ABSTRACT
Apicomplexa is a large monophyletic phylum of unicellular, parasitic organisms. Reptiles are hosts to both haemosporidian (Haemosporida) and hemogregarine (Eucoccidiorida) apicomplexan blood parasites. Within reptiles our understanding of their diversity remains limited, with a paucity of information from Australia, despite a high diversity of squamates (snakes and lizards). We provide a preliminary assessment of haemosporidian and hemogregarine diversity occurring in lizards across northern tropical Australia, building on existing data with results from a microscopy and genetic assessment. We screened total of 233 blood slides using microscopy and detected hemogregarines in 25 geckos, 2 skinks and 1 agamid, while haemosporidians were detected in 13 geckos. DNA sequencing of 28 samples of the hemogregarine 18S rRNA (â¼900 bp) nuclear gene revealed five lineages of Australian lizard hemogregarines within heteroxenous adeleids. We sequenced 10 samples of Haemosporida mtDNA (cytb & coI: â¼1313 bp) and phylogenetic analysis with 30 previously published sequences revealed that the Australian Haemosporida grouped within the Haemoproteidae but were not supported as a monophyletic clade. Our results demonstrate that there is significant undocumented evolutionary diversity in Australian lizard haemosporidian and hemogregarine parasites, with preliminary evidence of significantly higher infection rates in geckos.
Subject(s)
Haemosporida , Lizards , Parasites , Animals , Australia , Haemosporida/genetics , Lizards/genetics , PhylogenyABSTRACT
OBJECTIVE: Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe developmental and epileptic encephalopathies. We delineate the genetic causes and genotype-phenotype correlations of a large EIMFS cohort. METHODS: Phenotypic and molecular data were analyzed on patients recruited through an international collaborative study. RESULTS: We ascertained 135 patients from 128 unrelated families. Ninety-three of 135 (69%) had causative variants (42/55 previously reported) across 23 genes, including 9 novel EIMFS genes: de novo dominant GABRA1, GABRB1, ATP1A3; X-linked CDKL5, PIGA; and recessive ITPA, AIMP1, KARS, WWOX. The most frequently implicated genes were KCNT1 (36/135, 27%) and SCN2A (10/135, 7%). Mosaicism occurred in 2 probands (SCN2A, GABRB3) and 3 unaffected mothers (KCNT1). Median age at seizure onset was 4 weeks, with earlier onset in the SCN2A, KCNQ2, and BRAT1 groups. Epileptic spasms occurred in 22% patients. A total of 127 patients had severe to profound developmental impairment. All but 7 patients had ongoing seizures. Additional features included microcephaly, movement disorders, spasticity, and scoliosis. Mortality occurred in 33% at median age 2 years 7 months. INTERPRETATION: We identified a genetic cause in 69% of patients with EIMFS. We highlight the genetic heterogeneity of EIMFS with 9 newly implicated genes, bringing the total number to 33. Mosaicism was observed in probands and parents, carrying critical implications for recurrence risk. EIMFS pathophysiology involves diverse molecular processes from gene and protein regulation to ion channel function and solute trafficking. ANN NEUROL 2019;86:821-831.
Subject(s)
Genetic Predisposition to Disease/genetics , Seizures/diagnosis , Seizures/genetics , Spasms, Infantile/diagnosis , Spasms, Infantile/genetics , Adolescent , Child , Child, Preschool , Cohort Studies , Female , Humans , Infant , Male , Seizures/physiopathology , Spasms, Infantile/physiopathologyABSTRACT
Epilepsy of infancy with migrating focal seizures (EIMFS), one of the most severe developmental and epileptic encephalopathy syndromes, is characterized by seizures that migrate from one hemisphere to the other. EIMFS is genetically heterogeneous with 33 genes. We report five patients with EIMFS caused by recessive BRAT1 variants, identified via next generation sequencing. Recessive pathogenic variants in BRAT1 cause the rigidity and multifocal seizure syndrome, lethal neonatal with hypertonia, microcephaly, and intractable multifocal seizures. The epileptology of BRAT1 encephalopathy has not been well described. All five patients were profoundly impaired with seizure onset in the first week of life and focal seizure migration between hemispheres. We show that BRAT1 is an important recessive cause of EIMFS with onset in the first week of life, profound impairment, and early death. Early recognition of this genetic aetiology will inform management and reproductive counselling.
Subject(s)
Brain Diseases/genetics , Epilepsy/genetics , Epilepsy/pathology , Nuclear Proteins/genetics , Seizures/genetics , Seizures/pathology , Brain/pathology , Genes, Recessive , High-Throughput Nucleotide Sequencing , Humans , Infant, Newborn , Magnetic Resonance ImagingABSTRACT
Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.
Subject(s)
Gene Expression Regulation, Developmental , Genetic Variation , Histones/genetics , Promoter Regions, Genetic/genetics , Protozoan Proteins/genetics , Blotting, Western , Cells, Cultured , Chromatin Immunoprecipitation , DNA, Protozoan/genetics , Epigenomics , Euchromatin/genetics , Fluorescent Antibody Technique , Gene Silencing , Histone Deacetylases/metabolism , Humans , Immunoprecipitation , Malaria, Falciparum/genetics , Malaria, Falciparum/metabolism , Malaria, Falciparum/parasitology , Nucleosomes/genetics , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Initiation Site , Transcriptional ActivationABSTRACT
The Plasmodium mitochondrial electron transport chain has received considerable attention as a potential target for new antimalarial drugs. Atovaquone, a potent inhibitor of Plasmodium cytochrome bc(1), in combination with proguanil is recommended for chemoprophylaxis and treatment of malaria. The type II NADH:ubiquinone oxidoreductase (NDH2) is considered an attractive drug target, as its inhibition is thought to lead to the arrest of the mitochondrial electron transport chain and, as a consequence, pyrimidine biosynthesis, an essential pathway for the parasite. Using the rodent malaria parasite Plasmodium berghei as an in vivo infection model, we studied the role of NDH2 during Plasmodium life cycle progression. NDH2 can be deleted by targeted gene disruption and, thus, is dispensable for the pathogenic asexual blood stages, disproving the candidacy for an anti-malarial drug target. After transmission to the insect vector, NDH2-deficient ookinetes display an intact mitochondrial membrane potential. However, ndh2(-) parasites fail to develop into mature oocysts in the mosquito midgut. We propose that Plasmodium blood stage parasites rely on glycolysis as the main ATP generating process, whereas in the invertebrate vector, a glucose-deprived environment, the malaria parasite is dependent on an intact mitochondrial respiratory chain.
Subject(s)
Adenosine Triphosphate/biosynthesis , Electron Transport Complex I/metabolism , Glycolysis/physiology , Oocysts/enzymology , Plasmodium berghei/enzymology , Protozoan Proteins/metabolism , Animals , Cell Cycle/physiology , Culicidae/parasitology , Electron Transport/physiology , Electron Transport Complex I/genetics , Malaria/diet therapy , Malaria/enzymology , Malaria/genetics , Mice , Mitochondria/enzymology , Mitochondria/genetics , Oocysts/cytology , Organisms, Genetically Modified , Plasmodium berghei/genetics , Protozoan Proteins/geneticsABSTRACT
UNLABELLED: Malaria is transmitted when motile sporozoites are injected into the dermis by an infected female Anopheles mosquito. Inside the mosquito vector, sporozoites egress from midgut-associated oocysts and eventually penetrate the acinar cells of salivary glands. Parasite-encoded factors with exclusive vital roles in the insect vector can be studied by classical reverse genetics. Here, we characterized the in vivo roles of Plasmodium berghei falstatin/ICP (inhibitor of cysteine proteases). This protein was previously suggested to act as a protease inhibitor during erythrocyte invasion. We show by targeted gene disruption that loss of ICP function does not affect growth inside the mammalian host but causes a complete defect in sporozoite transmission. Sporogony occurred normally in icp(-) parasites, but hemocoel sporozoites showed a defect in continuous gliding motility and infectivity for salivary glands, which are prerequisites for sporozoite transmission to the mammalian host. Absence of ICP correlates with enhanced cleavage of circumsporozoite protein, in agreement with a role as a protease regulator. We conclude that ICP is essential for only the final stages of sporozoite maturation inside the mosquito vector. This study is the first genetic evidence that an ICP is necessary for the productive motility of a eukaryotic parasitic cell. IMPORTANCE: Cysteine proteases and their inhibitors are considered ideal drug targets for the treatment of a wide range of diseases, including cancer and parasitic infections. In protozoan parasites, including Leishmania, Trypanosoma, and Plasmodium, cysteine proteases play important roles in life cycle progression. A mouse malaria model provides an unprecedented opportunity to study the roles of a parasite-encoded inhibitor of cysteine proteases (ICP) over the entire parasite life cycle. By precise gene deletion, we found no evidence that ICP influences disease progression or parasite virulence. Instead, we discovered that this factor is necessary for parasite movement and malaria transmission from mosquitoes to mammals. This finding in a fast-moving unicellular protozoan has important implications for malaria intervention strategies and the roles of ICPs in the regulation of eukaryotic cell migration.