules. Les mécanismes impliqués dans la résistance sont partiellement élucidés et ont révélé l’importance de la famille des protéines ATP-binding cassette (ABC) dans la résistance. De plus, l’étude de mutants résistants sélectionnés en laboratoire indique la présence de mécanismes de résistance supplémentaires, pour lesquels les gènes impliqués n’ont pas encore été identifiés. Les objectifs de cette thèse étaient i) de définir la famille des protéines ABC chez Leishmania et d’en effectuer l’analyse phylogénique, pour ensuite ii) étudier l’implication de la sous-famille ABCC dans la résistance à l’antimoine chez ce parasite, et finalement iii) de profiter de la disponibilité de la séquence du génome de Leishmania pour évaluer le profil d’expression génique associé au phénotype de résistance à l’antimoine à l’échelle génomique. L’analyse phylogénique a d’abord permis de démontrer la diversité de la famille des protéines ABC chez Leishmania, qui semble avoir évoluée par des événements de duplication génique suite à la divergence évolutive du parasite. Ensuite, des études de localisation protéique, associées à des expériences de surexpression génique, ont permis de déterminer la localisation intracellulaire de l’ensemble des protéines appartenant à la sous-famille ABCC chez Leishmania et de démontrer l’implication de deux d’entre elles dans la résistance à l’antimoine chez ce parasite. Enfin, une étude transcriptomique a confirmé l’importance du gène MRPA dans la résistance à l’antimoine et a permis d’identifier les mécanismes de recombinaison homologue impliqués dans son amplification chez une souche de L. infantum hautement résistante. Finalement, l’analyse transcriptomique a également révélé la présence de chromosomes aneuploïdes chez différents mutants résistants à l’antimoine, alors que la sélection d’une souche révertante partielle a permis d’observer une bonne corrélation entre les niveaux de résistance et le nombre de copies des chromosomes aneuploïdes.The parasite Leishmania is responsible for considerable morbidity and mortality around the world. No effective vaccine is yet available against this parasite and treatment thus relies on chemotherapy. Few drugs are available and most of them are associated with limitations such as toxicity and high cost. Pentavalent antimonials have been used for decades in the treatment of leishmaniasis and remain the mainstay against all forms of Leishmania infections in most endemic regions. However, the efficacy of these compounds is compromised by the selection of resistant parasites that are now described on a frequent basis in several endemic regions. The mechanisms involved in antimony resistance are partly understood and have pinpointed the role of ATP-binding cassette (ABC) proteins. Moreover, drug resistance studies with different in vitro-selected mutants have suggested the presence of unidentified mechanisms involved in antimony resistance. The objectives of this thesis were i) to define the complete ABC protein family in Leishmania and to analaze their evolution by phylogenetic analyses, ii) to assess the role of the entire ABCC subfamily in antimony resistance, and iii) to take advantage of the availability of the Leishmania genome sequence to study the gene expression profile associated with an antimony resistance phenotype at the genomic level. Phylogenetic analyses revealed the magnitude of the ABC gene family in Leishmania, which seemed to have undergone gene duplication events following the divergence of the Leishmania lineage. Moreover, subcellular localization experiments indicated that the entire ABCC protein subfamily is located to intracellular compartments in Leishmania, and gene overexpression experiments revealed the involvement of two of these proteins in antimony resistance. Finally, a whole-genome transcriptomic study confirmed the involvement of MRPA in antimony resistance and revealed the recombination events associated with its amplification in the highly resistant L. infantum Sb2000.1 mutant. More importantly, the transcriptomic study revealed the presence of aneuploid chromosomes in at least two different antimony-resistant mutants and selection of a partial revertant strain allowed the observation of a good correlation between the antimony resistance levels and the copy number of the aneuploid chromosomes

A genomic approach to understand interactions between Streptococcus pneumoniae and its bacteriophages

Background: Bacteriophage replication depends on bacterial proteins and inactivation of genes coding for such host factors should interfere with phage infection. To gain further insights into the interactions between S. pneumoniae and its pneumophages, we characterized S. pneumoniae mutants selected for resistance to the virulent phages SOCP or Dp-1. Results: S. pneumoniae R6-SOCPR and R6-DP1R were highly resistant to the phage used for their selection and no cross-resistance between the two phages was detected. Adsorption of SOCP to R6-SOCPR was partly reduced whereas no difference in Dp-1 adsorption was noted on R6-DP1R . The replication of SOCP was completely inhibited in R6-SOCPR while Dp-1 was severely impaired in R6-DP1R . Genome sequencing identified 8 and 2 genes mutated in R6-SOCPR and R6-DP1R , respectively. Resistance reconstruction in phage-sensitive S. pneumoniae confirmed that mutations in a GntR-type regulator, in a glycerophosphoryl phosphodiesterase and in a Mur ligase were responsible for resistance to SOCP. The three mutations were additive to increase resistance to SOCP. In contrast, resistance to Dp-1 in R6-DP1R resulted from mutations in a unique gene coding for a type IV restriction endonuclease. Conclusion: The characterization of mutations conferring resistance to pneumophages highlighted that diverse host genes are involved in the replication of phages from different families

Measuring the quality of judgement and decision-making in nursing

Aim. This paper discusses measurement of the quality of judgement and decision-making in nursing research. It examines theoretical and research issues surrounding how to measure judgement accuracy as a component of evaluating decision-making in nursing practice. Discussion. Judgement accuracy is discussed with reference to different methods of measurement, including comparing judgements with independent criteria and inter-judge approaches. Existing research on how judgement accuracy has been measured in nursing practice is examined. Evaluation of decisions is then discussed, including consideration of the process of decision-making and evaluating decision outcomes. Finally, existing research on decision-making in nursing is assessed and the strengths and limitations of different types of measurement discussed. Conclusion. We suggests that researchers examining the quality of judgement and decision-making in nursing need to be aware of both the strengths and limitations of existing methods of measurement. We also suggest that researchers need to use a number of different methods, including normative approaches such as Bayes' Theorem and Subjective Expected Utility Theory

Different mutations in a P-type ATPase transporter in Leishmania parasites are associated with cross-resistance to two leading drugs by distinct mechanisms

Work in TKS’s lab is supported by the Wellcome Trust grant 093228 and European Community’s Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED).Leishmania infantum is an etiological agent of the life-threatening visceral form of leishmaniasis. Liposomal amphotericin B (AmB) followed by a short administration of miltefosine (MF) is a drug combination effective for treating visceral leishmaniasis in endemic regions of India. Resistance to MF can be due to point mutations in the miltefosine transporter (MT). Here we show that mutations in MT are also observed in Leishmania AmB-resistant mutants. The MF-induced MT mutations, but not the AmB induced mutations in MT, alter the translocation/uptake of MF. Moreover, mutations in the MT selected by AmB or MF have a major impact on lipid species that is linked to cross-resistance between both drugs. These alterations include changes of specific phospholipids, some of which are enriched with cyclopropanated fatty acids, as well as an increase in inositolphosphoceramide species. Collectively these results provide evidence of the risk of cross-resistance emergence derived from current AmB-MF sequential or co-treatments for visceral leishmaniasis.Publisher PDFPeer reviewe

Identification of resistance determinants for a promising antileishmanial oxaborole series

Current treatment options for visceral leishmaniasis have several drawbacks, and clinicians are confronted with an increasing number of treatment failures. To overcome this, the Drugs for Neglected Diseases initiative (DNDi) has invested in the development of novel antileishmanial leads, including a very promising class of oxaboroles. The mode of action/resistance of this series to Leishmania is still unknown and may be important for its further development and implementation. Repeated in vivo drug exposure and an in vitro selection procedure on both extracellular promastigote and intracellular amastigote stages were both unable to select for resistance. The use of specific inhibitors for ABC-transporters could not demonstrate the putative involvement of efflux pumps. Selection experiments and inhibitor studies, therefore, suggest that resistance to oxaboroles may not emerge readily in the field. The selection of a genome-wide cosmid library coupled to next-generation sequencing (Cos-seq) was used to identify resistance determinants and putative targets. This resulted in the identification of a highly enriched cosmid, harboring genes of chromosome 2 that confer a subtly increased resistance to the oxaboroles tested. Moderately enriched cosmids encompassing a region of chromosome 34 contained the cleavage and polyadenylation specificity factor (cpsf) gene, encoding the molecular target of several related benzoxaboroles in other organisms

Gene expression modulation is associated with gene amplification, supernumerary chromosomes and chromosome loss in antimony-resistant Leishmania infantum

Antimonials remain the first line drug against the protozoan parasite Leishmania but their efficacy is threatened by resistance. We carried out a RNA expression profiling analysis comparing an antimony-sensitive and -resistant (Sb2000.1) strain of Leishmania infantum using whole-genome 70-mer oligonucleotide microarrays. Several genes were differentially expressed between the two strains, several of which were found to be physically linked in the genome. MRPA, an ATP-binding cassette (ABC) gene known to be involved in antimony resistance, was overexpressed in the antimony-resistant mutant along with three other tandemly linked genes on chromosome 23. This four gene locus was flanked by 1.4 kb repeated sequences from which an extrachromosomal circular amplicon was generated in the resistant cells. Interestingly, gene expression modulation of entire chromosomes occurred in the antimony-resistant mutant. Southern blots analyses and comparative genomic hybridizations revealed that this was either due to the presence of supernumerary chromosomes or to the loss of one chromosome. Leishmania parasites with haploid chromosomes were viable. Changes in copy number for some of these chromosomes were confirmed in another antimony-resistant strain. Selection of a partial revertant line correlated antimomy resistance levels and the copy number of aneuploid chromosomes, suggesting a putative link between aneuploidy and drug resistance in Leishmania

A novel marker, <em>ARM58</em>, confers antimony resistance to <em>Leishmania</em> spp

AbstractProtozoa of the Leishmania genus cause a variety of disease forms that rank at the top of the list of neglected tropical diseases. Anti-leishmanial drugs based on pentavalent antimony have been the mainstay of therapy for over 60years and resistance against them is increasingly encountered in the field. The biochemical basis for this is poorly understood and likely diverse. No stringent correlation between genetic markers and antimony resistance has so far been shown, prompting us to use a functional cloning approach to identify markers of resistance. Using gene libraries derived from drug-resistant and drug-sensitive Leishmania braziliensis clinical isolates in a functional cloning strategy, we repeatedly selected one gene locus located on chromosome 20 whose amplification confers increased antimony (III) resistance in vitro to an otherwise sensitive L. braziliensis clone. The gene responsible for the effect encodes a previously hypothetical protein that we dubbed LbrARM58. It comprises four repeats of a domain of unknown function, DUF1935, one of them harbouring a potential trans-membrane domain. The gene is so far unique to the Leishmania genus, while a structurally related gene without antimony resistance functionality is also found in Trypanosoma spp. Overexpression of LbrARM58 also confers antimony resistance to promastigotes and intracellular amastigotes of the related species Leishmania infantum, indicating a conserved function in Old World and New World Leishmania species. Our results also show that in spite of their RNAi system, L. braziliensis promastigotes can serve as acceptor cells for episomally propagated cosmid libraries, at least for the initial stages of functional cloning efforts

The effect of nurses' preparedness and nurse practitioner status on triage call management in primary care: a secondary analysis of cross-sectional data from the ESTEEM Trial

Background: Nurse-led telephone triage is increasingly used to manage demand for GP consultations in UK general practice. Previous studies are equivocal about the relationship between clinical experience and the call outcomes of nurse triage. Most research is limited to investigating nurse telephone triage in out-of-hours settings. Objective: To investigate whether the professional characteristics of primary care nurses undertaking computer decision supported software telephone triage are related to call disposition. Design: Questionnaire survey of nurses delivering the nurse intervention arm of the ESTEEM trial, to capture role type (practice nurse or nurse practitioner), prescriber status, number of years’ nursing experience, graduate status, previous experience of triage, and perceived preparedness for triage. Our main outcome was the proportion of triaged patients recommended for follow-up within the practice (call disposition), including all contact types (face-to-face, telephone or home visit), by a GP or nurse. Settings: : 15 General Practices and 7012 patients receiving the nurse triage intervention in four regions of the U.K. Participants: : 45 Nurse Practitioners (NPs) and Practice Nurse (PNs) trained in the use of CDSS. Methods: We investigated the associations between nursing characteristics and triage call disposition for patient ‘same-day’ appointment requests in general practice using multivariable logistic regression modelling. Results: Valid responses from 35 nurses (78%) from 14 practices: 31/35 (89%) had ≥10 years’ experience with 24/35 (69%) having ≥20 years. Most patient contacts (3842/4605; 86%) were recommended for follow-up within the practice. Nurse practitioners were less likely to recommend patients for follow-up odds ratio (OR) 0.19, 95% confidence interval (CI) 0.07; 0.49 than practice nurses. Nurses who reported that their previous experience had prepared them less well for triage were more likely to recommend patients for follow-up (OR 3.17, 95% CI 1.18; 5.55). Conclusion: Nurse characteristics were associated with disposition of triage calls to within practice follow-up. Nurse practitioners or those who reported feeling ‘more prepared’ for the role were more likely to manage the call definitively. Practices considering nurse triage should ensure that nurses transitioning into new roles feel adequately prepared. While standardised training is necessary, it may not be sufficient to ensure successful implementation

Intrachromosomal tandem duplication and repeat expansion during attempts to inactivate the subtelomeric essential gene GSH1 in Leishmania

Gamma-glutamylcysteine synthetase encoded by GSH1 is the rate-limiting enzyme in the biosynthesis of glutathione and trypanothione in Leishmania. Attempts to generate GSH1 null mutants by gene disruption failed in Leishmania infantum. Removal of even a single allele invariably led to the generation of an extra copy of GSH1, maintaining two intact wild-type alleles. In the second and even third round of inactivation, the markers integrated at the homologous locus but always preserved two intact copies of GSH1. We probed into the mechanism of GSH1 duplication. GSH1 is subtelomeric on chromosome 18 and Southern blot analysis indicated that a 10-kb fragment flanked by 466-bp direct repeated sequences was duplicated in tandem on the same chromosomal allele each time GSH1 was targeted. Polymerase chain reaction analysis and sequencing confirmed the generation of novel junctions created at the level of the 466-bp repeats consequent to locus duplication. In loss of heterozygosity attempts, the same repeated sequences were utilized for generating extrachromosomal circular amplicons. Our results are consistent with break-induced replication as a mechanism for the generation of this regional polyploidy to compensate for the inactivation of an essential gene. This chromosomal repeat expansion through repeated sequences could be implicated in locus duplication in Leishmania