Biology subjects

Culleton, R.

Publications and source records attributed to Culleton, R..

8 recordsLinked to original sources

bioRxiv2026

A PCR-based assay for the sensitive and accurate determination of malaria parasite species that commonly infect humans, including discrimination of Plasmodium ovalecurtisi and Plasmodium ovalewallikeri

There are six Plasmodium species that commonly infect humans including Plasmodium ovalecurtisi, and Plasmodium ovalewallikeri. To ensure successful malaria control, it is important to track their prevalence and understand their epidemiological and clinical characteristics. Current PCR-based malaria diagnostic techniques targeting the highly sensitive mitochondrial genome do not distinguish between the two Plasmodium ovale species. Here we present a PCR-based species typing method targeting the mitochondrial COXIII involving a sensitive, nested PCR with species-specific primers and a simple enzyme digestion to distinguish P. ovalecurtisi and P. ovalewallikeri. The assay is highly specific to each species and highly sensitive, detecting 0.125 and 0.25 parasites per 1 {micro}l of blood from in vitro cultured Plasmodium knowlesi and Plasmodium falciparum respectively. Furthermore, using 30 field samples, we validate its effectiveness in discriminating the two P. ovale spp., identifying 18 P. ovalecurtisi, 16 P. ovalewallikeri, 23 P. falciparum and 17 Plasmodium malariae infections. This novel assay allows the sensitive and accurate diagnosis of P. falciparum, Plasmodium vivax, P. malariae, P. knowlesi, P. ovalecurtisi, and P. ovalewallikeri and can be used to monitor human and zoonotic malaria infections for both diagnosis and epidemiological studies.

molecular biology↗

bioRxiv2021

5-methylcytosine modification by Plasmodium NSUN2 stabilizes mRNA and mediates the development of gametocytes

5-methylcytosine (m5C) is an important epitranscriptomic modification involved in mRNA stability and translation efficiency in various biological processes. However, it remains unclear if m5C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m5C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (P. yoelii) and human (P. falciparum) malaria parasites. While different representations of m5C-modified mRNAs are associated with the different stages, the abundance of the m5C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m5C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m5C methyltransferase in malaria parasites. Upon knock-out of P. yoelii nsun2 (pynsun2), marked reductions of m5C modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in rodent and human malaria parasites. Restoration of the nsun2 gene in the knock-out parasites rescued the gametocyte production phenotype as well as m5C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m5C profiles for two species of Plasmodium, our findings demonstrate a major role for NSUN2-mediated m5C modifications in mRNA transcript stability and sexual differentiation in malaria parasites. SignificanceModifications of RNA including methylations of cytosine (m5C) and adenosine (m6A) have important roles in RNA metabolism, cellular responses to stress, and biological processes of differentiation and development. Here, we report on the profiles of m5C mRNA modifications in malaria parasites that infect rodents (Plasmodium yoelii) and humans (Plasmodium falciparum). These parasites have genes that encode homologs of human and plant NSUN2 methyltransferases (m5C "writers"). We show that one of these homologs, termed PyNSUN2, stabilizes mRNA transcripts in P. yoelii and mediates m5C-associated development of the parasite sexual stages (gametocytes). Further research on m5C and other epitranscriptomic modifications may yield new insights into molecular pathways of gametocyte development and mosquito infectivity that can be exploited to interrupt malaria transmission.

microbiology↗

bioRxiv2020

Plasmodium vinckei genomes provide insights into the pan-genome and evolution of rodent malaria parasites

BackgroundRodent malaria parasites (RMPs) serve as tractable tools to study malaria parasite biology and host-parasite-vector interactions. Plasmodium vinckei is the most geographically widespread of the four RMP species collected in sub-Saharan Central Africa. Several P. vinckei isolates are available but relatively less characterized than other RMPs, thus hindering their use in experimental studies. We have generated a comprehensive resource for P. vinckei comprising of high-quality reference genomes, genotypes, gene expression profiles and growth phenotypes for ten P. vinckei isolates. ResultsThe P. vinckei subspecies have diverged widely from their common ancestor and have undergone genomic structural variations. The subspecies from Katanga, P. v. vinckei, has a uniquely smaller genome, a reduced multigene family repertoire and is also amenable to genetic manipulation making it an ideal parasite for reverse genetics. Comparing P. vinckei genotypes reveals region-specific selection pressures particularly on genes involved in mosquito transmission. The erythrocyte membrane antigen 1 and fam-c families have expanded considerably among the lowland forest-dwelling P. vinckei parasites. Genetic crosses can be established in P. vinckei but are limited at present by low transmission success under the experimental conditions tested in this study. ConclusionsPlasmodium 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 genetic manipulation make them also suitable for classical and functional genetics to study Plasmodium biology.

genomics↗

bioRxiv2019

The genome of the zoonotic malaria parasite Plasmodium simium reveals adaptions to host-switching

BackgroundPlasmodium simium, a malaria parasite of non-human primates (NHP) was recently shown to cause zoonotic infections in humans in Brazil. We sequenced the P. simium genome to investigate its evolutionary history and to identify any genetic adaptions that may underlie the ability of this parasite to switch between host species. ResultsPhylogenetic analyses based on whole genome sequences of P. simium from humans and NHPs reveals that P. simium is monophyletic within the broader diversity of South American Plasmodium vivax, suggesting P. simium first infected NHPs as a result of a host-switch of P. vivax from humans. The P. simium isolates show the closest relationship to Mexican P. vivax isolates. Analysis of erythrocyte invasion genes reveals differences between P. vivax and P. simium, including large deletions in the Duffy Binding Protein 1 (DBP1) and Reticulocyte Binding Protein 2a genes of P. simium. Analysis of P. simium isolated from NHPs and humans revealed a deletion of 38 amino acids in DBP1 present in all human-derived isolates, whereas NHP isolates were multi-allelic. ConclusionsAnalysis of the P. simium genome confirmed a close phylogenetic relationship between P. simium and P. vivax, and suggests a very recent American origin for P. simium. The presence of the DBP1 deletion in all human-derived isolates tested suggests that this deletion, in combination with other genetic changes in P. simium, may facilitate the invasion of human red blood cells and may explain, at least in part, the basis of the recent zoonotic infections.

genomics↗

bioRxiv2019

Disruption of the coordination between host circadian rhythms and malaria parasite development alters the duration of the intraerythrocytic cycle

Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 hours (depending on the species), suggesting a circadian basis to the asexual cell cycle, but the mechanism controlling this periodicity is unknown. Combining in vivo and in vitro approaches using rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit 24 h \"circadian\" periodicity in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 9% of Plasmodium falciparum genes show circadian transcription under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a circadian transcription profile and disrupting it in rodent malaria parasites shortens the IDC by 2-3 hours; (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 show that malaria parasites are at least partly responsible for scheduling their IDCs explaining the fitness benefits of coordination with host rhythms.

genomics↗

bioRxiv2019

The consequences of mixed-species malaria parasite co-infections in mice and mosquitoes for disease severity, parasite fitness, and transmission success

The distributions of human malaria parasite species overlap in most malarious regions of the world, and co-infections involving two or more malaria parasite species are common. Little is known about the consequences of interactions between species during co-infection for disease severity and parasite transmission success. Anti-malarial interventions can have disproportionate effects on malaria parasite species, and may locally differentially reduce the number of species in circulation. Thus, it is important to have a clearer understanding of how the interactions between species affect disease and transmission dynamics. Controlled competition experiments using human malaria parasites are impossible, and thus we assessed the consequences of mixed-species infections on parasite fitness, disease severity, and transmission success using the rodent malaria parasite species Plasmodium chabaudi, P. yoelii yoelii, and P. vinckei lentum. We compared the fitness of individual species within single species and co-infections in mice. We also assessed the disease severity of single versus mixed infections in mice by measuring mortality rates, anaemia, and weight loss. Finally, we compared the transmission success of parasites in single or mixed species infections by quantifying oocyst development in Anopheles stephensi mosquitoes. We found that co-infections of P. yoelii with either P. vinckei or P. chabaudi led to a dramatic increase in infection virulence, with 100% mortality observed in mixed species infections, compared to no mortality for P. yoelii and P. vinckei single infections, and 40% mortality for P. chabaudi single infections. The increased mortality in the mixed infections was associated with an inability to clear parasitaemia, with the non-P. yoelii parasite species persisting at higher parasite densities than in single infections. P. yoelii growth was suppressed in all mixed infections compared to single infections. Transmissibility of P. vinckei and P. chabaudi to mosquitoes was also reduced in the presence of P. yoelii in co-infections compared to single infections. The increased virulence of co-infections containing P. yoelii (reticulocyte restricted) and P. chabaudi or P. vinckei (predominantly normocyte restricted) may be due to parasite cell tropism and/or immune modulation of the host. We explain the reduction in transmission success of species in co-infections in terms of inter-species gamete incompatibility.

microbiology↗

bioRxiv2018

A fast and cost-effective microsampling protocol incorporating reduced animal usage for time-series transcriptomics in rodent malaria parasites

The transcriptional regulation occurring in malaria parasites during the clinically important life stages within host erythrocytes can be studied in vivo with rodent malaria parasites propagated in mice. Time-series transcriptome profiling commonly involves the euthanasia of groups of mice at specific time points followed by the extraction of parasite RNA from whole blood samples. Current methodologies for parasite RNA extraction involve several steps and when multiple time points are profiled, these protocols are laborious, time consuming, and require the euthanisation of large cohorts of mice. We designed a simplified protocol for parasite RNA extraction from blood volumes as low as 20 microliters (microsamples), serially bled from mice via tail snips and directly lysed with TRIzol reagent. Gene expression data derived from microsampling using RNA-seq were closely matched to those derived from larger volumes of leucocyte-depleted and saponin-treated blood obtained from euthanized mice and also tightly correlated between biological replicates. Transcriptome profiling of microsamples taken at different time points during the intra-erythrocytic developmental cycle of the rodent malaria parasite Plasmodium vinckei revealed the transcriptional cascade commonly observed in malaria parasites. Microsampling is a quick, robust and cost-efficient approach to sample collection for in vivo time-series transcriptomic studies in rodent malaria parasites.

microbiology↗

bioRxiv2017

Plasmodium simium causing human malaria: a zoonosis with outbreak potential in the Rio de Janeiro Brazilian Atlantic forest

BackgroundMalaria was eliminated from Southern and Southeastern Brazil over 50 years ago. However, an increasing number of autochthonous episodes attributed to Plasmodium vivax have been recently reported in the Atlantic forest region of Rio de Janeiro State. As P. vivax-like non-human primate malaria parasite species Plasmodium simium is locally enzootic, we performed a molecular epidemiological investigation in order to determine whether zoonotic malaria transmission is occurring.\n\nMethodsBlood samples of humans presenting signs and/or symptoms suggestive of malaria as well as from local howler-monkeys were examined by microscopy and PCR. Additionally, a molecular assay based on sequencing of the parasite mitochondrial genome was developed to distinguish between P. vivax and P. simium, and applied to 33 cases from outbreaks occurred in 2015 and 2016.\n\nResultsOf 28 samples for which the assay was successfully performed, all were shown to be P. simium, indicating the zoonotic transmission of this species to humans in this region. Sequencing of the whole mitochondrial genome of three of these cases showed that P. simium is most closely related to P. vivax parasites from South American.\n\nFindingsThe explored malaria outbreaks were caused by P. simium, previously considered a monkey-specific malaria parasite, related to but distinct from P. vivax, and which has never conclusively been shown to infect humans before.\n\nInterpretationThis unequivocal demonstration of zoonotic transmission, 50 years after the only previous report of P. simium in man, leads to the possibility that this parasite has always infected humans in this region, but that it has been consistently misdiagnosed as P. vivax due to a lack of molecular typing techniques. Thorough screening of the local non-human primate and anophelines is required to evaluate the extent of this newly recognized zoonotic threat to public health and malaria eradication in Brazil.\n\nFundingFundacao Carlos Chagas Filho de Amparo a Pesquisa do Estado de Rio de Janeiro (Faperj), The Brazilian National Council for Scientific and Technological Development (CNPq), JSPS Grant-in-Aid for scientific research, Secretary for Health Surveillance (SVS) of the Ministry of Health, Global Fund, and PRONEX Program of the CNPq.

genetics↗