Physical aspects and cytoplasmic distribution of messenger RNA in mouse kidney.

As a prerequisite to examining mRNA metabolism in compensatory renal hypertrophy, polyadenylated RNA has been purified from normal mouse kidney polysomal RNA by selection on oligo(dT)-cellulose. Poly(A)-containing RNA dissociated from polysomes by treatment with 10 mM EDTA and sedimented heterogeneously in dodecyl sulfate-containing sucrose density gradients with a mean sedimentation coefficient of 20 S. Poly(A) derived from this RNA migrated at the rate of 6-7 S RNA in dodecyl sulfate-containing 10% polyacrylamide gels. Coelectrophoresis of poly(A) labeled for 90 min with poly(A) labeled for 24 h indicated the long-term labeled poly(A) migrated faster than pulse-labeled material. Twenty percent of the cytoplasmic poly(A)-containing mRNA was not associated with the polysomes, but sedimented in the 40-80 S region (post-polysomal). Messenger RNA from the post-polysomal region had sedimentation properties similar to those of mRNA prepared from polysomes indicating post-polysomal mRNA was not degraded polysomal mRNA. Preliminary labeling experiments indicated a rapid equilibration of radioactivity between the polysomal and post-polysomal mRNA populations, suggesting the post-polysomal mRNA may consist of mRNA in transit to the polysomes.     

Leishmania major LmACR2 is a pentavalent antimony reductase that confers sensitivity to the drug pentostam

Arsenicals and antimonials are first line drugs for the treatment of trypanosomal and leishmanial diseases. To create the active form of the drug, Sb(V) must be reduced to Sb(III). Because arsenic and antimony are related metalloids, and arsenical resistant Leishmania strains are frequently cross-resistant to antimonials, we considered the possibility that Sb(V) is reduced by a leishmanial As(V) reductase. The sequence for the arsenate reductase of Saccharomyces cerevisiae, ScAcr2p, was used to clone the gene for a homologue, LmACR2, from Leishmania major. LmACR2 was able to complement the arsenate-sensitive phenotype of an arsC deletion strain of Escherichia coli or an ScACR2 deletion strain of Saccharomyces cerevisiae. Transfection of Leishmania infantum with LmACR2 augmented Pentostam sensitivity in intracellular amastigotes. LmACR2 was purified and shown to reduce both As(V) and Sb(V). This is the first report of an enzyme that confers Pentostam sensitivity in intracellular amastigotes of Leishmania. We propose that LmACR2 is responsible for reduction of the pentavalent antimony in Pentostam to the active trivalent form of the drug in Leishmania.     

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.     

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.     

Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid

The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.     

Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes

Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV‐1 co‐infections. We have delineated the mechanism of cell death induced by the HIV‐1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir–Leishmania interactions, we selected Nelfinavir‐resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir‐resistant and ‐sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir‐resistant and ‐sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time‐dependent manner. Furthermore, high‐resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir‐resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug‐induced intracellular vesicles.     

Comparative proteomics analyses reveal a potential biomarker for the detection of vancomycin-intermediate Staphylococcus aureus strains

Vancomycin-intermediate Staphylococcus aureus (VISA) strains tend to develop during glycopeptide treatment of infections caused by methicillin-resistant S. aureus (MRSA). Rapid and effective detection methods for VISA strains are lacking, and mechanisms of resistance are unclear. Here, global comparative proteomic approaches have been used to identify potential biomarkers of intermediate vancomycin resistance. With the use of high-resolution two-dimensional gels and iTRAQ mass tagging, numerous proteins were found to be differentially expressed between clinical MRSA and VISA isolates of the same multilocus sequence type. One of these, the predicted lytic transglycosylase SAV2095 (SceD-like protein), was selected for further study based on both its high level of induction in Mu50 and its predicted role in modeling the cell wall, which is the target of vancomycin. Relative SAV2095 mRNA expression levels were compared between 25 MRSA and VISA/heterogeneous VISA clinical isolates by real-time RT-PCR. The SAV2095 mRNA was significantly induced in all VISA isolates relative to all MRSA strains ( p < 0.001), and significant induction of SAV2095 was also seen for several potential heterogeneous VISA strains that appear vancomycin-sensitive by standard minimum inhibitory concentration-determining methods. Furthermore, strains selected in vitro for increasing levels of resistance from four unrelated clinical MRSA isolates displayed concomitant increases in levels of SAV2095 expression. Together, these results suggest that SAV2095 expression level could serve as a molecular diagnostic marker for the rapid detection of VISA.     

Selection of Specific Endophytic Bacterial Genotypes by Plants in Response to Soil Contamination

Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.