Toxoplasma ceramide synthases: a curious case of gene duplication, divergence and key functionality

Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause foetal damage and abortion in both animals and humans. Sphingolipids have indispensable functions as signaling molecules and are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Ceramide is the precursor for all sphingolipids, and here we report the identification, localisation and analyses of the Toxoplasma ceramide synthases Tg CerS1 and Tg CerS2 and, using a conditional gene regulation approach, establish their roles in pathogenicity and parasite fitness. Interestingly, we observed that whilst Tg CerS1 was a fully functional orthologue of the yeast Lag1p capable of catalysing the conversion of sphinganine to ceramide, in contrast Tg CerS2 was catalytically inactive. Furthermore, genomic deletion of Tg CerS1 using CRISPR/Cas-9 led to viable but slow growing parasites indicating its importance but not indispensability. In contrast, genomic knock out of Tg CerS2 was only accessible utilising the rapamycin-inducible Cre recombinase system. Surprisingly, the results demonstrated that this ‘pseudo’ ceramide synthase, Tg CerS2, has an even greater role in parasite fitness than its catalytically active orthologue (Tg CerS1). Phylogenetic analyses indicated that, as in humans and plants, the ceramide synthase isoforms found in Toxoplasma and other Apicomplexa arose through gene duplication. However, in the Apicomplexa the duplicated copy subsequently evolved into a non-functional ‘pseudo’ ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the unusual biology that characterize these protozoan parasites, a feature that could potentially be exploited in the development of new antiprotozoals.

Parasitic manipulation or by-product of infection: an experimental approach using trematode-infected snails

Natural selection should favour parasite genotypes that manipulate hosts in ways that enhance parasite fitness. However, it is also possible that the effects of infection are not adaptive. Here we experimentally examined the phenotypic effects of infection in a snail–trematode system. These trematodes (Atriophallophorus winterbourni) produce larval cysts within the snail's shell (Potamopyrgus antipodarum); hence the internal shell volume determines the total number of parasite cysts produced. Infected snails in the field tend to be larger than uninfected snails, suggesting the hypothesis that parasites manipulate host growth so as to increase the space available for trematode reproduction. To test the hypothesis, we exposed juvenile snails to trematode eggs. Snails were then left to grow for about one year in 800-l outdoor mesocosms. We found that uninfected males were smaller than uninfected females (sexual dimorphism). We also found that infection did not affect the shell dimensions of males. However, infected females were smaller than uninfected females. Hence, infection stunts the growth of females, and (contrary to the hypothesis) it results in a smaller internal volume for larval cysts. Finally, infected females resembled males in size and shape, suggesting the possibility that parasitic castration prevents the normal development of females. These results thus indicate that the parasite is not manipulating the growth of infected hosts so as to increase the number of larval cysts, although alternative adaptive explanations are possible.

Virulence constrains transmission even in the absence of a genetic trade-off

The virulence-transmission trade-off predicts that parasite fitness peaks at intermediate virulence. However, whether this relationship is driven by the environment or genetically determined and if it depends on transmission opportunities remains unclear. We tackled these issues using inbred lines of the macro-parasitic spider-mite Tetranychus urticae. When transmission was not possible during the infection period, we observed a hump-shaped relationship between virulence and parasite fitness, as predicted by theory. This was environmentally driven, as no genetic correlation between traits was detected. However, when transmission to uninfected hosts occurred during the infection period, virulence was positively, environmentally and genetically correlated with parasite fitness. Therefore, the virulence-transmission trade-off depends on within-host dynamics and on the timing of transmission, rather than on a genetic correlation. This fundamental correlation may thus be easier to manipulate than previously thought.

Anthropization Affects the Assembly of Bat-Bat Fly Interaction Networks

Increasing anthropization is detrimental to the natural environment and the quality of life, affecting populations, communities, and the relationships between organisms. One of the most unique relationships in the animal world is parasitism, which often involves tightly specialized interactions between pairs of species. Bat flies, for example, are obligate ectoparasites represented by two highly adapted dipteran families that usually parasite a single bat species or genus. Recent studies have shown that bat flies could carry pathogens such as bacteria and viruses, transmitting them among bat individuals in a colony. Because host roost characteristics can influence bat-fly parasitism, we aimed to assess whether the ecological networks between parasites and their host bats are influenced by the degree of habitat anthropization. Our hypothesis was that bat-fly interaction networks would be less specialized and more nested in highly anthropized sites. We collected bat fly individuals from bats captured at 21 sampling sites located in the Federal District of Brazil and quantified the amount of natural and anthropized area within a 3-km buffer from the sampling site. Areas consisting of agriculture, construction, mining, roads, or any man-made structure were considered anthropized. Sites presented different degrees of anthropization, with areas ranging from 100% anthropized to areas retaining full natural cover. We built bat-bat fly networks for each of the sites and excluded those with less than 0.7% of sampling completeness. We calculated key weighted structural metrics for each network, such as nestedness, specialization, and modularity. The effect of the reduction in natural cover on structural metrics was assessed through GLMMs, controlling for network size and ectoparasite diversity. Nestedness increased with the amount of anthropization, while specialization and modularity did not change and were overall high in all networks. This result suggests that anthropization may influence the assembly of bat-bat fly networks, leading to the emergence of a hierarchical assembly of interactions as parasites become less specialized and interact with a wider variety of hosts. Less specialized relationships could influence parasite fitness or even increase the likelihood of transmitting pathogens between populations of different bat species.

Common host variation drives malaria parasite fitness in healthy human red cells

The replication of Plasmodium falciparum parasites within red blood cells (RBCs) causes severe disease in humans, especially in Africa. Deleterious alleles like hemoglobin S are well-known to confer strong resistance to malaria, but the effects of common RBC variation are largely undetermined. Here we collected fresh blood samples from 121 healthy donors, most with African ancestry, and performed exome sequencing, detailed RBC phenotyping, and parasite fitness assays. Over one third of healthy donors unknowingly carried alleles for G6PD deficiency or hemoglobinopathies, which were associated with characteristic RBC phenotypes. Among non-carriers alone, variation in RBC hydration, membrane deformability, and volume was strongly associated with P. falciparum growth rate. Common genetic variants in PIEZO1, SPTA1/SPTB, and several P. falciparum invasion receptors were also associated with parasite growth rate. Interestingly, we observed little or negative evidence for divergent selection on non-pathogenic RBC variation between Africans and Europeans. These findings suggest a model in which globally widespread variation in a moderate number of genes and phenotypes modulates P. falciparum fitness in RBCs.

Plasmodium falciparum K13 mutations in Africa and Asia impact artemisinin resistance and parasite fitness

The emergence of mutant K13-mediated artemisinin (ART) resistance in Plasmodium falciparum malaria parasites has led to widespread treatment failure across Southeast Asia. In Africa, K13-propeller genotyping confirms the emergence of the R561H mutation in Rwanda and highlights the continuing dominance of wild-type K13 elsewhere. Using gene editing, we show that R561H, along with C580Y and M579I, confer elevated in vitro ART resistance in some African strains, contrasting with minimal changes in ART susceptibility in others. C580Y and M579I cause substantial fitness costs, which may slow their dissemination in high-transmission settings, in contrast with R561H that in African 3D7 parasites is fitness neutral. In Cambodia, K13 genotyping highlights the increasing spatio-temporal dominance of C580Y. Editing multiple K13 mutations into a panel of Southeast Asian strains reveals that only the R561H variant yields ART resistance comparable to C580Y. In Asian Dd2 parasites C580Y shows no fitness cost, in contrast with most other K13 mutations tested, including R561H. Editing point mutations in ferredoxin or mdr2, earlier associated with resistance, has no impact on ART susceptibility or parasite fitness. These data underline the complex interplay between K13 mutations, parasite survival, growth and genetic background in contributing to the spread of ART resistance.

Balanced impacts of fitness and drug pressure on the evolution of PfMDR1 polymorphisms in Plasmodium falciparum

Background Anti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1. Methods Impacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted. Results Co-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184 F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact. Conclusions These results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum.

Elevated nest temperature has opposing effects on host species infested with parasitic nest flies

Hosts have developed or evolved defense strategies, including tolerance and resistance, to reduce damage caused by parasites. Environmental factors, such as elevated temperature, can influence the effectiveness of these different host defenses but also can directly affect parasite fitness. Therefore, the net effect of elevated temperature on host-parasite relationships are determined by its direct effects on the host and the parasite. Furthermore, because host species can defend themselves differently against their parasites, the net effect of temperature might differ across each hosts interaction with the same parasite. Few studies have determined the net effects of temperature on both host defenses and parasites in a multi-host system. To address this gap, we experimentally manipulated temperature and parasite presence in the nests of two host species who defend themselves differently to the same parasitic nest fly (Protocalliphora sialia). Specifically, we conducted a factorial experiment by increasing temperature (or not) and removing all parasitic nest flies (or not) in the nests of tolerant eastern bluebirds (Sialia sialis) and resistant tree swallows (Tachycineta bicolor). We then quantified parasite load in nests and measured nestling body size metrics, blood loss, and survival. If temperature predominately affected parasite fitness, then elevated temperature would cause similar directional effects on parasite abundance across species. If temperature has different effects on hosts, then parasite abundance would differ in response to elevated temperature across host species. In contrast to previous years, we found that bluebird nests had half as many parasites as compared to swallow nests. Elevated temperature affected parasite abundance differently in each host species. Swallows from heated nests had fewer parasites compared to non-heated nests, suggesting that they were more resistant to the parasites. Interestingly, swallows from heated nests were also more tolerant to the effects of parasites than controls. In contrast, bluebirds from heated nests had more parasites and lower body mass compared to controls, suggesting that they lost tolerance, and resistance, to the parasites. Our results suggest that a changing climate could have complex net effects on host-parasite interactions, including on host defenses, with implications for host health and parasite survival.

Treatment of canine visceral leishmaniasis with Milteforan™ induces Leishmania infantum resistance to miltefosine and amphotericin B

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is caused by Leishmania infantum in the Americas. Since the use of Milteforam™ was authorized to treat canine visceral leishmaniasis (CVL) in Brazil in 2017, there has also been fear of the emergence of parasites resistant to this drug and, through cross-resistance mechanisms, to meglumine antimoniate and amphotericin B. Additionally, the literature shows that acquisition of resistance is followed by increased parasite fitness, with higher rates of proliferation, infectivity and metacyclogenesis, which are determining factors for parasite virulence. In this context, this study aims to analyze the impact of treating a dog with Milteforan™ on the generation of parasites resistant to miltefosine, meglumine antimoniate, and amphotericin B. To this end, in vitro susceptibility tests were conducted against these drugs with T0 (parasites isolated from the dog before treatment with Milteforan™), T1 (after one course of treatment), and T2 (after two courses of treatment) isolates. The rates of cell proliferation, infectivity, and metacyclogenesis of the isolates were also evaluated. The results indicate a gradual increase in parasite resistance to miltefosine and amphotericin B with increasing the number of treatment courses. A trend increase in the metacyclogenesis rate of the parasites was also observed as drug resistance increased. Therefore, treatment of CVL with Milteforan™ induces resistance to miltefosine and amphotericin B as well as changes in parasite fitness, and may have an impact on animal and human public health.

Paraphysoderma sedebokerense Infection in Three Economically Valuable Microalgae: Host Preference Correlates with Parasite Fitness

The blastocladialean fungus Paraphysoderma sedebokerense parasitizes three microalgae species of economic interest: Haematococcus pluvialis, Chromochloris zofingiensis and Scenedesmus dimorphus. For the first time, we characterized the developmental stages of isolated fungal propagules in H. pluvialis co-culture, finding a generation time of 16 h. We established a patho-system to compare the infection in the three different host species for 48 h, with two different setups to quantify parameters of the infection and parameters of the parasite fitness. The prevalence of the parasite in H. pluvialis and C. zofingiensis cultures was 100%, but only 20% in S. dimorphus culture. The infection of S. dimorphus not only reached lower prevalence but was also qualitatively different; the infection developed preferentially on senescent cells and more resting cysts were produced, being consistent with a reservoir host. In addition, we carried out cross infection experiments and the inoculation of a mixed algal culture containing the three microalgae, to determine the susceptibility of the host species and to investigate the preference of P. sedebokerense for these microalgae. The three tested microalgae showed different susceptibility to P. sedebokerense, which correlates with blastoclad’s preference to the host in the following order: H. pluvialis > C. zofingiensis > S. dimorphus.