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The virulence of parasites is expected to reflect an evolutionary tradeoff between increasing proliferation rates that enhance transmission and host mortality which curtails transmission. However, host resource availability may also limit parasites' proliferation rate. To understand the role of resource limitation as a driver of virulence evolution, Pak et al. (2024) use a within-host model of red blood cell (RBC) invasion by Plasmodium chabaudi. They find that within-host resource consumption limits the evolution of the parasite's proliferation rate, as the depletion of RBCs during infection results in intermediate optimal virulence. These results suggest that resource limitation, rather than host mortality, may drive the evolution of virulence., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE). All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

For parasites, robust proliferation within hosts is crucial for establishing the infection and creating opportunities for onward transmission. While faster proliferation enhances transmission rates, it is often assumed to curtail transmission duration by killing the host (virulence), a trade-off constraining parasite evolution. Yet in many diseases, including malaria, the preponderance of infections with mild or absent symptoms suggests that host mortality is not a sufficient constraint, raising the question of what restrains evolution toward faster proliferation. In malaria infections, the maximum rate of proliferation is determined by the burst size, the number of daughter parasites produced per infected red blood cell. Larger burst sizes should expand the pool of infected red blood cells that can be used to produce the specialized transmission forms needed to infect mosquitoes. We use a within-host model parameterized for rodent malaria parasites (Plasmodium chabaudi) to project the transmission consequences of burst size, focusing on initial acute infection where resource limitation and risk of host mortality are greatest. We find that resource limitation restricts evolution toward higher burst sizes below the level predicted by host mortality alone. Our results suggest resource limitation could represent a more general constraint than virulence-transmission trade-offs, preventing evolution towards faster proliferation., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE).)

Objective: To analyse the transcriptional profiles of the pir multigene family of Plasmodium chabaudi chabaudi in male and female gametocytes isolated from the blood of infected mice., Results: Infected red blood cells containing female and male P. chabaudi gametocytes transcribe a distinct set of genes encoded by the multigene family pir. The overall patterns are similar to what has been observed in the close relative P. berghei, but here we show that gametocyte-associated pir genes are distinct from those involved in chronic blood-stage infection and highlight a male-associated pir gene which should be the focus of future studies., (© 2023. The Author(s).)

Malaria is a devastating disease that still claims over half a million lives every year, mostly in sub-Saharan Africa. One of the main barriers to malaria control is the evolution and propagation of drug-resistant mutant parasites. Knowing the genes and respective mutations responsible for drug resistance facilitates the design of drugs with novel modes of action and allows predicting and monitoring drug resistance in natural parasite populations in real-time. The best way to identify these mutations is to experimentally evolve resistance to the drug in question and then comparing the genomes of the drug-resistant mutants to that of the sensitive progenitor parasites. This simple evolutive concept was the starting point for the development of a paradigm over the years, based on the use of the rodent malaria parasite Plasmodium chabaudi to unravel the genetics of drug resistance in malaria. It involves the use of a cloned parasite isolate (P. chabaudi AS) whose genome is well characterized, to artificially select resistance to given drugs through serial passages in mice under slowly increasing drug pressure. The end resulting parasites are cloned and the genetic mutations are then discovered through Linkage Group Selection, a technique conceived by Prof. Richard Carter and his group, and/or Whole Genome Sequencing. The precise role of these mutations can then be interrogated in malaria parasites of humans through allelic replacement experiments and/or genotype-phenotype association studies in natural parasite populations. Using this paradigm, all the mutations underlying resistance to the most important antimalarial drugs were identified, most of which were pioneering and later shown to also play a role in drug resistance in natural infections of human malaria parasites. This supports the use of P. chabaudi a fast-track predictive model to identify candidate genetic markers of resistance to present and future antimalarial drugs and improving our understanding of the biology of resistance., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Plasmodium interspersed repeat (pir) is the largest multigene family in the genomes of most Plasmodium species. A variety of functions for the PIR proteins which they encode have been proposed, including antigenic variation, immune evasion, sequestration and rosetting. However, direct evidence for these is lacking. The repetitive nature of the family has made it difficult to determine function experimentally. However, there has been some success in using gene expression studies to suggest roles for some members in virulence and chronic infection., Methods: Here pir gene expression was examined across the life cycle of Plasmodium berghei using publicly available RNAseq data-sets, and at high resolution in the intraerythrocytic development cycle using new data from Plasmodium chabaudi., Results: Expression of pir genes is greatest in stages of the parasite which invade and reside in red blood cells. The marked exception is that liver merozoites and male gametocytes produce a very large number of pir gene transcripts, notably compared to female gametocytes, which produce relatively few. Within the asexual blood stages different subfamilies peak at different times, suggesting further functional distinctions. Representing a subfamily of its own, the highly conserved ancestral pir gene warrants further investigation due to its potential tractability for functional investigation. It is highly transcribed in multiple life cycle stages and across most studied Plasmodium species and thus is likely to play an important role in parasite biology., Conclusions: The identification of distinct expression patterns for different pir genes and subfamilies is likely to provide a basis for the design of future experiments to uncover their function., (© 2021. The Author(s).)

Malaria parasites exhibit a complex lifecycle, requiring extensive asexual replication in the liver and blood of the vertebrate host, and in the haemocoel of the insect vector. Yet, they must also undergo a single round of sexual reproduction, which occurs in the vector's midgut upon uptake of a blood meal. Sexual reproduction is obligate for infection of the vector and thus, is essential for onwards transmission to new hosts. Sex in malaria parasites involves several bottlenecks in parasite number, making the stages involved attractive targets for blocking disease transmission. Malaria parasites have evolved a suite of adaptations ("strategies") to maximise the success of sexual reproduction and transmission, which could undermine transmission-blocking interventions. Yet, understanding parasite strategies may also reveal novel opportunities for such interventions. Here, we outline how evolutionary and ecological theories, developed to explain reproductive strategies in multicellular taxa, can be applied to explain two reproductive strategies (conversion rate and sex ratio) expressed by malaria parasites within the vertebrate host., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The deadly symptoms of malaria occur as Plasmodium parasites replicate within blood cells. Members of several variant surface protein families are expressed on infected blood cell surfaces. Of these, the largest and most ubiquitous are the Plasmodium -interspersed repeat (PIR) proteins, with more than 1,000 variants in some genomes. Their functions are mysterious, but differential pir gene expression associates with acute or chronic infection in a mouse malaria model. The membership of the PIR superfamily, and whether the family includes Plasmodium falciparum variant surface proteins, such as RIFINs and STEVORs, is controversial. Here we reveal the structure of the extracellular domain of a PIR from Plasmodium chabaudi We use structure-guided sequence analysis and molecular modeling to show that this fold is found across PIR proteins from mouse- and human-infective malaria parasites. Moreover, we show that RIFINs and STEVORs are not PIRs. This study provides a structure-guided definition of the PIRs and a molecular framework to understand their evolution., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

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.

Malarial rhythmic fevers are the consequence of the synchronous bursting of red blood cells (RBCs) on completion of the malaria parasite asexual cell cycle. Here, we hypothesized that an intrinsic clock in the parasite Plasmodium chabaudi underlies the 24-hour-based rhythms of RBC bursting in mice. We show that parasite rhythms are flexible and lengthen to match the rhythms of hosts with long circadian periods. We also show that malaria rhythms persist even when host food intake is evenly spread across 24 hours, suggesting that host feeding cues are not required for synchrony. Moreover, we find that the parasite population remains synchronous and rhythmic even in an arrhythmic clock mutant host. Thus, we propose that parasite rhythms are generated by the parasite, possibly to anticipate its circadian environment., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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, Plasmodium yoelii , and Plasmodium vinckei . We compared the fitness of individual species within single species and co-infections in mice. We also assessed the disease severity of single vs. mixed infections in mice by measuring mortality rates, anemia, 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., (Copyright © 2020 Tang, Templeton, Cao and Culleton.)

Background: The intraerythrocytic development cycle (IDC) of the rodent malaria Plasmodium chabaudi is coordinated with host circadian rhythms. When this coordination is disrupted, parasites suffer a 50% reduction in both asexual stages and sexual stage gametocytes over the acute phase of infection. Reduced gametocyte density may not simply follow from a loss of asexuals because investment into gametocytes ("conversion rate") is a plastic trait; furthermore, the densities of both asexuals and gametocytes are highly dynamic during infection. Hence, the reasons for the reduction of gametocytes in infections that are out-of-synch with host circadian rhythms remain unclear. Here, two explanations are tested: first, whether out-of-synch parasites reduce their conversion rate to prioritize asexual replication via reproductive restraint; second, whether out-of-synch gametocytes experience elevated clearance by the host's circadian immune responses., Methods: First, conversion rate data were analysed from a previous experiment comparing infections of P. chabaudi that were in-synch or 12 h out-of-synch with host circadian rhythms. Second, three new experiments examined whether the inflammatory cytokine TNF varies in its gametocytocidal efficacy according to host time-of-day and gametocyte age., Results: There was no evidence that parasites reduce conversion or that their gametocytes become more vulnerable to TNF when out-of-synch with host circadian rhythms., Conclusions: The factors causing the reduction of gametocytes in out-of-synch infections remain mysterious. Candidates for future investigation include alternative rhythmic factors involved in innate immune responses and the rhythmicity in essential resources required for gametocyte development. Explaining why it matters for gametocytes to be synchronized to host circadian rhythms might suggest novel approaches to blocking transmission.

Background: The ability of malaria (Plasmodium) parasites to adjust investment into sexual transmission stages versus asexually replicating stages is well known, but plasticity in other traits underpinning the replication rate of asexual stages in the blood has received less attention. Such traits include burst size (the number of merozoites produced per schizont), the duration of the asexual cycle, and invasion preference for different ages of red blood cell (RBC)., Methods: Here, plasticity [environment (E) effects] and genetic variation [genotype (G) effects] in traits relating to asexual replication rate are examined for 4 genotypes of the rodent malaria parasite Plasmodium chabaudi. An experiment tested whether asexual dynamics differ between parasites infecting control versus anaemic hosts, and whether variation in replication rate can be explained by differences in burst size, asexual cycle, and invasion rates., Results: The within-host environment affected each trait to different extents but generally had similar impacts across genotypes. The dynamics of asexual densities exhibited a genotype by environment effect (G×E), in which one of the genotypes increased replication rate more than the others in anaemic hosts. Burst size and cycle duration varied between the genotypes (G), while burst size increased and cycle duration became longer in anaemic hosts (E). Variation in invasion rates of differently aged RBCs was not explained by environmental or genetic effects. Plasticity in burst size and genotype are the only traits making significant contributions to the increase in asexual densities observed in anaemic hosts, together explaining 46.4% of the variation in replication rate., Conclusions: That host anaemia induces several species of malaria parasites to alter conversion rate is well documented. Here, previously unknown plasticity in other traits underpinning asexual replication is revealed. These findings contribute to mounting evidence that malaria parasites deploy a suite of sophisticated strategies to maximize fitness by coping with, or exploiting the opportunities provided by, the variable within-host conditions experienced during infections. That genetic variation and genotype by environment interactions also shape these traits highlights their evolutionary potential. Asexual replication rate is a major determinant of virulence and so, understanding the evolution of virulence requires knowledge of the ecological (within-host environment) and genetic drivers of variation among parasites.

Antimalarial drug resistance hampers effective malaria treatment. Critical SNPs in a particular, putative amino acid transporter were recently linked to chloroquine (CQ) resistance in malaria parasites. Here, we show that this conserved protein (PF3D7_0629500 in Plasmodium falciparum; AAT1 in P. chabaudi) is a structural homologue of the yeast amino acid transporter Tat2p, which is known to mediate quinine uptake and toxicity. Heterologous expression of PF3D7_0629500 in yeast produced CQ hypersensitivity, coincident with increased CQ uptake. PF3D7_0629500-expressing cultures were also sensitized to related antimalarials; amodiaquine, mefloquine and particularly quinine. Drug sensitivity was reversed by introducing a SNP linked to CQ resistance in the parasite. Like Tat2p, PF3D7_0629500-dependent quinine hypersensitivity was suppressible with tryptophan, consistent with a common transport mechanism. A four-fold increase in quinine uptake by PF3D7_0629500 expressing cells was abolished by the resistance SNP. The parasite protein localised primarily to the yeast plasma membrane. Its expression varied between cells and this heterogeneity was used to show that high-expressing cell subpopulations were the most drug sensitive. The results reveal that the PF3D7_0629500 protein can determine the level of sensitivity to several major quinine-related antimalarials through an amino acid-inhibitable drug transport function. The potential clinical relevance is discussed.

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.

Background: Parasite cytoadherence within the microvasculature of tissues and organs of infected individuals is implicated in the pathogenesis of several malaria syndromes. Multiple host receptors may mediate sequestration. The identity of the host receptor(s), or the parasite ligand(s) responsible for sequestration of Plasmodium species other than Plasmodium falciparum is largely unknown. The rodent malaria parasites may be useful to model interactions of parasite species, which lack the var genes with their respective hosts, as other multigene families are shared between the species. The role of the endothelial receptors ICAM-1 and CD36 in cytoadherence and in the development of pathology was investigated in a Plasmodium chabaudi infection in C57BL/6 mice lacking these receptors. The schizont membrane-associated cytoadherence (SMAC) protein of Plasmodium berghei has been shown to exhibit reduced CD36-associated cytoadherence in P. berghei ANKA-infected mice., Methods: Parasite tissue sequestration and the development of acute stage pathology in P. chabaudi infections of mice lacking CD36 or ICAM-1, their respective wild type controls, and in infections with mutant P. chabaudi parasites lacking the smac gene were compared. Peripheral blood parasitaemia, red blood cell numbers and weight change were monitored throughout the courses of infection. Imaging of bioluminescent parasites in isolated tissues (spleen, lungs, liver, kidney and gut) was used to measure tissue parasite load., Results: This study shows that neither the lack of CD36 nor the deletion of the smac gene from P. chabaudi significantly impacted on acute-stage pathology or parasite sequestration. By contrast, in the absence of ICAM-1, infected animals experience less anaemia and weight loss, reduced parasite accumulation in both spleen and liver and higher peripheral blood parasitaemia during acute stage malaria. The reduction in parasite tissue sequestration in infections of ICAM-1 null mice is maintained after mosquito transmission., Conclusions: These results indicate that ICAM-1-mediated cytoadherence is important in the P. chabaudi model of malaria and suggest that for rodent malarias, as for P. falciparum, there may be multiple host and parasite molecules involved in sequestration.

A major challenge in disease ecology is to understand how co-infecting parasite species interact. We manipulate in vivo resources and immunity to explain interactions between two rodent malaria parasites, Plasmodium chabaudi and P. yoelii. These species have analogous resource-use strategies to the human parasites Plasmodium falciparum and P. vivax: P. chabaudi and P. falciparum infect red blood cells (RBC) of all ages (RBC generalist); P. yoelii and P. vivax preferentially infect young RBCs (RBC specialist). We find that: (1) recent infection with the RBC generalist facilitates the RBC specialist (P. yoelii density is enhanced ~10 fold). This occurs because the RBC generalist increases availability of the RBC specialist's preferred resource; (2) co-infections with the RBC generalist and RBC specialist are highly virulent; (3) and the presence of an RBC generalist in a host population can increase the prevalence of an RBC specialist. Thus, we show that resources shape how parasite species interact and have epidemiological consequences., (© 2016 The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd.)

Plasmodium multigene families play a central role in the pathogenesis of malaria. The Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium spp. However their function(s) remains unknown. Using the rodent model of malaria, Plasmodium chabaudi, we show that individual CIR proteins have differential localizations within infected red cell (iRBC), suggesting different functional roles in a blood-stage infection. Some CIRs appear to be located on the surface of iRBC and merozoites and are therefore well placed to interact with host molecules. In line with this hypothesis, we show for the first time that a subset of recombinant CIRs bind mouse RBCs suggesting a role for CIR in rosette formation and/or invasion. Together, our results unravel differences in subcellular localization and ability to bind mouse erythrocytes between the members of the cir family, which strongly suggest different functional roles in a blood-stage infection.

Resistant malaria parasites are frequently found in mixed infections with drug-sensitive parasites. Particularly early in the evolutionary process, the frequency of these resistant mutants can be extremely low and below the level of molecular detection. We tested whether the rarity of resistance in infections impacted the health outcomes of treatment failure and the potential for onward transmission of resistance. Mixed infections of different ratios of resistant and susceptible Plasmodium chabaudi parasites were inoculated in laboratory mice and dynamics tracked during the course of infection using highly sensitive genotype-specific quantitative polymerase chain reaction (qPCR). Frequencies of resistant parasites ranged from 10% to 0.003% at the onset of treatment. We found that the rarer the resistant parasites were, the lower the likelihood of their onward transmission, but the worse the treatment failure was in terms of parasite numbers and disease severity. Strikingly, drug resistant parasites had the biggest impact on health outcomes when they were too rare to be detected by any molecular methods currently available for field samples. Indeed, in the field, these treatment failures would not even have been attributed to resistance., (© The American Society of Tropical Medicine and Hygiene.)

The emergence of drug-resistant pathogens is often considered a canonical case of evolution by natural selection. Here we argue that the strength of selection can be a poor predictor of the rate of resistance emergence. It is possible for a resistant strain to be under negative selection and still emerge in an infection or spread in a population. Measuring the right parameters is a necessary first step toward the development of evidence-based resistance-management strategies. We argue that it is the absolute fitness of the resistant strains that matters most and that a primary determinant of the absolute fitness of a resistant strain is the ecological context in which it finds itself., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Investment in the production of transmissible stages (gametocytes) and their sex ratio are malaria parasite traits that underpin mosquito infectivity and are therefore central to epidemiology. Malaria parasites adjust their levels of investment into gametocytes and sex ratio in response to changes in the in-host environment (including red blood cell resource availability, host immune responses, competition from con-specific genotypes in mixed infections, and drug treatment). This plasticity appears to be adaptive (strategic) because parasites prioritize investment (in sexual versus asexual stages and male versus female stages) in manners predicted to maximize fitness. However, the information, or 'cues' that parasites use to detect environmental changes and make appropriate decisions about investment into gametocytes and their sex ratio are unknown., Methods: Single genotype Plasmodium chabaudi infections were exposed to 'cue' treatments consisting of intact or lysed uninfected red blood cells, lysed parasitized RBCs of the same clone or an unrelated clone, and an unmanipulated control. Infection dynamics (proportion of reticulocytes, red blood cell and asexual stage parasite densities) were monitored, and changes in gametocyte investment and sex ratio in response to cue treatments, applied either pre- or post-peak of infection were examined., Results and Conclusions: A significant reduction in gametocyte density was observed in response to the presence of lysed parasite material and a borderline significant increase in sex ratio (proportion of male gametocytes) upon exposure to lysed red blood cells (both uninfected and infected) was observed. Furthermore, the changes in gametocyte density and sex ratio in response to these cues depend on the age of infection. Demonstrating that variation in gametocyte investment and sex ratio observed during infections are a result of parasite strategies (rather than the footprint of host physiology), provides a foundation to investigate the fitness consequences of plasticity and explore whether drugs could be developed to trick parasites into making suboptimal decisions.

Background: A series of elegant experiments was recently published which demonstrated that transmission of malaria parasites through mosquitoes elicited an attenuated growth phenotype, whereby infections grew more slowly and reached peak parasitaemia at least five-fold lower than parasites which had not been mosquito transmitted. To assess the implications of these results it is essential to understand whether the attenuated infection phenotype is a general phenomenon across parasites genotypes and conditions., Methods: Using previously published data, the impact of mosquito transmission on parasite growth rates and virulence of six Plasmodium chabaudi lines was analysed., Results: The effect of mosquito transmission varied among strains, but did not lead to pronounced or consistent reductions in parasite growth rate., Conclusions: Mosquito-induced attenuated growth phenotype is sensitive to experimental conditions.

Within-host competition among parasite genotypes affects epidemiology as well as the evolution of virulence. In the rodent malaria Plasmodium chabaudi, competition among genotypes, as well as clone-specific and clone-transcending immunity are well documented. However, variation among genotypes in the induction of antibodies is not well understood, despite the important role of antibodies in the clearance of malaria infection. Here, we quantify the potential for antibodies induced by one clone to bind another (i.e., to cause antibody-mediated apparent competition) for nine genetically distinct P. chabaudi clones. We hypothesised that clones would vary in the strength of antibody induction, and that the propensity for clone-transcending immunity between a pair of clones would increase with increasing genetic relatedness at key antigenic loci. Using serum collected from mice 35 days post-infection, we measured titres of antibody to an unrelated antigen, Keyhole Limpet Haemocyanin (KLH), and two malaria antigens: recombinant Apical Membrane Antigen-1 (AMA-1) and Merozoite Surface Protein-119 (MSP-119). Amino acid sequence homology within each antigenic locus was used as a measure of relatedness. We found significant parasite genetic variation for the strength of antibody induction. We also found that relatedness at MSP-119 but not AMA-1 predicted clone-transcending binding. Our results help explain the outcome of chronic-phase mixed infections and generate testable predictions about the pairwise competitive ability of P. chabaudi clones., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

All eukaryotic genomes have genes with introns in variable sizes. As far as spliceosomal introns are concerned, there are at least three basic parameters to stratify introns across diverse eukaryotic taxa: size, number, and sequence context. The number parameter is highly variable in lower eukaryotes, especially among protozoan and fungal species, which ranges from less than 4% to 78% of the genes. Over greater evolutionary time scales, the number parameter undoubtedly increases as observed in higher plants and higher vertebrates, reaching greater than 12.5 exons per gene in average among mammalian genomes. The size parameter is more complex, where multiple modes appear at work. Aside from intronless genes, there are three other types of intron-containing genes: half-sized, minimal, and size-expandable introns. The half-sized introns have only been found in a limited number of genomes among protozoan and fungal lineages and the other two types are prevalent in all animal and plant genomes. Among the size-expandable introns, the sizes of plant introns are expansion-limited in that the large introns exceeding 1000 bp are fewer in numbers and transposon-free as compared to the large introns among animals, where the larger introns are filled with transposable elements and appear expansion-flexible, reaching several kilobasepairs (kbp) and even thousands of kbp in size. Most of the intron parameters can be studied as signatures of the specific splicing machineries of different eukaryotic lineages and are highly relevant to the regulation of gene expression and functionality. In particular, the transcription-splicing-export coupling of eukaryotic intron dispensing leads to a working hypothesis that all intron parameters are evolved to be efficient and function-related in processing and routing the spliced transcripts.

Drug resistant pathogens are one of the key public health challenges of the 21st century. There is a widespread belief that resistance is best managed by using drugs to rapidly eliminate target pathogens from patients so as to minimize the probability that pathogens acquire resistance de novo. Yet strong drug pressure imposes intense selection in favor of resistance through alleviation of competition with wild-type populations. Aggressive chemotherapy thus generates opposing evolutionary forces which together determine the rate of drug resistance emergence. Identifying treatment regimens which best retard resistance evolution while maximizing health gains and minimizing disease transmission requires empirical analysis of resistance evolution in vivo in conjunction with measures of clinical outcomes and infectiousness. Using rodent malaria in laboratory mice, we found that less aggressive chemotherapeutic regimens substantially reduced the probability of onward transmission of resistance (by >150-fold), without compromising health outcomes. Our experiments suggest that there may be cases where resistance evolution can be managed more effectively with treatment regimens other than those which reduce pathogen burdens as fast as possible.

The apicoplast, a non-photosynthetic plastid of apicomplexan species, has an extremely reduced but highly conserved genome. Here, the apicoplast genome of the rodent malaria parasite Plasmodium chabaudi chabaudi (Pcc) isolate CB was characterized. Although the set of genes in the genome is identical, the copy number of some tRNA genes differs between Pcc and other Plasmodium species because the Pcc DNA has only one rRNA/tRNA gene cluster, which is normally duplicated in other species. The location of the duplicated trnR(ACG) and trnM implies that one of the duplicated clusters in the ancestral molecule has been lost due to an intramolecular recombination event. The Pcc DNA occurs in two isoforms with an internal inversion between them. The presence of a unique variant in the duplicated trnT gene suggests that the two isoforms are interconvertible. This is the first report of the complete nucleotide sequence of a Plasmodium apicoplast DNA.

Background: The control of malaria, caused by Plasmodium falciparum, is hampered by the relentless evolution of drug resistance. Because artemisinin derivatives are now used in the most effective anti-malarial therapy, resistance to artemisinin would be catastrophic. Indeed, studies suggest that artemisinin resistance has already appeared in natural infections. Understanding the mechanisms of resistance would help to prolong the effective lifetime of these drugs. Genetic markers of resistance are therefore required urgently. Previously, a mutation in a de-ubiquitinating enzyme was shown to confer artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi., Methods: Here, for a mutant P. chabaudi malaria parasite and its immediate progenitor, the in vivo artemisinin resistance phenotypes and the mutations arising using Illumina whole-genome re-sequencing were compared., Results: An increased artemisinin resistance phenotype is accompanied by one non-synonymous substitution. The mutated gene encodes the μ-chain of the AP2 adaptor complex, a component of the endocytic machinery. Homology models indicate that the mutated residue interacts with a cargo recognition sequence. In natural infections of the human malaria parasite P. falciparum, 12 polymorphisms (nine SNPs and three indels) were identified in the orthologous gene., Conclusion: An increased artemisinin-resistant phenotype occurs along with a mutation in a functional element of the AP2 adaptor protein complex. This suggests that endocytosis and trafficking of membrane proteins may be involved, generating new insights into possible mechanisms of resistance. The genotypes of this adaptor protein can be evaluated for its role in artemisinin responses in human infections of P. falciparum.

If drug-resistant malaria mutants are less fit than sensitive forms, they will wane over time when active drug pressure is removed and the overall sensitivity to the drug may be restored. However, most studies addressing this issue have been largely retrospective. Here, we undertook a predictive study, using mutant rodent malaria parasites resistant to the Artemisinin combination treatment (ACT) version of artesunate+mefloquine (ATN+MF) to gain insights about their ability to compete with ATN+MF-sensitive forms in untreated hosts. Previously, Plasmodium chabaudi parasites resistant to ATN+MF were selected in vivo through prolonged passaging in mice under increasing doses of the two drugs, and shown to harbour duplication of the mdr1 gene. Here, the resistant parasite, AS-ATNMF1, was mixed with its progenitor AS-ATN in different proportions and each mixture was injected into mice that were left untreated. Absolute percentage parasitaemias and the proportion of each parasite were then monitored by microscopy and proportional sequencing, respectively, every two days for a period of 14days. AS-ATNMF1 outperformed its progenitor AS-ATN over the whole sampling period regardless of the relative starting proportion of each parasite clone. In order to assess if consecutive sub-inoculations could have been responsible for the apparent fitness gain of the resistant parasite, its growth was compared to that of AS-ATN27P, a parasite which was passaged the same number of times as AS-ATNMF1, but left untreated. Although small fluctuations in the proportion of each parasite were observed through time, the relative abundance of each on the last day of sampling (Day 14) was virtually identical to that of the starting inoculum. We conclude that there is no fitness cost associated with MDR1-associated ATN+MF resistance in vivo. These observations offer the first insights about the within-host dynamics between ACT-resistant and -sensitive parasites in absence of drug pressure., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Genetically modified Plasmodium parasites are central gene function reagents in malaria research. The Rodent Malaria genetically modified DataBase (RMgmDB) ( www.pberghei.eu ) is a manually curated Web - based repository that contains information on genetically modified rodent malaria parasites. It provides easy and rapid access to information on the genotype and phenotype of genetically modified mutant and reporter parasites. Here, we provide guidelines for generating and describing rodent malaria parasite mutants. Standardization in describing mutant genotypes and phenotypes is important not only to enhance publication quality but also to facilitate cross-linking and mining data from multiple sources, and should permit information derived from mutant parasites to be used in integrative system biology approaches. We also provide guidelines on how to submit information to RMgmDB on non-published mutants, mutants that do not exhibit a clear phenotype, as well as negative attempts to disrupt/mutate genes. Such information helps to prevent unnecessary duplication of experiments in different laboratories, and can provide indirect evidence that these genes are essential for blood-stage development.

Here, we test the hypothesis that virulent malaria parasites are less susceptible to drug treatment than less virulent parasites. If true, drug treatment might promote the evolution of more virulent parasites (defined here as those doing more harm to hosts). Drug-resistance mechanisms that protect parasites through interactions with drug molecules at the sub-cellular level are well known. However, parasite phenotypes associated with virulence might also help parasites survive in the presence of drugs. For example, rapidly replicating parasites might be better able to recover in the host if drug treatment fails to eliminate parasites. We quantified the effects of drug treatment on the in-host survival and between-host transmission of rodent malaria (Plasmodium chabaudi) parasites which differed in virulence and had never been previously exposed to drugs. In all our treatment regimens and in single- and mixed-genotype infections, virulent parasites were less sensitive to pyrimethamine and artemisinin, the two antimalarial drugs we tested. Virulent parasites also achieved disproportionately greater transmission when exposed to pyrimethamine. Overall, our data suggest that drug treatment can select for more virulent parasites. Drugs targeting transmission stages (such as artemisinin) may minimize the evolutionary advantage of virulence in drug-treated infections.

Background: Over the last 6 decades, rodent Plasmodium species have become key model systems for understanding the basic biology of malaria parasites. Cell and molecular parasitology have made much progress in identifying genes underpinning interactions between malaria parasites, hosts, and vectors. However, little attention has been paid to the evolutionary genetics of parasites, which provides context for identifying potential therapeutic targets and for understanding the selective forces shaping parasites in natural populations. Additionally, understanding the relationships between species, subspecies, and strains, is necessary to maximize the utility of rodent malaria parasites as medically important infectious disease models, and for investigating the evolution of host-parasite interactions., Results: Here, we collected multi-locus sequence data from 58 rodent malaria genotypes distributed throughout 13 subspecies belonging to P. berghei, P. chabaudi, P. vinckei, and P. yoelii. We employ multi-locus methods to infer the subspecies phylogeny, and use population-genetic approaches to elucidate the selective patterns shaping the evolution of these organisms. Our results reveal a time-line for the evolution of rodent Plasmodium and suggest that all the subspecies are independently evolving lineages (i.e. species). We show that estimates of species-level polymorphism are inflated if subspecies are not explicitly recognized, and detect purifying selection at most loci., Conclusions: Our work resolves previous inconsistencies in the phylogeny of rodent malaria parasites, provides estimates of important parameters that relate to the parasite's natural history and provides a much-needed evolutionary context for understanding diverse biological aspects from the cross-reactivity of immune responses to parasite mating patterns.

Apical membrane Ag 1 (AMA1) is one of the leading candidate Ags for inclusion in a subunit vaccine against blood-stage malaria. However, the efficacy of Ab-inducing recombinant AMA1 protein vaccines in phase IIa/b clinical trials remains disappointing. In this article, we describe the development of recombinant human adenovirus serotype 5 and modified vaccinia virus Ankara vectors encoding AMA1 from the Plasmodium chabaudi chabaudi strain AS. These vectors, when used in a heterologous prime-boost regimen in BALB/c mice, are capable of inducing strong transgene-specific humoral and cellular immune responses. We show that this vaccination regimen is protective against a nonlethal P. chabaudi chabaudi strain AS blood-stage challenge, resulting in reduced peak parasitemias. The role of vaccine-induced, AMA1-specific Abs and T cells in mediating the antiparasite effect was investigated by in vivo depletion of CD4(+) T cells and adoptive-transfer studies into naive and immunodeficient mice. Depletion of CD4(+) T cells led to a loss of vaccine-induced protection. Adoptive-transfer studies confirmed that efficacy is mediated by both CD4(+) T cells and Abs functioning in the context of an intact immune system. Unlike previous studies, these results confirm that Ag-specific CD4(+) T cells, induced by a clinically relevant vaccine-delivery platform, can make a significant contribution to vaccine blood-stage efficacy in the P. chabaudi model. Given that cell-mediated immunity may also contribute to parasite control in human malaria, these data support the clinical development of viral-vectored vaccines that induce both T cell and Abs against Plasmodium falciparum blood-stage malaria Ags like AMA1.

Loop-mediated isothermal amplification (LAMP) has been increasingly used for diagnosis and quantification of pathogens. Since the Bst DNA polymerase used in this assay is highly resistant to PCR inhibitors present in blood, direct analysis of blood samples without DNA or RNA extraction is possible. Indeed, the presence of Plasmodium chabaudi specific nucleic acids was easily detectable using primer sets for P. chabaudi 18S rRNA and the cir 1 mRNA. Despite the fact that primers for cir 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and actin II mRNAs were used that spanned an intron, selective amplification of mRNA in the presence of contaminating genomic DNA was not possible. Optimization of the reaction temperature could only improve discrimination when low complexity templates (target sequences cloned in a plasmid vector) were used. Placing different LAMP primers across intron exon boundaries did not prevent amplification in the absence of reverse transcriptase. Probably due to the high A+T content and low number of introns only a very limited number of possible primer sets spanning introns could be identified in the target genes and no reaction conditions could be established that would allow quantification of RNA levels in the presence of DNA directly from blood samples., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The global spread of drug-resistant parasites is a serious problem for the treatment of malaria. Although identifying drug-resistance genes is crucial for the efforts against resistant parasites, an effective approach has not yet been developed. Here, we report a robust method for identifying resistance genes from parasites by using a Plasmodium artificial chromosome (PAC). Large genomic DNA fragments (10-50 kb) from the drug-resistant rodent malaria parasite Plasmodium berghei were ligated into the PAC and directly introduced into the drug-sensitive (i.e., wild-type) parasite by electroporation, resulting in a PAC library that encompassed the whole genomic sequence of the parasite. Subsequently, the transformed parasites that acquired resistance were selected by screening with the drug, and the resistance gene in the PAC was successfully identified. Furthermore, the drug-resistance gene was identified from a PAC library that was made from the pyrimethamine-resistant parasite Plasmodium chabaudi, further demonstrating the utility of our method. This method will promote the identification of resistance genes and contribute to the global fight against drug-resistant parasites.

Background: The pir genes comprise the largest multi-gene family in Plasmodium, with members found in P. vivax, P. knowlesi and the rodent malaria species. Despite comprising up to 5% of the genome, little is known about the functions of the proteins encoded by pir genes. P. chabaudi causes chronic infection in mice, which may be due to antigenic variation. In this model, pir genes are called cirs and may be involved in this mechanism, allowing evasion of host immune responses. In order to fully understand the role(s) of CIR proteins during P. chabaudi infection, a detailed characterization of the cir gene family was required., Results: The cir repertoire was annotated and a detailed bioinformatic characterization of the encoded CIR proteins was performed. Two major sub-families were identified, which have been named A and B. Members of each sub-family displayed different amino acid motifs, and were thus predicted to have undergone functional divergence. In addition, the expression of the entire cir repertoire was analyzed via RNA sequencing and microarray. Up to 40% of the cir gene repertoire was expressed in the parasite population during infection, and dominant cir transcripts could be identified. In addition, some differences were observed in the pattern of expression between the cir subgroups at the peak of P. chabaudi infection. Finally, specific cir genes were expressed at different time points during asexual blood stages., Conclusions: In conclusion, the large number of cir genes and their expression throughout the intraerythrocytic cycle of development indicates that CIR proteins are likely to be important for parasite survival. In particular, the detection of dominant cir transcripts at the peak of P. chabaudi infection supports the idea that CIR proteins are expressed, and could perform important functions in the biology of this parasite. Further application of the methodologies described here may allow the elucidation of CIR sub-family A and B protein functions, including their contribution to antigenic variation and immune evasion.

Background: Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance.A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions., Results: Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter., Conclusions: Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.

Malaria kills close to a million people every year, mostly children under the age of five. In the drive towards the development of an effective vaccine and new chemotherapeutic targets for malaria, field-based studies on human malaria infection and laboratory-based studies using animal models of malaria offer complementary opportunities to further our understanding of the mechanisms behind malaria infection and pathology. We outline here the parallels between the Plasmodium chabaudi mouse model of malaria and human malaria. We will highlight the contribution of P. chabaudi to our understanding of malaria in particular, how the immune response in malaria infection is initiated and regulated, its role in pathology, and how immunological memory is maintained. We will also discuss areas where new tools have opened up potential areas of exploration using this invaluable model system., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The evolution of drug resistant Plasmodium parasites is a major challenge to effective malaria control. In theory, competitive interactions between sensitive parasites and resistant parasites within infections are a major determinant of the rate at which parasite evolution undermines drug efficacy. Competitive suppression of resistant parasites in untreated hosts slows the spread of resistance; competitive release following treatment enhances it. Here we report that for the murine model Plasmodium chabaudi, co-infection with drug-sensitive parasites can prevent the transmission of initially rare resistant parasites to mosquitoes. Removal of drug-sensitive parasites following chemotherapy enabled resistant parasites to transmit to mosquitoes as successfully as sensitive parasites in the absence of treatment. We also show that the genetic composition of gametocyte populations in host venous blood accurately reflects the genetic composition of gametocytes taken up by mosquitoes. Our data demonstrate that, at least for this mouse model, aggressive chemotherapy leads to very effective transmission of highly resistant parasites that are present in an infection, the very parasites which undermine the long term efficacy of front-line drugs.

Malaria vaccine developers are concerned that antigenic escape will erode vaccine efficacy. Evolutionary theorists have raised the possibility that some types of vaccine could also create conditions favoring the evolution of more virulent pathogens. Such evolution would put unvaccinated people at greater risk of severe disease. Here we test the impact of vaccination with a single highly purified antigen on the malaria parasite Plasmodium chabaudi evolving in laboratory mice. The antigen we used, AMA-1, is a component of several candidate malaria vaccines currently in various stages of trials in humans. We first found that a more virulent clone was less readily controlled by AMA-1-induced immunity than its less virulent progenitor. Replicated parasites were then serially passaged through control or AMA-1 vaccinated mice and evaluated after 10 and 21 rounds of selection. We found no evidence of evolution at the ama-1 locus. Instead, virulence evolved; AMA-1-selected parasites induced greater anemia in naïve mice than both control and ancestral parasites. Our data suggest that recombinant blood stage malaria vaccines can drive the evolution of more virulent malaria parasites., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: The author has declared that no competing interests exist.

Genes underlying important phenotypic differences between Plasmodium species, the causative agents of malaria, are frequently found in only a subset of species and cluster at dynamically evolving subtelomeric regions of chromosomes. We hypothesized that chromosome-internal regions of Plasmodium genomes harbour additional species subset-specific genes that underlie differences in human pathogenicity, human-to-human transmissibility, and human virulence. We combined sequence similarity searches with synteny block analyses to identify species subset-specific genes in chromosome-internal regions of six published Plasmodium genomes, including Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. To improve comparative analysis, we first revised incorrectly annotated gene models using homology-based gene finders and examined putative subset-specific genes within syntenic contexts. Confirmed subset-specific genes were then analyzed for their role in biological pathways and examined for molecular functions using publicly available databases. We identified 16 genes that are well conserved in the three primate parasites but not found in rodent parasites, including three key enzymes of the thiamine (vitamin B1) biosynthesis pathway. Thirteen genes were found to be present in both human parasites but absent in the monkey parasite P. knowlesi, including genes specifically upregulated in sporozoites or gametocytes that could be linked to parasite transmission success between humans. Furthermore, we propose 15 chromosome-internal P. falciparum-specific genes as new candidate genes underlying increased human virulence and detected a currently uncharacterized cluster of P. vivax-specific genes on chromosome 6 likely involved in erythrocyte invasion. In conclusion, Plasmodium species harbour many chromosome-internal differences in the form of protein-coding genes, some of which are potentially linked to human disease and thus promising leads for future laboratory research.

Parasite strategies for exploiting host resources are key determinants of disease severity (i.e., virulence) and infectiousness (i.e., transmission between hosts). By iterating the development of theory and empirical tests, we investigated whether variation in parasite traits across two genetically distinct clones of the rodent malaria parasite, Plasmodium chabaudi, explains differences in within-host infection dynamics and virulence. First, we experimentally tested key predictions of our earlier modeling work. As predicted, the more virulent genotype produced more progeny parasites per infected cell (burst size), but in contrast to predictions, invasion rates of red blood cells (RBCs) did not differ between the genotypes studied. Second, we further developed theory by confronting our earlier model with these new data, testing a new set of models that incorporate more biological realism, and developing novel theoretical tools for identifying differences between parasite genotypes. Overall, we found robust evidence that differences in burst sizes contribute to variation in dynamics and that differential interactions between parasites and host immune responses also play a role. In contrast to previous work, our model predicts that RBC age structure is not important for explaining dynamics. Integrating theory and empirical tests is a potentially powerful way of progressing understanding of disease biology.

Multidrug-resistant Plasmodium falciparum malaria parasites pose a threat to effective drug control, even to artemisinin-based combination therapies (ACTs). Here we used linkage group selection and Solexa whole-genome resequencing to investigate the genetic basis of resistance to component drugs of ACTs. Using the rodent malaria parasite P. chabaudi, we analyzed the uncloned progeny of a genetic backcross between the mefloquine-, lumefantrine-, and artemisinin-resistant mutant AS-15MF and a genetically distinct sensitive clone, AJ, following drug treatment. Genomewide scans of selection showed that parasites surviving each drug treatment bore a duplication of a segment of chromosome 12 (translocated to chromosome 04) present in AS-15MF. Whole-genome resequencing identified the size of the duplicated segment and its position on chromosome 4. The duplicated fragment extends for ∼393 kbp and contains over 100 genes, including mdr1, encoding the multidrug resistance P-glycoprotein homologue 1. We therefore show that resistance to chemically distinct components of ACTs is mediated by the same genetic mutation, highlighting a possible limitation of these therapies.

Background: Variant antigens expressed on the surface of parasitized red blood cells (pRBCs) are important virulence factors of malaria parasites. Whereas Plasmodium falciparum erythrocyte membrane proteins 1 (PfEMP1) are responsible for sequestration of mature parasites, little is known about putative ligands mediating cytoadherence to host receptors in other Plasmodium species. Candidates include members of the pir superfamily found in the human parasite Plasmodium vivax (vir), in the simian pathogen Plasmodium knowlesi (kir) and in the rodent malarias Plasmodium yoelii (yir), Plasmodium berghei (bir) and Plasmodium chabaudi (cir). The aim of this study was to reveal a potential involvement of cir genes in P. chabaudi sequestration., Methods: Subfamilies of cir genes were identified by bioinformatic analyses of annotated sequence data in the Plasmodium Genome Database. In order to examine tissue-specific differences in the expression of cir mRNAs, RT-PCR with subfamily-specific primers was used. In total, 432 cDNA clones derived from six different tissues were sequenced to characterize the transcribed cir gene repertoire. To confirm differences in transcription profiles of cir genes, restriction fragment length polymorphism (RFLP) analyses were performed to compare different host tissues and to identify changes during the course of P. chabaudi infections in immunocompetent mice., Results: The phylogenetic analysis of annotated P. chabaudi putative CIR proteins identified two major subfamilies. Comparison of transcribed cir genes from six different tissues revealed significant differences in the frequency clones belonging to individual cir gene subgroups were obtained from different tissues. Further hints of difference in the transcription of cir genes in individual tissues were obtained by RFLP. Whereas only minimal changes in the transcription pattern of cir genes could be detected during the developmental cycle of the parasites, switching to expression of other cir genes during the course of an infection was observed around or after peak parasitemia., Conclusions: The tissue-specific expression of cir mRNAs found in this study indicates correlation between expression of CIR antigens and distribution of parasites in inner organs. Together with comparable results for other members of the pir superfamily this suggests a role of cir and other pir genes in antigenic variation and sequestration of malaria parasites.

Second-generation sequencing technologies have made large-scale sequencing projects commonplace. However, making use of these datasets often requires gene function to be ascribed genome wide. Although tool development has kept pace with the changes in sequence production, for tasks such as mapping, de novo assembly or visualization, genome annotation remains a challenge. We have developed a method to rapidly provide accurate annotation for new genomes using previously annotated genomes as a reference. The method, implemented in a tool called RATT (Rapid Annotation Transfer Tool), transfers annotations from a high-quality reference to a new genome on the basis of conserved synteny. We demonstrate that a Mycobacterium tuberculosis genome or a single 2.5 Mb chromosome from a malaria parasite can be annotated in less than five minutes with only modest computational resources. RATT is available at http://ratt.sourceforge.net., (© The Author(s) 2011. Published by Oxford University Press.)

The rodent malaria parasite Plasmodium chabaudi chabaudi shares many features with human malaria species, including P. falciparum, and is the in vivo model of choice for many aspects of malaria research in the mammalian host, from sequestration of parasitized erythrocytes, to antigenic variation and host immunity and immunopathology. This protocol describes an optimized method for the transformation of mature blood-stage P.c. chabaudi and a description of a vector that targets efficient, single crossover integration into the P.c. chabaudi genome. Transformed lines are reproducibly generated and selected within 14-20 d, and show stable long-term protein expression even in the absence of drug selection. This protocol, therefore, provides the scientific community with a robust and reproducible method to generate transformed P.c. chabaudi parasites expressing fluorescent, bioluminescent and model antigens that can be used in vivo to dissect many of the fundamental principles of malaria infection.

All organisms must trade off resource allocation between different life processes that determine their survival and reproduction. Malaria parasites replicate asexually in the host but must produce sexual stages to transmit between hosts. Because different specialized stages are required for these functions, the division of resources between these life-history components is a key problem for natural selection to solve. Despite the medical and economic importance of these parasites, their reproductive strategies remain poorly understood and often seem counterintuitive. Here, we tested recent theory predicting that in-host competition shapes how parasites trade off investment in in-host replication relative to between-host transmission. We demonstrate, across several genotypes, that Plasmodium chabaudi parasites detect the presence of competing genotypes and facultatively respond by reducing their investment in sexual stages in the manner predicted to maximize their competitive ability. Furthermore, we show that genotypes adjust their allocation to sexual stages in line with the availability of exploitable red blood cell resources. Our findings are predicted by evolutionary theory developed to explain life-history trade-offs in more traditionally studied multicellular taxa and suggest that the answer to the long-standing question of why so few transmission stages are produced is that in most natural infections heavy investment in reproduction may compromise in-host survival.

In malaria parasites, mutations in two genes of folate biosynthesis encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) modify responses to antifolate therapies which target these enzymes. However, the involvement of other genes which modify the availability of exogenous folate, for example, has been proposed. Here, we used short-read whole-genome re-sequencing to determine the mutations in a clone of the rodent malaria parasite, Plasmodium chabaudi, which has altered susceptibility to both sulphadoxine and pyrimethamine. This clone bears a previously identified S106N mutation in dhfr and no mutation in dhps. Instead, three additional point mutations in genes on chromosomes 2, 13 and 14 were identified. The mutated gene on chromosome 13 (mdr2 K392Q) encodes an ABC transporter. Because Quantitative Trait Locus analysis previously indicated an association of genetic markers on chromosome 13 with responses to individual and combined antifolates, MDR2 is proposed to modulate antifolate responses, possibly mediated by the transport of folate intermediates., (Copyright © 2010 Australian Society for Parasitology Inc. All rights reserved.)

Background: Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum., Results: A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme., Conclusions: This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.

Background: Lacking suitable alternatives, the control of malaria increasingly depends upon Artemisinin Combination Treatments (ACT): resistance to these drugs would therefore be disastrous. For ACTs, the biology of resistance to the individual components has been investigated, but experimentally induced resistance to component drugs in combination has not been generated., Methodology/principal Findings: We have used the rodent malaria parasite Plasmodium chabaudi to select in vivo resistance to the artesunate (ATN)+mefloquine (MF) version of ACT, through prolonged exposure of parasites to both drugs over many generations. The selection procedure was carried out over twenty-seven consecutive sub-inoculations under increasing ATN+MF doses, after which a genetically stable resistant parasite, AS-ATNMF1, was cloned. AS-ATNMF1 showed increased resistance to ATN+MF treatment and to artesunate or mefloquine administered separately. Investigation of candidate genes revealed an mdr1 duplication in the resistant parasites and increased levels of mdr1 transcripts and protein. There were no point mutations in the atpase6 or ubp1genes., Conclusion: Resistance to ACTs may evolve even when the two drugs within the combination are taken simultaneously and amplification of the mdr1 gene may contribute to this phenotype. However, we propose that other gene(s), as yet unidentified, are likely to be involved.

Despite many decades of research, no registered vaccine against the pathogenic blood stages of the malaria parasite exists, translating into the loss of many hundreds of thousands of young lives each year in tropical Africa. Although many parasite proteins have been shown to induce immune responses in the host, proof for their induction of protective immunity is still lacking. We previously reported a novel genetic approach called linkage group selection (LGS) for rapid identification of target antigens of strain-specific protective immunity (SSPI) against malaria. In preliminary LGS experiments, we crossed two genetically distinct strains of Plasmodium chabaudi chabaudi and subjected their progeny to selection in strain-specifically immunised mice, measuring the effects of SSPI selection with low coverage/resolution genetic markers. In the present study, through application of high coverage/resolution, single nucleotide polymorphism (SNP) markers spanning all 14 parasite chromosomes, we analysed 35 SSPI selection events on different populations of progeny parasites. Here we report a comprehensive high resolution genome-wide analysis of the effects of strain-specific immune selection on blood stage parasites. Our analyses consistently identify a single genomic region spanning approximately 79kb on chromosome 8 as the region controlling SSPI. Within this region, one gene (that of merozoite surface protein 1, MSP-1) accounted for >60% of genetic polymorphism and was most frequently under greatest reduction under SSPI. These results, combined with those of an independent LGS analysis of a different genetic cross with different parental strains, demonstrate that more than any other locus, the gene for MSP-1 determines the effect of strain-specific protective immunity against malaria in these host-parasite combinations. Our results provide unique insight into the precise timing of the parasite killing immune response against progeny parasites carrying specific alleles of MSP-1; these findings pave the way for investigating which part(s) of this highly polymorphic molecule mediate the protective immune response., (2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)