Properties of Plasmodium falciparum with a deleted apicoplast DNA gyrase

Malaria parasites have three genomes: a nuclear genome, a mitochondrial genome, and an apicoplast genome. Since the apicoplast is a plastid organelle of prokaryotic origin and has no counterpart in the human host, it can be a source of novel targets for antimalarials. Plasmodium falciparum DNA gyrase ( Pf Gyr) A and B subunits both have apicoplast-targeting signals. First, to test the predicted localization of this enzyme in the apicoplast and the breadth of its function at the subcellular level, nuclear encoded Pf GyrA was disrupted using CRISPR/Cas9 gene-editing. Isopentenyl pyrophosphate (IPP) is known to rescue parasites from apicoplast inhibitors. Indeed, successful growth and characterization of Pf ΔGyrA was possible in the presence of IPP. Pf GyrA disruption was accompanied by loss of plastid acyl-carrier protein (ACP) immunofluorescence and the plastid genome. Second, ciprofloxacin, an antibacterial gyrase inhibitor, has been used for malaria prophylaxis but there is a need for a more detailed description of the mode-of-action of ciprofloxacin in malaria parasites. As predicted Pf ΔGyrA clone supplemented with IPP was less sensitive to ciprofloxacin, but not the nuclear topoisomerase inhibitor etoposide. At high concentrations, however, ciprofloxacin continued to inhibit IPP-rescued Pf ΔGyrA possibly suggesting that ciprofloxacin may have an additional non-apicoplast target in P. falciparum . Overall, we confirm that Pf GyrA is an apicoplast enzyme in the malaria parasite, essential for blood-stage parasites, and a possible target of ciprofloxacin but perhaps not the only target.

Phylogenomics identifies a new major subgroup of apicomplexans, Marosporida class. nov., with extreme apicoplast genome reduction

The phylum Apicomplexa consists largely of obligate animal parasites that include the causative agents of human diseases such as malaria. Apicomplexans have also emerged as models to study the evolution of non-photosynthetic plastids, as they contain a relict chloroplast known as the apicoplast. The apicoplast offers important clues into how apicomplexan parasites evolved from free-living ancestors and can provide insights into reductive organelle evolution. Here, we sequenced the transcriptomes and apicoplast genomes of three deep-branching apicomplexans, Margolisiella islandica, Aggregata octopiana and Merocystis kathae. Phylogenomic analyses show that these taxa, together with Rhytidocystis, form a new lineage of apicomplexans that is sister to the Coccidia and Hematozoa (the lineages including most medically significant taxa). Members of this clade retain plastid genomes and the canonical apicomplexan plastid metabolism. However, the apicoplast genomes of Margolisiella and Rhytidocystis are the most reduced of any apicoplast, are extremely GC-poor, and have even lost genes for the canonical plastidial RNA polymerase. This new lineage of apicomplexans, for which we propose the class Marosporida class nov., occupies a key intermediate position in the apicomplexan phylogeny, and adds a new complexity to the models of stepwise reductive evolution of genome structure and organelle function in these parasites.

Nephromyces represents a diverse and novel lineage of the Apicomplexa that has retained apicoplasts

A most interesting exception within the parasitic Apicomplexa is Nephromyces, an extracellular, probably mutualistic, endosymbiont found living inside molgulid ascidian tunicates (i.e., sea squirts). Even though Nephromyces is now known to be an apicomplexan, many other questions about its nature remain unanswered. To gain further insights into the biology and evolutionary history of this unusual apicomplexan, we aimed to (1) find the precise phylogenetic position of Nephromyces within the Apicomplexa, (2) search for the apicoplast genome of Nephromyces, and (3) infer the major metabolic pathways in the apicoplast of Nephromyces. To do this, we sequenced a metagenome and a metatranscriptome from the molgulid renal sac, the specialized habitat where Nephromyces thrives. Our phylogenetic analyses of conserved nucleus-encoded genes robustly suggest that Nephromyces is a novel lineage sister to the Hematozoa, which comprises both the Haemosporidia (e.g., Plasmodium) and the Piroplasmida (e.g., Babesia and Theileria). Furthermore, a survey of the renal sac metagenome revealed 13 small contigs that closely resemble the genomes of the non-photosynthetic reduced plastids, or apicoplasts, of other apicomplexans. We show that these apicoplast genomes correspond to a diverse set of most closely related but genetically divergent Nephromyces lineages that co-inhabit a single tunicate host. In addition, the apicoplast of Nephromyces appears to have retained all biosynthetic pathways inferred to have been ancestral to parasitic apicomplexans. Our results shed light on the evolutionary history of the only probably mutualistic apicomplexan known, Nephromyces, and provide context for a better understanding of its life style and intricate symbiosis.

Detection and quantification of house mouse Eimeria at the species level – challenges and solutions for the assessment of Coccidia in wildlife

Detection and quantification of coccidia in studies of wildlife can be challenging. Therefore, the prevalence of coccidia is often not assessed at the parasite species level in non-livestock animals. Parasite species-specific prevalences are especially important when studying evolutionary questions in wild populations. We tested whether increased host population density increases the prevalence of individual Eimeria species at the farm level, as predicted by epidemiological theory. We studied free-living commensal populations of the house mouse (Mus musculus) in Germany and established a strategy to detect and quantify Eimeria infections. We show that a novel diagnostic primer targeting the apicoplast genome (Ap5) and coprological assessment after flotation provide complementary detection results increasing sensitivity. Genotyping PCRs confirm detection in a subset of samples and cross-validation of different PCR markers does not indicate a bias towards a particular parasite species in genotyping. We were able to detect double infections and to determine the preferred niche of each parasite species along the distal-proximal axis of the intestine. Parasite genotyping from tissue samples provides an additional indication for the absence of species bias in genotyping amplifications. Three Eimeria species were found infecting house mice at different prevalences: Eimeria ferrisi (16.7%; 95% CI 13.2 – 20.7), E. falciformis (4.2%; 95% CI 2.6 – 6.8) and E. vermiformis (1.9%; 95% CI 0.9 – 3.8). We also find that mice in dense populations are more likely to be infected with E. falciformis and E. ferrisi.We provide methods for the assessment of prevalences of coccidia at the species level in rodent systems. We show and discuss how such data can help to test hypotheses in ecology, evolution and epidemiology on a species level.

Atg8 is essential specifically for an autophagy-independent function in apicoplast biogenesis in blood-stage malaria parasites

Plasmodium parasites and related pathogens contain an essential non-photosynthetic plastid organelle, the apicoplast, derived from secondary endosymbiosis. Intriguingly, a highly conserved eukaryotic protein, autophagy-related protein 8 (Atg8), has an autophagy-independent function in the apicoplast. Little is known about the novel apicoplast function of Atg8 and its importance in blood-stage P. falciparum. Using a P. falciparum strain in which Atg8 expression was conditionally regulated, we showed that PfAtg8 is essential for parasite replication. Significantly, growth inhibition caused by the loss of PfAtg8 was reversed by addition of isopentenyl pyrophosphate (IPP), which was previously shown to rescue apicoplast defects in P. falciparum. Parasites deficient in PfAtg8, but growth rescued by IPP, had lost their apicoplast. We designed a suite of functional assays, including a new fluorescence in situ hybridization (FISH) method for detection of the low-copy apicoplast genome, to interrogate specific steps in apicoplast biogenesis and detect apicoplast defects which preceded the block in parasite replication. Though protein import and membrane expansion of the apicoplast were unaffected, the apicoplast was not inherited by daughter parasites. Our findings demonstrate that, though multiple autophagy-dependent and independent functions have been proposed for PfAtg8, only its role in apicoplast biogenesis is essential. We propose that PfAtg8 is required for fission or segregation of the apicoplast during parasite replication.