The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for rapid and sensitive protein detection and quantification in simple and robust formats for widespread point-of-care ...applications. Here, we report on nanobody-functionalized organic electrochemical transistors with a modular architecture for the rapid quantification of single-molecule-to-nanomolar levels of specific antigens in complex bodily fluids. The sensors combine a solution-processable conjugated polymer in the transistor channel and high-density and orientation-controlled bioconjugation of nanobody-SpyCatcher fusion proteins on disposable gate electrodes. The devices provide results after 10 min of exposure to 5 μl of unprocessed samples, maintain high specificity and single-molecule sensitivity in human saliva and serum, and can be reprogrammed to detect any protein antigen if a corresponding specific nanobody is available. We used the sensors to detect green fluorescent protein, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) spike proteins, and for the COVID-19 screening of unprocessed clinical nasopharyngeal swab and saliva samples with a wide range of viral loads.
Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of ...drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ~92 k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ~7 k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type.
The mitochondrial F-type ATP synthase, a multisubunit nanomotor, is critical for maintaining cellular ATP levels. In T. gondii and other apicomplexan parasites, many subunit components necessary for ...proper assembly and functioning of this enzyme appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomeric (approximately 600 kDa) and dimeric (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits a, b, and d can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid, and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex could facilitate the development of novel antiparasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.
Volatile organic compounds (VOCs) comprise a diverse range of metabolites with high vapour pressure and low boiling points. Although they have received attention, they are a largely unexplored part ...of the metabolome. Previous studies have shown that malaria infections produce characteristic, definitive, and detectable volatile signatures. Many transcriptional and metabolic differences are observed at different stages of the parasite Intraerythrocytic Developmental Cycle (IDC) as well as when artemisinin-resistant parasites are put under drug pressure. This prompted our research to characterize whether these responses are reflected at a volatile level in malaria during the IDC stages using gas chromatography-mass spectrometry. We investigated whether the resistant P. falciparum parasites would produce their own characteristic volatilome profile compared to near-isogenic wild-type parasite in vitro; firstly at three different stages of the IDC and secondly in the presence or absence of artemisinin drug treatment. Finally, we explored the VOC profiles from two media environments (Human serum and Albumax) of recently lab-adapted field parasite isolates, from Southeast Asia and West/East Africa, compared to long-term lab-adapted parasites. Recognizable differences were observed between IDC stages, with schizonts having the largest difference between wild type and resistant parasites, and with cyclohexanol and 2,5,5-trimethylheptane only present for resistant schizonts. Artemisinin treatment had little effect on the resistant parasite VOC profile, whilst for the wild type parasites compounds ethylbenzene and nonanal were greatly affected. Lastly, differing culturing conditions had an observable impact on parasite VOC profile and clustering patterns of parasites were specific to geographic origin. The results presented here provide the foundation for future studies on VOC based characterization of P. falciparum strains differing in abilities to tolerate artemisinin.
Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 h suggesting a circadian basis, but the mechanism controlling this periodicity is unknown. Combining ...in vivo and in vitro approaches utilizing rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit daily rhythms in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 6% of Plasmodium falciparum genes show 24 h rhythms in expression under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a 24 h transcriptional rhythm and disrupting it in rodent malaria parasites shortens the IDC by 2-3 h; (v) Multiple processes including DNA replication, and the ubiquitin and proteasome pathways, are affected by loss of coordination with host rhythms and by disruption of SR10. Our results reveal malaria parasites are at least partly responsible for scheduling the IDC and coordinating their development with host daily rhythms.
For decades, the American palm weevil (APW), Rhynchophorus palmarum, has been a threat to coconut and oil palm production in the Americas. It has recently spread towards North America, endangering ...ornamental palms, and the expanding date palm production. Its behavior presents several parallelisms with a closely related species, R. ferrugineus, the red palm weevil (RPW), which is the biggest threat to palms in Asia and Europe. For both species, semiochemicals have been used for management. However, their control is far from complete. We generated an adult antennal transcriptome from APW and annotated chemosensory related gene families to obtain a better understanding of these species' olfaction mechanism. We identified unigenes encoding 37 odorant-binding proteins (OBPs), ten chemosensory proteins (CSPs), four sensory neuron membrane proteins (SNMPs), seven gustatory receptors (GRs), 63 odorant receptors (ORs), and 28 ionotropic receptors (IRs). Noticeably, we find out the R. ferrugineus pheromone-binding protein and pheromone receptor orthologs from R. palmarum. Candidate genes identified and annotated in this study allow us to compare these palm weevils' chemosensory gene sets. Most importantly, this study provides the foundation for functional studies that could materialize as novel pest management strategies.
Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum , has benefitted enormously from the ability to culture and genetically manipulate ...blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax , and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Human-adapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as species-specific features of an emerging pathogen.
Rodent malaria parasites (RMPs) serve as tractable tools to study malaria parasite biology and host-parasite-vector interactions. Among the four RMPs originally collected from wild thicket rats in ...sub-Saharan Central Africa and adapted to laboratory mice, Plasmodium vinckei is the most geographically widespread with isolates collected from five separate locations. However, there is a lack of extensive phenotype and genotype data associated with this species, thus hindering its use in experimental studies.
We have generated a comprehensive genetic resource for P. vinckei comprising of five reference-quality genomes, one for each of its subspecies, blood-stage RNA sequencing data for five P. vinckei isolates, and genotypes and growth phenotypes for ten isolates. Additionally, we sequenced seven isolates of the RMP species Plasmodium chabaudi and Plasmodium yoelii, thus extending genotypic information for four additional subspecies enabling a re-evaluation of the genotypic diversity and evolutionary history of RMPs. The five subspecies of P. vinckei have diverged widely from their common ancestor and have undergone large-scale genome rearrangements. Comparing P. vinckei genotypes reveals region-specific selection pressures particularly on genes involved in mosquito transmission. Using phylogenetic analyses, we show that RMP multigene families have evolved differently across the vinckei and berghei groups of RMPs and that family-specific expansions in P. chabaudi and P. vinckei occurred in the common vinckei group ancestor prior to speciation. The erythrocyte membrane antigen 1 and fam-c families in particular show considerable expansions among the lowland forest-dwelling P. vinckei parasites. The subspecies from the highland forests of Katanga, P. v. vinckei, has a uniquely smaller genome, a reduced multigene family repertoire and is also amenable to transfection making it an ideal parasite for reverse genetics. We also show that P. vinckei parasites are amenable to genetic crosses.
Plasmodium vinckei isolates display a large degree of phenotypic and genotypic diversity and could serve as a resource to study parasite virulence and immunogenicity. Inclusion of P. vinckei genomes provide new insights into the evolution of RMPs and their multigene families. Amenability to genetic crossing and transfection make them also suitable for classical and functional genetics to study Plasmodium biology.
Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division ...cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that
CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single
-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during
male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.
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