Colistin Resistance Mediated by Mcr-3-Related Phosphoethanolamine Transferase Genes in Aeromonas Species Isolated from Aquatic Environments in Avaga and Pakro Communities in the Eastern Region of Ghana.

Purpose: Colistin is classified by the World Health Organization (WHO) as a critically important and last-resort antibiotic for the treatment of infections caused by carbapenem-resistant bacteria. However, colistin resistance mediated by chromosomal mutations or plasmid-linked mobilized colistin resistance (mcr) genes has emerged. Methods: Thirteen mcr-positive Aeromonas species isolated from water samples collected in Eastern Ghana were analyzed using whole-genome sequencing (WGS). Antimicrobial susceptibility was tested using the broth microdilution method. Resistome analysis was performed in silico using a web-based platform. Results: The minimum inhibitory concentration (MIC) of colistin for all except three isolates was >4 µg/mL. Nine new sequence types were identified and whole-genome analysis revealed that the isolates harbored genes (mcr-3-related genes) that code for Lipid A phosphoethanolamine transferases on their chromosomes. BLAST analysis indicated that the amino acid sequences of the mcr-3-related genes detected varied from those previously reported and shared 79.04-99.86% nucleotide sequence identity with publicly available mcr-3 variants and mcr-3-related phosphoethanolamine transferases. Analysis of the genetic context of mcr-3-related genes revealed that the genetic environment surrounding mcr-3-related genes was diverse among the different species of Aeromonas but conserved among isolates of the same species. Mcr-3-related-gene-IS-mcr-3-related-gene segment was identified in three Aeromonas caviae strains. Conclusion: The presence of mcr-3-related genes close to insertion elements is important for continuous monitoring to better understand how to control the mobilization and dissemination of antibiotic resistance genes.

Identification of a novel protein localized to the crystalloid of the Plasmodium ookinete.

Reducing Plasmodium parasite transmission via the mosquito vector is a promising strategy for malaria control and elimination in endemic regions. In the mosquito midgut after the ingestion of an infected blood meal, malaria parasite gametes egress from erythrocytes and fertilize to develop into motile ookinetes that traverse midgut epithelial cells and transform into oocysts adjacent the basal lamina. Plasmodium ookinetes and young oocysts possess a unique organelle called the crystalloid; which has a honeycomb-like matrix structure and is indicated to be involved in sporozoite formation and maturation. In this study, we identified a novel crystalloid protein, PY17X_1113800, that is exclusively expressed in developing ookinetes. The protein possesses a signal peptide sequence, but lacks a transmembrane domain or GPI anchor signal sequence, as well as predicted adhesive domains which are characterisitic of many crystalloid proteins. The protein is highly conserved across the phylum Apicomplexa and within the greater clade Alveolata, such as Vitrella and the ciliates Paramecium and Tetrahymena, but is absent in cryptosporidia.

Skeleton binding protein 1 localizes to the Maurer's cleft and interacts with PfHSP70-1 and PfHSP70-x in Plasmodium falciparum gametocyte-infected erythrocytes.

Plasmodium falciparum accounts for the majority of malaria deaths, due to pathology provoked by the ability of infected erythrocytes to adhere to vascular endothelium within deep tissues. The parasite recognizes endothelium by trafficking and displaying protein ligands on the surface of asexual stage infected erythrocytes, such as members of the large family of pathogenic proteins, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Parasite-encoded skeleton binding protein 1 (SBP1) plays an important role in the transport of these binding-related surface proteins, via cleft-like membranous structures termed Maurer's clefts, which are present within the cytoplasm of infected erythrocytes. Erythrocytes infected with gametocyte stages accumulate in the extravascular compartment of bone marrow; and it was suggested that their surface-expressed adhesion molecule profile and protein trafficking mechanisms might differ from those in asexual stage parasites. Protein trafficking mechanisms via Maurer's clefts have been well investigated in asexual stage parasite-infected erythrocytes; but little is known regarding the gametocyte stages. In this study, we characterized SBP1 during gametocyte maturation and demonstrated that SBP1 is expressed and localizes to dot-like Maurer's cleft structures in the cytoplasm of gametocyte-infected erythrocytes. Co-immunoprecipitation and mass spectrometry assays indicated that SBP1 interacts with the molecular chaperones PfHSP70-1 and PfHSP70-x. Localization analysis suggested that some PfHSP70-1 and/or PfHSP70-x localize in a dot-like pattern within the cytoplasm of immature gametocyte-infected erythrocytes. These findings suggest that SBP1 may interact with HSP70 chaperones in the infected erythrocyte cytoplasm during the immature gametocyte stages.

Naturally Acquired Transmission-Blocking Immunity Against Different Strains of Plasmodium vivax in a Malaria-Endemic Area in Thailand.

BACKGROUND: Human immunity triggered by natural malaria infections impedes parasite transmission from humans to mosquitoes, leading to interest in transmission-blocking vaccines. However, immunity characteristics, especially strain specificity, remain largely unexplored. We investigated naturally acquired transmission-blocking immunity (TBI) against Plasmodium vivax, a major malaria parasite. METHODS: Using the direct membrane-feeding assay, we assessed TBI in plasma samples and examined the role of antibodies by removing immunoglobulins through protein G/L adsorption before mosquito feeding. Strain specificity was evaluated by conducting a direct membrane-feeding assay with plasma exchange. RESULTS: Blood samples from 47 patients with P vivax were evaluated, with 37 plasma samples successfully infecting mosquitoes. Among these, 26 showed inhibition before immunoglobulin depletion. Despite substantial immunoglobulin removal, 4 samples still exhibited notable inhibition, while 22 had reduced blocking activity. Testing against heterologous strains revealed some plasma samples with broad TBI and others with strain-specific TBI. CONCLUSIONS: Our findings indicate that naturally acquired TBI is mainly mediated by antibodies, with possible contributions from other serum factors. The transmission-blocking activity of plasma samples varied by the tested parasite strain, suggesting single polymorphic or multiple targets for naturally acquired TBI. These observations improve understanding of immunity against P vivax and hold implications for transmission-blocking vaccine development.

A Pvs25 mRNA vaccine induces complete and durable transmission-blocking immunity to Plasmodium vivax.

Plasmodium vivax (P. vivax) is the major malaria parasite outside of Africa and no vaccine is available against it. A vaccine that interrupts parasite transmission (transmission-blocking vaccine, TBV) is considered highly desirable to reduce the spread of P. vivax and to accelerate its elimination. However, the development of a TBV against this pathogen has been hampered by the inability to culture the parasite as well as the low immunogenicity of the vaccines developed to date. Pvs25 is the most advanced TBV antigen candidate for P. vivax. However, in previous phase I clinical trials, TBV vaccines based on Pvs25 yielded low antibody responses or had unacceptable safety profiles. As the nucleoside-modified mRNA-lipid nanoparticle (mRNA-LNP) vaccine platform proved to be safe and effective in humans, we generated and tested mRNA-LNP vaccines encoding several versions of Pvs25 in mice. We found that in a prime-boost vaccination schedule, all Pvs25 mRNA-LNP vaccines elicited robust antigen-specific antibody responses. Furthermore, when compared with a Pvs25 recombinant protein vaccine formulated with Montanide ISA-51 adjuvant, the full-length Pvs25 mRNA-LNP vaccine induced a stronger and longer-lasting functional immunity. Seven months after the second vaccination, vaccine-induced antibodies retained the ability to fully block P. vivax transmission in direct membrane feeding assays, whereas the blocking activity induced by the protein/ISA-51 vaccine dropped significantly. Taken together, we report on mRNA vaccines targeting P. vivax and demonstrate that Pvs25 mRNA-LNP outperformed an adjuvanted Pvs25 protein vaccine suggesting that it is a promising candidate for further testing in non-human primates.

Roles of retrovirus-derived PEG10 and PEG11/RTL1 in mammalian development and evolution and their involvement in human disease.

PEG10 and PEG11/RTL1 are paternally expressed, imprinted genes that play essential roles in the current eutherian developmental system and are therefore associated with developmental abnormalities caused by aberrant genomic imprinting. They are also presumed to be retrovirus-derived genes with homology to the sushi-ichi retrotransposon GAG and POL, further expanding our comprehension of mammalian evolution via the domestication (exaptation) of retrovirus-derived acquired genes. In this manuscript, we review the importance of PEG10 and PEG11/RTL1 in genomic imprinting research via their functional roles in development and human disease, including neurodevelopmental disorders of genomic imprinting, Angelman, Kagami-Ogata and Temple syndromes, and the impact of newly inserted DNA on the emergence of newly imprinted regions. We also discuss their possible roles as ancestors of other retrovirus-derived RTL/SIRH genes that likewise play important roles in the current mammalian developmental system, such as in the placenta, brain and innate immune system.

Retrovirus-Derived RTL9 Plays an Important Role in Innate Antifungal Immunity in the Eutherian Brain.

Retrotransposon Gag-like (RTL) genes play a variety of essential and important roles in the eutherian placenta and brain. It has recently been demonstrated that RTL5 and RTL6 (also known as sushi-ichi retrotransposon homolog 8 (SIRH8) and SIRH3) are microglial genes that play important roles in the brain's innate immunity against viruses and bacteria through their removal of double-stranded RNA and lipopolysaccharide, respectively. In this work, we addressed the function of RTL9 (also known as SIRH10). Using knock-in mice that produce RTL9-mCherry fusion protein, we examined RTL9 expression in the brain and its reaction to fungal zymosan. Here, we demonstrate that RTL9 plays an important role, degrading zymosan in the brain. The RTL9 protein is localized in the microglial lysosomes where incorporated zymosan is digested. Furthermore, in Rtl9 knockout mice expressing RTL9ΔC protein lacking the C-terminus retroviral GAG-like region, the zymosan degrading activity was lost. Thus, RTL9 is essentially engaged in this reaction, presumably via its GAG-like region. Together with our previous study, this result highlights the importance of three retrovirus-derived microglial RTL genes as eutherian-specific constituents of the current brain innate immune system: RTL9, RTL5 and RTL6, responding to fungi, viruses and bacteria, respectively.

Retrovirus-Derived RTL/SIRH: Their Diverse Roles in the Current Eutherian Developmental System and Contribution to Eutherian Evolution.

Eutherians have 11 retrotransposon Gag-like (RTL)/sushi-ichi retrotransposon homolog (SIRH) genes presumably derived from a certain retrovirus. Accumulating evidence indicates that the RTL/SIRH genes play a variety of roles in the current mammalian developmental system, such as in the placenta, brain, and innate immune system, in a eutherian-specific manner. It has been shown that the functional role of Paternally Expressed 10 (PEG10) in placental formation is unique to the therian mammals, as are the eutherian-specific roles of PEG10 and PEG11/RTL1 in maintaining the fetal capillary network and the endocrine regulation of RTL7/SIRH7 (aka Leucine Zipper Down-Regulated in Cancer 1 (LDOCK1)) in the placenta. In the brain, PEG11/RTL1 is expressed in the corticospinal tract and hippocampal commissure, mammalian-specific structures, and in the corpus callosum, a eutherian-specific structure. Unexpectedly, at least three RTL/SIRH genes, RTL5/SIRH8, RTL6/SIRH3, and RTL9/SIRH10, play important roles in combating a variety of pathogens, namely viruses, bacteria, and fungi, respectively, suggesting that the innate immunity system of the brain in eutherians has been enhanced by the emergence of these new components. In this review, we will summarize the function of 10 out of the 11 RTL/SIRH genes and discuss their roles in eutherian development and evolution.

Antifungal Efficacy of Luliconazole in an Experimental Rabbit Model of Fungal Keratitis Caused by Fusarium solani.

Fungal keratitis is a corneal fungal infection that potentially leads to blindness and is mainly caused by filamentous fungi, such as Fusarium, with limited drug options available, such as natamycin and voriconazole. Therefore, this study aimed to evaluate the therapeutic effects of the imidazole antifungal drug-luliconazole-using a rabbit experimental model of fungal keratitis caused by Fusarium solani, which is the dominant causative agent of fungal keratitis. F. solani was inoculated into rabbit corneas. luliconazole 1% suspension or natamycin 5% eye drops were administered four times a day (N = 6 for each group) 3 days after inoculation. Signs were scored up to 14 days after inoculation to evaluate the efficacy of the drugs. Compared with the peak mean sign scores of the placebo control group, there was a significant decrease in the mean sign scores of both the treatment groups (P < 0.05). Sign score trends were similar between the two treatment groups. In conclusion, luliconazole demonstrated therapeutic efficacy comparable to that of natamycin in treating experimental fungal keratitis. This suggests that luliconazole can be a novel therapeutic agent for human fungal keratitis.

Rhoptry neck protein 4 plays important roles during Plasmodium sporozoite infection of the mammalian liver.

During invasion, Plasmodium parasites secrete proteins from rhoptry and microneme apical end organelles, which have crucial roles in attaching to and invading target cells. A sporozoite stage-specific gene silencing system revealed that rhoptry neck protein 2 (RON2), RON4, and RON5 are important for sporozoite invasion of mosquito salivary glands. Here, we further investigated the roles of RON4 during sporozoite infection of the liver in vivo. Following intravenous inoculation of RON4-knockdown sporozoites into mice, we demonstrated that sporozoite RON4 has multiple functions during sporozoite traversal of sinusoidal cells and infection of hepatocytes. In vitro infection experiments using a hepatoma cell line revealed that secreted RON4 is involved in sporozoite adhesion to hepatocytes and has an important role in the early steps of hepatocyte infection. In addition, in vitro motility assays indicated that RON4 is required for sporozoite attachment to the substrate and the onset of migration. These findings indicate that RON4 is crucial for sporozoite migration toward and invasion of hepatocytes via attachment ability and motility.IMPORTANCEMalarial parasite transmission to mammals is established when sporozoites are inoculated by mosquitoes and migrate through the bloodstream to infect hepatocytes. Many aspects of the molecular mechanisms underpinning migration and cellular invasion remain largely unelucidated. By applying a sporozoite stage-specific gene silencing system in the rodent malarial parasite, Plasmodium berghei, we demonstrated that rhoptry neck protein 4 (RON4) is crucial for sporozoite infection of the liver in vivo. Combined with in vitro investigations, it was revealed that RON4 functions during a crossing of the sinusoidal cell layer and invading hepatocytes, at an early stage of liver infection, by mediating the sporozoite capacity for adhesion and the onset of motility. Since RON4 is also expressed in Plasmodium merozoites and Toxoplasma tachyzoites, our findings contribute to understanding the conserved invasion mechanisms of Apicomplexa parasites.

A possible circulation of a dominant Dibothriocephalus nihonkaiensis haplotype in Japan revealed by molecular analysis of clinical tapeworm samples.

Human diphyllobothriasis, caused by Dibothriocephalus nihonkaiensis, is prevalent globally, especially in regions where raw fish is consumed. Recent molecular diagnostic techniques have made species identification of tapeworm parasites and the determination of genetic variations among parasite populations possible. However, only a few studies done over a decade ago, have reported on the genetic variation among D. nihonkaiensis in Japan. The present study employed PCR-based mitochondrial DNA analysis to specifically detect D. nihonkaiensis from archived clinical samples, and to determine any genetic variation that may exist among the Japanese broad tapeworms from patients of Kanagawa Prefecture, Japan. Target genes were amplified from DNA extracted from the ethanol- or formaldehyde-fixed samples by PCR. Further sequencing and comparative phylogenetic analyses based on mitochondrial COI and ND1 sequences were also performed. In our results, all PCR-amplified and sequenced samples were identified as D. nihonkaiensis. Analysis of COI sequences revealed two haplotype lineages. However, clustering of almost all COI (and ND1) sample sequences into one of the two haplotype clades, together with reference sequences from different countries worldwide, revealed a common haplotype among D. nihonkaiensis samples in our study. Our results suggest a possible presence of a dominant D. nihonkaiensis haplotype, with a global distribution circulating in Japan. Results from this study have the potential to improve the management of clinical cases and establish robust control measures to reduce the burden of human diphyllobothriasis in Japan.

Cysteine Residues in Region 6 of the Plasmodium yoelii Erythrocyte-Binding-like Ligand That Are Related to Its Localization and the Course of Infection.

Plasmodium malaria parasites use erythrocyte-binding-like (EBL) ligands to invade erythrocytes in their vertebrate host. EBLs are released from micronemes, which are secretory organelles located at the merozoite apical end and bind to erythrocyte surface receptors. Because of their essential nature, EBLs have been studied as vaccine candidates, such as the Plasmodium vivax Duffy binding protein. Previously, we showed through using the rodent malaria parasite Plasmodium yoelii that a single amino acid substitution within the EBL C-terminal Cys-rich domain (region 6) caused mislocalization of this molecule and resulted in alteration of the infection course and virulence between the non-lethal 17X and lethal 17XL strains. In the present study, we generated a panel of transgenic P. yoelii lines in which seven of the eight conserved Cys residues in EBL region 6 were independently substituted to Ala residues to observe the consequence of these substitutions with respect to EBL localization, the infection course, and virulence. Five out of seven transgenic lines showed EBL mislocalizations and higher parasitemias. Among them, three showed increased virulence, whereas the other two did not kill the infected mice. The remaining two transgenic lines showed low parasitemias similar to their parental 17X strain, and their EBL localizations did not change. The results indicate the importance of Cys residues in EBL region 6 for EBL localization, parasite infection course, and virulence and suggest an association between EBL localization and the parasite infection course.

Evaluating the mosquito host range of Getah virus and the vector competence of selected medically important mosquitoes in Getah virus transmission.

BACKGROUND: The Getah virus (GETV) is a mosquito-borne Alphavirus (family Togaviridae) that is of significant importance in veterinary medicine. It has been associated with major polyarthritis outbreaks in animals, but there are insufficient data on its clinical symptoms in humans. Serological evidence of GETV exposure and the risk of zoonotic transmission makes GETV a potentially medically relevant arbovirus. However, minimal emphasis has been placed on investigating GETV vector transmission, which limits current knowledge of the factors facilitating the spread and outbreaks of GETV. METHODS: To examine the range of the mosquito hosts of GETV, we selected medically important mosquitoes, assessed them in vitro and in vivo and determined their relative competence in virus transmission. The susceptibility and growth kinetics of GETVs in various mosquito-derived cell lines were also determined and quantified using plaque assays. Vector competency assays were also conducted, and quantitative reverse transcription-PCR and plaque assays were used to determine the susceptibility and transmission capacity of each mosquito species evaluated in this study. RESULTS: GETV infection in all of the investigated mosquito cell lines resulted in detectable cytopathic effects. GETV reproduced the fastest in Culex tritaeniorhynchus- and Aedes albopictus-derived cell lines, as evidenced by the highest exponential titers we observed. Regarding viral RNA copy numbers, mosquito susceptibility to infection, spread, and transmission varied significantly between species. The highest vector competency indices for infection, dissemination and transmission were obtained for Cx. tritaeniorhynchus. This is the first study to investigate the ability of Ae. albopictus and Anopheles stephensi to transmit GETV, and the results emphasize the role and capacity of other mosquito species to transmit GETV upon exposure to GETV, in addition to the perceived vectors from which GETV has been isolated in nature. CONCLUSIONS: This study highlights the importance of GETV vector competency studies to determine all possible transmission vectors, especially in endemic regions.

Klebsiella Species and Enterobacter cloacae Isolates Harboring blaOXA-181 and blaOXA-48: Resistome, Fitness Cost, and Plasmid Stability.

IncX3 and IncL plasmids have been named as catalysts advancing dissemination of blaOXA-181 and blaOXA-48 genes. However, their impact on the performance of host cells is vastly understudied. Genetic characteristics of blaOXA-48- and blaOXA-181-containing Klebsiella pneumoniae (EFN299), Klebsiella quasipneumoniae (EFN262), and Enterobacter cloacae (EFN743) isolated from clinical samples in a Ghanaian hospital were investigated by whole-genome sequencing. Transfer of plasmids by conjugation and electroporation, plasmid stability, fitness cost, and genetic context of blaOXA-48, blaOXA-181, and blaDHA-1 were assessed. blaOXA-181 was carried on two IncX3 plasmids, an intact 51.5-kb IncX3 plasmid (p262-OXA-181) and a 45.3-kb IncX3 plasmid (p743-OXA-181) without replication protein sequence. The fluoroquinolone-resistant gene qnrS1 region was also excised, and unlike in p262-OXA-181, the blaOXA-181 drug-resistant region was not found on a composite transposon. blaOXA-48 was carried on a 74.6-kb conjugative IncL plasmid with unknown ~10.9-kb sequence insertion. This IncL plasmid proved to be highly transferable, with a conjugation efficiency of 1.8 × 10-2. blaDHA-1 was present on an untypeable 22.2 kb genetic structure. Plasmid stability test revealed plasmid loss rate between 4.3% and 12.4%. The results also demonstrated that carriage of IncX3-blaOXA-181 or IncL-blaOXA-48 plasmids was not associated with any fitness defect, but rather an enhanced competitive ability of host cells. This study underscores the significant contribution of IncX3 and IncL plasmids in the dissemination of resistance genes and their efficient transfer calls for regular monitoring to control the expansion of resistant strains. IMPORTANCE The growing rate of antibiotic resistance is an important global health threat. This threat is exacerbated by the lack of safe and potent alternatives to carbapenems in addition to the slow developmental process of newer and effective antibiotics. Infections by carbapenem-resistant Gram-negative bacteria are becoming almost untreatable, leading to poor clinical outcomes and high mortality rates. OXA-48-like carbapenemases are one of the most widespread carbapenemases accounting for resistance among Enterobacteriaecae. We characterized OXA-48- and OXA-181-producing Enterobacteriaecae to gain insights into the genetic basis and mechanism of resistance to carbapenems. Findings from the study showed that the genes encoding these enzymes were carried on highly transmissible plasmids, one of which had sequences absent in other similar plasmids. This implies that mobile genetic elements are important players in the dissemination of resistance genes. Further characterization of this plasmid is warranted to determine the role of this sequence in the spread of resistance genes.

Retrovirus-derived RTL5 and RTL6 genes are novel constituents of the innate immune system in the eutherian brain.

Retrotransposon Gag-like 5 [RTL5, also known as sushi-ichi-related retrotransposon homolog 8 (SIRH8)] and RTL6 (also known as SIRH3) are eutherian-specific genes presumably derived from a retrovirus and phylogenetically related to each other. They, respectively, encode a strongly acidic and extremely basic protein, and are well conserved among the eutherians. Here, we report that RTL5 and RTL6 are microglial genes with roles in the front line of innate brain immune response. Venus and mCherry knock-in mice exhibited expression of RTL5-mCherry and RTL6-Venus fusion proteins in microglia and appeared as extracellular dots and granules in the central nervous system. These proteins display a rapid response to pathogens such as lipopolysaccharide (LPS), double-stranded (ds) RNA analog and non-methylated CpG DNA, acting both cooperatively and/or independently. Experiments using Rtl6 or Rtl5 knockout mice provided additional evidence that RTL6 and RTL5 act as factors against LPS and dsRNA, respectively, in the brain, providing the first demonstration that retrovirus-derived genes play a role in the eutherian innate immune system. Finally, we propose a model emphasizing the importance of extra-embryonic tissues as the origin site of retrovirus-derived genes. This article has an associated 'The people behind the papers' interview.

Endogenous Retroviruses and Placental Evolution, Development, and Diversity.

The main roles of placentas include physical protection, nutrient and oxygen import, export of gasses and fetal waste products, and endocrinological regulation. In addition to physical protection of the fetus, the placentas must provide immune protection throughout gestation. These basic functions are well-conserved; however, placentas are undoubtedly recent evolving organs with structural and cellular diversities. These differences have been explained for the last two decades through co-opting genes and gene control elements derived from transposable elements, including endogenous retroviruses (ERVs). However, the differences in placental structures have not been explained or characterized. This manuscript addresses the sorting of ERVs and their integration into the mammalian genomes and provides new ways to explain why placental structures have diverged.

Vaccine co-display of CSP and Pfs230 on liposomes targeting two Plasmodium falciparum differentiation stages.

A vaccine targeting multiple stages of the Plasmodium falciparum parasite life cycle is desirable. The sporozoite surface Circumsporozoite Protein (CSP) is the target of leading anti-infective P. falciparum pre-erythrocytic vaccines. Pfs230, a sexual-stage P. falciparum surface protein, is currently in trials as the basis for a transmission-blocking vaccine, which inhibits parasite development in the mosquito vector. Here, recombinant full-length CSP and a Pfs230 fragment (Pfs230D1+) are co-displayed on immunogenic liposomes to induce immunity against both infection and transmission. Liposomes contain cobalt-porphyrin phospholipid (CoPoP), monophosphoryl lipid A and QS-21, and rapidly bind His-tagged CSP and Pfs230D1+ upon admixture to form bivalent particles that maintain reactivity with conformational monoclonal antibodies. Use of multicolor fluorophore-labeled antigens reveals liposome binding upon admixture, stability in serum and enhanced uptake in murine macrophages in vitro. Bivalent liposomes induce humoral and cellular responses against both CSP and Pfs230D1+. Vaccine-induced antibodies reduce parasite numbers in mosquito midguts in a standard membrane feeding assay. Mice immunized with liposome-displayed antigens or that passively receive antibodies from immunized rabbits have reduced parasite liver burden following challenge with transgenic sporozoites expressing P. falciparum CSP.

Evaluation of the Effect of Gene Duplication by Genome Editing on Drug Resistance in Plasmodium falciparum.

The emergence and spread of drug-resistant Plasmodium falciparum have compromised antimalarial efficacy and threatened the global malaria elimination campaign using artemisinin combination therapies. The impacts of amino acid substitutions in antimalarial drug resistance-associated genes on drug susceptibility have been investigated; however, the effects of amplification of those genes remain unexplored due to the lack of robust genetic approaches. Here, we generated transgenic P. falciparum parasites with an additional copy of a drug resistance-associated gene using the highly efficient CRISPR/Cas9 system and investigated their drug response. Insertion of a drug resistance-associated gene expression cassette in the genome resulted in a roughly twofold increase of mRNA levels of the target gene mdr1, which encodes multidrug resistance protein 1. The gene duplication event contributed to resistance to mefloquine, lumefantrine, and dihydroartemisinin, while the duplication of a genomic region encoding plasmepsin 2 and plasmepsin 3 did not affect resistance to antimalarial drugs, including piperaquine. We further demonstrated that mdr1 mRNA expression levels are strongly associated with mefloquine resistance in several field-derived P. falciparum lines with various genetic backgrounds. This study provides compelling evidence that mdr1 could serve as a molecular marker for the surveillance of mefloquine-resistant parasites. Long DNA integration into parasite genomes using the CRISPR/Cas9 system provides a useful tool for the evaluation of the effect of copy number variation on drug response.