Development of copy number assays for detection and surveillance of piperaquine resistance associated plasmepsin 2/3 copy number variation in Plasmodium falciparum.

BACKGROUND: Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the plasmepsin 2 and plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance. RESULTS: To accurately and quickly determine the presence of copy number variations in the plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene (pfmdr1), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of plasmepsin 2 in Pursat in 2013 to three copies in 2014-2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015. CONCLUSIONS: The multiplex TaqMan assay is a robust tool for monitoring both plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring plasmepsin 2/3 amplifications. This study shows increasing levels of plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.

Histone H3 K79 methylation states play distinct roles in UV-induced sister chromatid exchange and cell cycle checkpoint arrest in Saccharomyces cerevisiae.

Histone post-translational modifications have been shown to contribute to DNA damage repair. Prior studies have suggested that specific H3K79 methylation states play distinct roles in the response to UV-induced DNA damage. To evaluate these observations, we examined the effect of altered H3K79 methylation patterns on UV-induced G1/S checkpoint response and sister chromatid exchange (SCE). We found that the di- and trimethylated states both contribute to activation of the G1/S checkpoint to varying degrees, depending on the synchronization method, although methylation is not required for checkpoint in response to high levels of UV damage. In contrast, UV-induced SCE is largely a product of the trimethylated state, which influences the usage of gene conversion versus popout mechanisms. Regulation of H3K79 methylation by H2BK123 ubiquitylation is important for both checkpoint function and SCE. H3K79 methylation is not required for the repair of double-stranded breaks caused by transient HO endonuclease expression, but does play a modest role in survival from continuous exposure. The overall results provide evidence for the participation of H3K79 methylation in UV-induced recombination repair and checkpoint activation, and further indicate that the di- and trimethylation states play distinct roles in these DNA damage response pathways.

Distinct transcriptional profiles of maternal and fetal placental macrophages at term are associated with gravidity.

Maternal intervillous monocytes (MIMs) and fetal Hofbauer cells (HBCs) are myeloid-derived immune cells at the maternal-fetal interface. Little is known regarding the molecular phenotypes and roles of these distinct monocyte/macrophage populations. Here, we used RNA sequencing to investigate the transcriptional profiles of MIMs and HBCs in six normal term pregnancies. Our analyses revealed distinct transcriptomes of MIMs and HBCs. Genes involved in differentiation and cell organization pathways were more highly expressed in MIMs vs. HBCs. In contrast, HBCs had higher expression of genes involved in inflammatory responses and cell surface receptor signaling. Maternal gravidity influenced monocyte programming, as expression of pro-inflammatory molecules was significantly higher in MIMs from multigravidas compared to primigravidas. In HBCs, multigravidas displayed enrichment of gene pathways involved in cell-cell signaling and differentiation. In summary, our results demonstrated that MIMs and HBCs have highly divergent transcriptional signatures, reflecting their distinct origins, locations, functions, and roles in inflammatory responses. Our data further suggested that maternal gravidity influences the gene signatures of MIMs and HBCs, potentially modulating the interplay between tolerance and trained immunity. The phenomenon of reproductive immune memory may play a novel role in the differential susceptibility of primigravidas to pregnancy complications.

Mapping the genomic landscape of multidrug resistance in Plasmodium falciparum and its impact on parasite fitness.

Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance in vitro and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping using 34 recombinant haplotypes, and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.

Drug-dependent inhibition of nucleotide hydrolysis in the heterodimeric ABC multidrug transporter PatAB from Streptococcus pneumoniae.

The bacterial heterodimeric ATP-binding cassette (ABC) multidrug exporter PatAB has a critical role in conferring antibiotic resistance in multidrug-resistant infections by Streptococcus pneumoniae. As with other heterodimeric ABC exporters, PatAB contains two transmembrane domains that form a drug translocation pathway for efflux and two nucleotide-binding domains that bind ATP, one of which is hydrolysed during transport. The structural and functional elements in heterodimeric ABC multidrug exporters that determine interactions with drugs and couple drug binding to nucleotide hydrolysis are not fully understood. Here, we used mass spectrometry techniques to determine the subunit stoichiometry in PatAB in our lactococcal expression system and investigate locations of drug binding using the fluorescent drug-mimetic azido-ethidium. Surprisingly, our analyses of azido-ethidium-labelled PatAB peptides point to ethidium binding in the PatA nucleotide-binding domain, with the azido moiety crosslinked to residue Q521 in the H-like loop of the degenerate nucleotide-binding site. Investigation into this compound and residue's role in nucleotide hydrolysis pointed to a reduction in the activity for a Q521A mutant and ethidium-dependent inhibition in both mutant and wild type. Most transported drugs did not stimulate or inhibit nucleotide hydrolysis of PatAB in detergent solution or lipidic nanodiscs. However, further examples for ethidium-like inhibition were found with propidium, novobiocin and coumermycin A1, which all inhibit nucleotide hydrolysis by a non-competitive mechanism. These data cast light on potential mechanisms by which drugs can regulate nucleotide hydrolysis by PatAB, which might involve a novel drug binding site near the nucleotide-binding domains.

Modulation of Triple Artemisinin-Based Combination Therapy Pharmacodynamics by Plasmodium falciparum Genotype.

The first-line treatments for uncomplicated Plasmodium falciparum malaria are artemisinin-based combination therapies (ACTs), consisting of an artemisinin derivative combined with a longer acting partner drug. However, the spread of P. falciparum with decreased susceptibility to artemisinin and partner drugs presents a significant challenge to malaria control efforts. To stem the spread of drug resistant parasites, novel chemotherapeutic strategies are being evaluated, including the implementation of triple artemisinin-based combination therapies (TACTs). Currently, there is limited knowledge on the pharmacodynamic and pharmacogenetic interactions of proposed TACT drug combinations. To evaluate these interactions, we established an in vitro high-throughput process for measuring the drug concentration-response to three distinct antimalarial drugs present in a TACT. Sixteen different TACT combinations were screened against 15 parasite lines from Cambodia, with a focus on parasites with differential susceptibilities to piperaquine and artemisinins. Analysis revealed drug-drug interactions unique to specific genetic backgrounds, including antagonism between piperaquine and pyronaridine associated with gene amplification of plasmepsin II/III, two aspartic proteases that localize to the parasite digestive vacuole. From this initial study, we identified parasite genotypes with decreased susceptibility to specific TACTs, as well as potential TACTs that display antagonism in a genotype-dependent manner. Our assay and analysis platform can be further leveraged to inform drug implementation decisions and evaluate next-generation TACTs.

Overexpression of plasmepsin II and plasmepsin III does not directly cause reduction in Plasmodium falciparum sensitivity to artesunate, chloroquine and piperaquine.

Artemisinin derivatives and their partner drugs in artemisinin combination therapies (ACTs) have played a pivotal role in global malaria mortality reduction during the last two decades. The loss of artemisinin efficacy due to evolving drug-resistant parasites could become a serious global health threat. Dihydroartemisinin-piperaquine is a well tolerated and generally highly effective ACT. The implementation of a partner drug in ACTs is critical in the control of emerging artemisinin resistance. Even though artemisinin is highly effective in parasite clearance, it is labile in the human body. A partner drug is necessary for killing the remaining parasites when the pulses of artemisinin have ceased. A population of Plasmodium falciparum parasites in Cambodia and adjacent countries has become resistant to piperaquine. Increased copy number of the genes encoding the haemoglobinases Plasmepsin II and Plasmepsin III has been linked with piperaquine resistance by genome-wide association studies and in clinical trials, leading to the use of increased plasmepsin II/plasmepsin III copy number as a molecular marker for piperaquine resistance. Here we demonstrate that overexpression of plasmepsin II and plasmepsin III in the 3D7 genetic background failed to change the susceptibility of P. falciparum to artemisinin, chloroquine and piperaquine by both a standard dose-response analysis and a piperaquine survival assay. Whilst plasmepsin copy number polymorphism is currently implemented as a molecular surveillance resistance marker, further studies to discover the molecular basis of piperaquine resistance and potential epistatic interactions are needed.

Screening compounds with a novel high-throughput ABCB1-mediated efflux assay identifies drugs with known therapeutic targets at risk for multidrug resistance interference.

ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [(125)I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC50 value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.