Gene expression profiling and molecular characterization of antimony resistance in Leishmania amazonensis

Background

Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis.

Methods/Principal Findings

We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated.

Conclusions/Significance

Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers.     

Gene Amplification and Point Mutations in Pyrimidine Metabolic Genes in 5-Fluorouracil Resistant Leishmania infantum

Background

The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines.

Methodology/Principal Findings

Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import.

Conclusion/Significance

This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.     

Proteomic and Genomic Analyses of Antimony Resistant Leishmania infantum Mutant

Background

Antimonials remain the primary antileishmanial drugs in most developing countries. However, drug resistance to these compounds is increasing and our understanding of resistance mechanisms is partial.

Methods/Principal Findings

In the present study, quantitative proteomics using stable isotope labelling of amino acids in cell culture (SILAC) and genome next generation sequencing were used in order to better characterize in vitro generated Leishmania infantum antimony resistant mutant (Sb2000.1). Using the proteomic method, 58 proteins were found to be differentially regulated in Sb2000.1. The ABC transporter MRPA (ABCC3), a known marker of antimony resistance, was observed for the first time in a proteomic screen. Furthermore, transfection of its gene conferred antimony resistance in wild-type cells. Next generation sequencing revealed aneuploidy for 8 chromosomes in Sb2000.1. Moreover, specific amplified regions derived from chromosomes 17 and 23 were observed in Sb2000.1 and a single nucleotide polymorphism (SNP) was detected in a protein kinase (LinJ.33.1810-E629K).

Conclusion/Significance

Our results suggest that differentially expressed proteins, chromosome number variations (CNVs), specific gene amplification and SNPs are important features of antimony resistance in Leishmania.     

Subtelomeric chromosomal rearrangements in a large cohort of unexplained intellectually disabled individuals in Indonesia: A clinical and molecular study

CONTEXT:

Unbalanced subtelomeric chromosomal rearrangements are often associated with intellectual disability (ID) and malformation syndromes. The prevalence of such rearrangements has been reported to be 5-9% in ID populations.

AIMS:

To study the prevalence of subtelomeric rearrangements in the Indonesian ID population.

MATERIALS AND METHODS:

We tested 436 subjects with unexplained ID using multiplex ligation dependent probe amplification (MLPA) using the specific designed sets of probes to detect human subtelomeric chromosomal imbalances (SALSA P070 and P036D). If necessary, abnormal findings were confirmed by other MLPA probe kits, fluorescent in situ hybridization or Single Nucleotide Polymorphism array.

RESULTS:

A subtelomeric aberration was identified in 3.7% of patients (16/436). Details on subtelomeric aberrations and confirmation analyses are discussed.

CONCLUSION:

This is the first study describing the presence of subtelomeric rearrangements in individuals with ID in Indonesia. Furthermore, it shows that also in Indonesia such abnormalities are a prime cause of ID and that in developing countries with limited diagnostic services such as Indonesia, it is important and feasible to uncover the genetic etiology in a significant number of cases with ID.     

Multiple mutations in heterogeneous miltefosine-resistant Leishmania major population as determined by whole genome sequencing

Background

Miltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance.

Methods/Principal Findings

Analysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins—pyridoxal kinase and α-adaptin like protein—were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes.

Conclusions/Significance

Whole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes.     

Evolution of BCR/ABL Gene Mutation in CML Is Time Dependent and Dependent on the Pressure Exerted by Tyrosine Kinase Inhibitor

Background

Mutations in the ABL kinase domain and SH3-SH2 domain of the BCR/ABL gene and amplification of the Philadelphia chromosome are the two important BCR/ABL dependent mechanisms of imatinib resistance. Here, we intended to study the role played by TKI, imatinib, in selection of gene mutations and development of chromosomal abnormalities in Indian CML patients.

Methods

Direct sequencing methodology was employed to detect mutations and conventional cytogenetics was done to identify Philadelphia duplication.

Results

Among the different mechanisms of imatinib resistance, kinase domain mutations (39%) of the BCR/ABL gene were seen to be more prevalent, followed by mutations in the SH3-SH2 domain (4%) and then BCR/ABL amplification with the least frequency (1%). The median duration of occurrence of mutation was significantly shorter for patients with front line imatinib than those pre-treated with hydroxyurea. Patients with high Sokal score (p = 0.003) showed significantly higher incidence of mutations, as compared to patients with low/intermediate score. Impact of mutations on the clinical outcome in AP and BC was observed to be insignificant. Of the 94 imatinib resistant patients, only 1 patient exhibited duplication of Philadelphia chromosome, suggesting a less frequent occurrence of this abnormality in Indian CML patients.

Conclusion

Close monitoring at regular intervals and proper analysis of the disease resistance would facilitate early detection of resistance and thus aid in the selection of the most appropriate therapy.     

Telomeric gene deletion and intrachromosomal amplification in antimony‐resistant Leishmania

Antimonials are still the mainstay of treatment against leishmaniasis but drug resistance is increasing. We carried out short read next‐generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony‐resistant mutants. Copy number variations were consistently detected with both NGS and CGH. A major attribute of antimony resistance was a novel terminal deletion of variable length (67 kb to 204 kb) of the polyploid chromosome 31 in the three mutants. Terminal deletions in two mutants occurred at the level of inverted repeated sequences. The AQP1 gene coding for an aquaglyceroporin was part of the deleted region and its transfection into resistant mutants reverted resistance to SbIII. We also highlighted an intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded for ascorbate‐dependent peroxidase (APX) and glucose‐6‐phosphate dehydrogenase (G6PDH). Overexpression of these genes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS). Generation of a G6PDH null mutant in one revertant exhibited SbIII sensitivity and a decreased protection of ROS. Our genomic analyses and functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania.     

High Prevalence and Clinical Relevance of Genes Affected by Chromosomal Breaks in Colorectal Cancer

Background

Cancer is caused by somatic DNA alterations such as gene point mutations, DNA copy number aberrations (CNA) and structural variants (SVs). Genome-wide analyses of SVs in large sample series with well-documented clinical information are still scarce. Consequently, the impact of SVs on carcinogenesis and patient outcome remains poorly understood. This study aimed to perform a systematic analysis of genes that are affected by CNA-associated chromosomal breaks in colorectal cancer (CRC) and to determine the clinical relevance of recurrent breakpoint genes.

Methods

Primary CRC samples of patients with metastatic disease from CAIRO and CAIRO2 clinical trials were previously characterized by array-comparative genomic hybridization. These data were now used to determine the prevalence of CNA-associated chromosomal breaks within genes across 352 CRC samples. In addition, mutation status of the commonly affected APC, TP53, KRAS, PIK3CA, FBXW7, SMAD4, BRAF and NRAS genes was determined for 204 CRC samples by targeted massive parallel sequencing. Clinical relevance was assessed upon stratification of patients based on gene mutations and gene breakpoints that were observed in >3% of CRC cases.

Results

In total, 748 genes were identified that were recurrently affected by chromosomal breaks (FDR <0.1). MACROD2 was affected in 41% of CRC samples and another 169 genes showed breakpoints in >3% of cases, indicating that prevalence of gene breakpoints is comparable to the prevalence of well-known gene point mutations. Patient stratification based on gene breakpoints and point mutations revealed one CRC subtype with very poor prognosis.

Conclusions

We conclude that CNA-associated chromosomal breaks within genes represent a highly prevalent and clinically relevant subset of SVs in CRC.     

Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions

Background

Identifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. Plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. Post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. Identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. Currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes.

Results

We used a whole-genome sequencing (WGS) approach using Illumina HiSeq 2000 technology to investigate DNA tag insertion points and chromosomal deletion events in mutagenised, reduced virulence F. graminearum isolates identified in disease tests on wheat (Triticum aestivum). We developed the FindInsertSeq workflow to localise the DNA tag insertions to the nucleotide level. The workflow was tested using four mutants showing evidence of single and multi-copy insertions in DNA blot analysis. FindInsertSeq was able to identify both single and multi-copy concatenation insertion sites. By comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and DNA amplification were observed in three of the analysed mutants. A random data sampling approach revealed the minimum genome coverage required to survey the F. graminearum genome for alterations.

Conclusions

This study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete Fusarium graminearum. In some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. The FindInsertSeq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1412-9) contains supplementary material, which is available to authorized users.     

Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah,Malaysia

Background

Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission.

Methods

The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket.

Results

Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates.

Conclusion

Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.     

A Look into the Melting Pot: The mecC-Harboring Region Is a Recombination Hot Spot in Staphylococcus stepanovicii

Introduction

Horizontal gene transfer (HGT) is an important driver for resistance- and virulence factor accumulation in pathogenic bacteria such as Staphylococcus aureus.

Methods

Here, we have investigated the downstream region of the bacterial chromosomal attachment site (attB) for the staphylococcal cassette chromosome mec (SCCmec) element of a commensal mecC-positive Staphylococcus stepanovicii strain (IMT28705; ODD4) with respect to genetic composition and indications of HGT. S. stepanovicii IMT28705 was isolated from a fecal sample of a trapped wild bank vole (Myodes glareolus) during a screening study (National Network on “Rodent-Borne Pathogens”) in Germany. Whole genome sequencing (WGS) of IMT28705 together with the mecC-negative type strain CM7717 was conducted in order to comparatively investigate the genomic region downstream of attB (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KR732654","term_id":"922979931"}}KR732654 and {"type":"entrez-nucleotide","attrs":{"text":"KR732653","term_id":"922979910"}}KR732653).

Results

The bank vole isolate (IMT28705) harbors a mecC gene which shares 99.2% nucleotide (and 98.5% amino acid) sequence identity with mecC of MRSA_LGA251. In addition, the mecC-encoding region harbors the typical blaZ-mecC-mecR1-mecI structure, corresponding with the class E mec complex. While the sequences downstream of attB in both S. stepanovicii isolates (IMT28705 and CM7717) are partitioned by 15 bp direct repeats, further comparison revealed a remarkable low concordance of gene content, indicating a chromosomal “hot spot” for foreign DNA integration and exchange.

Conclusion

Our data highlight the necessity for further research on transmission routes of resistance encoding factors from the environmental and wildlife resistome.     

Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

Background

Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs).

Methodology/Principal Findings

Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid.

Conclusion/Significance

The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis.     

Performance of Four Transport and Storage Systems for Molecular Detection of Multidrug-Resistant Tuberculosis

Background

Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus.

Methods

Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing.

Results

For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%.

Conclusion

The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings.     

Identification of a Novel G2073A Mutation in 23S rRNA in Amphenicol-Selected Mutants of Campylobacter jejuni

Objectives

This study was conducted to examine the development and molecular mechanisms of amphenicol resistance in Campylobacter jejuni by using in vitro selection with chloramphenicol and florfenicol. The impact of the resistance development on growth rates was also determined using in vitro culture.

Methods

Chloramphenicol and florfenicol were used as selection agents to perform in vitro stepwise selection. Mutants resistant to the selective agents were obtained from the selection process. The mutant strains were compared with the parent strain for changes in MICs and growth rates. The 23S rRNA gene and the L4 and L22 ribosomal protein genes in the mutant strains and the parent strain were amplified and sequenced to identify potential resistance-associated mutations.

Results

C. jejuni strains that were highly resistant to chloramphenicol and florfenicol were obtained from in vitro selection. A novel G2073A mutation in all three copies of the 23S rRNA gene was identified in all the resistant mutants examined, which showed resistance to both chloramphenicol and florfenicol. In addition, all the mutants selected by chloramphenicol also exhibited the G74D modification in ribosomal protein L4, which was previously shown to confer a low-level erythromycin resistance in Campylobacter species. The mutants selected by florfenicol did not have the G74D mutation in L4. Notably, the amphenicol-resistant mutants also exhibited reduced susceptibility to erythromycin, suggesting that the selection resulted in cross resistance to macrolides.

Conclusions

This study identifies a novel point mutation (G2073A) in 23S rRNA in amphenicol-selected mutants of C. jejuni. Development of amphenicol resistance in Campylobacter likely incurs a fitness cost as the mutant strains showed slower growth rates in antibiotic-free media.     

Detection of MET Gene Copy Number in Cancer Samples Using the Droplet Digital PCR Method

Purpose

The analysis of MET gene copy number (CN) has been considered to be a potential biomarker to predict the response to MET-targeted therapies in various cancers. However, the current standard methods to determine MET CN are SNP 6.0 in the genomic DNA of cancer cell lines and fluorescence in situ hybridization (FISH) in tumor models, respectively, which are costly and require advanced technical skills and result in relatively subjective judgments. Therefore, we employed a novel method, droplet digital PCR (ddPCR), to determine the MET gene copy number with high accuracy and precision.

Methods

The genomic DNA of cancer cell lines or tumor models were tested and compared with the MET gene CN and MET/CEN-7 ratio determined by SNP 6.0 and FISH, respectively.

Results

In cell lines, the linear association of the MET CN detected by ddPCR and SNP 6.0 is strong (Pearson correlation = 0.867). In tumor models, the MET CN detected by ddPCR was significantly different between the MET gene amplification and non-amplification groups according to FISH (mean: 15.4 vs 2.1; P = 0.044). Given that MET gene amplification is defined as MET CN >5.5 by ddPCR, the concordance rate between ddPCR and FISH was 98.0%, and Cohen''s kappa coefficient was 0.760 (95% CI, 0.498–1.000; P <0.001).

Conclusions

The results demonstrated that the ddPCR method has the potential to quantify the MET gene copy number with high precision and accuracy as compared with the results from SNP 6.0 and FISH in cancer cell lines and tumor samples, respectively.     

Dolabelladienetriol, a Compound from Dictyota pfaffii Algae, Inhibits the Infection by Leishmania amazonensis

Background

Chemotherapy for leishmaniasis, a disease caused by Leishmania parasites, is expensive and causes side effects. Furthermore, parasite resistance constitutes an increasing problem, and new drugs against this disease are needed. In this study, we examine the effect of the compound 8,10,18-trihydroxy-2,6-dolabelladiene (Dolabelladienetriol), on Leishmania growth in macrophages. The ability of this compound to modulate macrophage function is also described.

Methodology/Principal Findings

Leishmania-infected macrophages were treated with Dolabelladienetriol, and parasite growth was measured using an infectivity index. Nitric oxide (NO), TNF-α and TGF-β production were assayed in macrophages using specific assays. NF-kB nuclear translocation was analyzed by western blot. Dolabelladienetriol inhibited Leishmania in a dose-dependent manner; the IC₅₀ was 44 µM. Dolabelladienetriol diminished NO, TNF-α and TGF-β production in uninfected and Leishmania-infected macrophages and reduced NF-kB nuclear translocation. Dolabelladienetriol inhibited Leishmania infection even when the parasite growth was exacerbated by either IL-10 or TGF-β. In addition, Dolabelladienetriol inhibited Leishmania growth in HIV-1-co-infected human macrophages.

Conclusion

Our results indicate that Dolabelladienetriol significantly inhibits Leishmania in macrophages even in the presence of factors that exacerbate parasite growth, such as IL-10, TGF-β and HIV-1 co-infection. Our results suggest that Dolabelladienetriol is a promising candidate for future studies regarding treatment of leishmaniasis, associated or not with HIV-1 infection.