RESUMEN
Toxoplasma gondii is a zoonotic protist pathogen that infects up to one third of the human population. This apicomplexan parasite contains three genome sequences: nuclear (65 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear integrants of mitochondrial DNA) and NUPTs (nuclear integrants of plastid DNA) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome-the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma ~28 mya, revealed that the movement and fixation of five NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb), and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together, these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.
Asunto(s)
Toxoplasma , Humanos , Toxoplasma/genética , Genoma , ADN Mitocondrial/genética , Mitocondrias/genética , Evolución Molecular , Núcleo Celular/genética , Análisis de Secuencia de ADNRESUMEN
Malaria remains one of the deadliest infectious diseases. Transcriptional regulation effects of noncoding variants in this unusual genome of malaria parasites remain elusive. We developed a sequence-based, ab initio deep learning framework, MalariaSED, for predicting chromatin profiles in malaria parasites. The MalariaSED performance was validated by published ChIP-qPCR and TF motifs results. Applying MalariaSED to ~ 1.3 million variants shows that geographically differentiated noncoding variants are associated with parasite invasion and drug resistance. Further analysis reveals chromatin accessibility changes at Plasmodium falciparum rings are partly associated with artemisinin resistance. MalariaSED illuminates the potential functional roles of noncoding variants in malaria parasites.
Asunto(s)
Antimaláricos , Aprendizaje Profundo , Malaria Falciparum , Malaria , Parásitos , Animales , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Parásitos/genética , Plasmodium falciparum/genética , Malaria/tratamiento farmacológico , Malaria/parasitología , Cromatina , Resistencia a Medicamentos/genética , Antimaláricos/farmacología , Proteínas Protozoarias/genéticaRESUMEN
Toxoplasma gondii is a zoonotic protist pathogen that infects up to 1/3 of the human population. This apicomplexan parasite contains three genome sequences: nuclear (63 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear DNA of mitochondrial origin) and NUPTs (nuclear DNA of plastid origin) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome; the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma ~28 MY ago, revealed that the movement and fixation of 5 NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb) and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.
RESUMEN
Transmission of the deadly malaria parasite Plasmodium falciparum from humans to mosquitoes is achieved by specialized intraerythrocytic sexual forms called gametocytes. Though the crucial regulatory mechanisms leading to gametocyte commitment have recently come to light, networks of genes that control sexual development remain to be elucidated. Here, we report a pooled-mutant screen to identify genes associated with gametocyte development in P. falciparum. Our results categorized genes that modulate gametocyte progression as hypoproducers or hyperproducers of gametocytes, and the in-depth analysis of individual clones confirmed phenotypes in sexual commitment rates and putative functions in gametocyte development. We present a new set of genes that have not been implicated in gametocytogenesis before and demonstrate the potential of forward genetic screens in isolating genes impacting parasite sexual biology, an exciting step toward the discovery of new antimalarials for a globally significant pathogen. IMPORTANCE Blocking human-to-vector transmission is an essential step toward malaria elimination. Gametocytes are solely responsible for achieving this transmission and represent an opportunity for therapeutic intervention. While these falciform-shaped parasite stages were first discovered in the 1880s, our understanding of the genetic determinants responsible for their formation and molecular mechanisms that drive their development is limited. In this work, we developed a scalable screening methodology with piggyBac mutants to identify genes that influence the development of gametocytes in the most lethal human malaria parasite, P. falciparum. By doing so, we lay the foundation for large-scale functional genomic studies specifically designed to address remaining questions about sexual commitment, maturation, and mosquito infection in P. falciparum. Such functional genetic screens will serve to expedite the identification of essential pathways and processes for the development of novel transmission-blocking agents.
Asunto(s)
Culicidae , Malaria Falciparum , Malaria , Parásitos , Animales , Humanos , Plasmodium falciparum/genética , Mosquitos Vectores/genética , Malaria Falciparum/parasitología , FenotipoRESUMEN
The implementation of artemisinin (ART) combination therapies (ACTs) has greatly decreased deaths caused by Plasmodium falciparum malaria, but increasing ACT resistance in Southeast Asia and Africa could reverse this progress. Parasite population genetic studies have identified numerous genes, single-nucleotide polymorphisms (SNPs), and transcriptional signatures associated with altered artemisinin activity with SNPs in the Kelch13 (K13) gene being the most well-characterized artemisinin resistance marker. However, there is an increasing evidence that resistance to artemisinin in P. falciparum is not related only to K13 SNPs, prompting the need to characterize other novel genes that can alter ART responses in P. falciparum. In our previous analyses of P. falciparum piggyBac mutants, several genes of unknown function exhibited increased sensitivity to artemisinin that was similar to a mutant of K13. Further analysis of these genes and their gene co-expression networks indicated that the ART sensitivity cluster was functionally linked to DNA replication and repair, stress responses, and maintenance of homeostatic nuclear activity. In this study, we have characterized PF3D7_1136600, another member of the ART sensitivity cluster. Previously annotated as a conserved Plasmodium gene of unknown function, we now provide putative annotation of this gene as a Modulator of Ring Stage Translation (MRST). Our findings reveal that the mutagenesis of MRST affects gene expression of multiple translation-associated pathways during the early ring stage of asexual development via putative ribosome assembly and maturation activity, suggesting an essential role of MRST in protein biosynthesis and another novel mechanism of altering the parasite's ART drug response.IMPORTANCEPlasmodium falciparum malaria killed more than 600,000 people in 2021, though ACTs have been critical in reducing malaria mortality as a first-line treatment for infection. However, ACT resistance in Southeast Asia and emerging resistance in Africa are detrimental to this progress. Mutations to Kelch13 (K13) have been identified to confer increased artemisinin tolerance in field isolates, however, genes other than K13 are implicated in altering how the parasite responds to artemisinin prompts additional analysis. Therefore, in this study we have characterized a P. falciparum mutant clone with altered sensitivity to artemisinin and identified a novel gene (PF3D7_1136600) that is associated with alterations to parasite translational metabolism during critical timepoints for artemisinin drug response. Many genes of the P. falciparum genome remain unannotated, posing a challenge for drug-gene characterizations in the parasite. Therefore, through this study, we have putatively annotated PF3D7_1136600 as a novel MRST gene and have identified a potential link between MRST and parasite stress response mechanisms.
Asunto(s)
Antimaláricos , Artemisininas , Malaria Falciparum , Humanos , Plasmodium falciparum/metabolismo , Antimaláricos/farmacología , Antimaláricos/metabolismo , Artemisininas/farmacología , Malaria Falciparum/parasitologíaRESUMEN
The antimalarial activity of the frontline drug artemisinin involves generation of reactive oxygen species (ROS) leading to oxidative damage of parasite proteins. To achieve homeostasis and maintain protein quality control in the overwhelmed parasite, the ubiquitin-proteasome system kicks in. Even though molecular markers for artemisinin resistance like pfkelch13 have been identified, the intricate network of mechanisms driving resistance remains to be elucidated. Here, we report a forward genetic screening strategy that enables a broader identification of genetic factors responsible for altering sensitivity to dihydroartemisinin (DHA) and a proteasome inhibitor, bortezomib (BTZ). Using a library of isogenic piggyBac mutants in P. falciparum, we defined phenotype-genotype associations influencing drug responses and highlighted shared mechanisms between the two processes, which mainly included proteasome-mediated degradation and the lipid metabolism genes. Additional transcriptomic analysis of a DHA/BTZ-sensitive piggyBac mutant showed it is possible to find differences between the two response mechanisms on the specific components for regulation of the exportome. Our results provide further insight into the molecular mechanisms of antimalarial drug resistance. IMPORTANCE Malaria control is seriously threatened by the emergence and spread of Plasmodium falciparum resistance to the leading antimalarial, artemisinin. The potent killing activity of artemisinin results from oxidative damage unleashed by free heme activation released by hemoglobin digestion. Although the ubiquitin-proteasome system is considered critical for parasite survival of this toxicity, the diverse genetic changes linked to artemisinin resistance are complex and, so far, have not included the ubiquitin-proteasome system. In this study, we use a systematic forward genetic approach by screening a library of P. falciparum random piggyBac mutants to decipher the genetic factors driving malaria parasite responses to the oxidative stress caused by antimalarial drugs. This study compares phenotype-genotype associations influencing dihydroartemisinin responses with the proteasome inhibitor bortezomib to delineate the role of ubiquitin-proteasome system. Our study highlights shared and unique pathways from the complex array of molecular processes critical for P. falciparum survival resulting from the oxidative damage of artemisinin.
Asunto(s)
Antimaláricos , Artemisininas , Malaria Falciparum , Malaria , Humanos , Plasmodium falciparum , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Bortezomib/farmacología , Bortezomib/metabolismo , Bortezomib/uso terapéutico , Metabolismo de los Lípidos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/farmacología , Inhibidores de Proteasoma/metabolismo , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Proteínas Protozoarias/genética , Artemisininas/farmacología , Malaria Falciparum/tratamiento farmacológico , Resistencia a Medicamentos/genética , Ubiquitina/metabolismoRESUMEN
Artemisinin combination therapies (ACTs) have led to a significant decrease in Plasmodium falciparum malaria mortality. This progress is now threatened by emerging artemisinin resistance (ART-R) linked originally in SE Asia to polymorphisms in the Kelch propeller protein (K13) and more recently to several other seemingly unrelated genetic mutations. To better understand the parasite response to ART, we are characterizing a P. falciparum mutant with altered sensitivity to ART that was created via piggyBac transposon mutagenesis. The transposon inserted near the putative transcription start site of a gene defined as a "Plasmodium-conserved gene of unknown function," now functionally linked to K13 as the Kelch13 Interacting Candidate 5 protein (KIC5). Phenotype analysis of the KIC5 mutant during intraerythrocytic asexual development identified transcriptional changes associated with DNA stress response and altered mitochondrial metabolism, linking dysregulation of the KIC5 gene to the parasite's ability to respond to ART exposure. Through characterization of the KIC5 transcriptome, we hypothesize that this gene may be essential under ART exposure to manage gene expression of the wild-type stress response at early ring stage, thereby providing a better understanding of the parasite's processes that can alter ART sensitivity.
Asunto(s)
Antimaláricos , Artemisininas , Plasmodium falciparum , Antimaláricos/farmacología , Artemisininas/uso terapéutico , Resistencia a Medicamentos/genética , Mutación , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Atrial fibrillation (AF), the most common abnormal heart rhythm, is a major cause for stroke. Aging is a significant risk factor for AF; however, specific ionic pathways that can elucidate how aging leads to AF remain elusive. We used young and old wild-type and PKC epsilon- (PKCϵ) knockout mice, whole animal, and cellular electrophysiology, as well as whole heart, and cellular imaging to investigate how aging leads to the aberrant functioning of a potassium current, and consequently to AF facilitation. Our experiments showed that knocking out PKCϵ abrogates the effects of aging on AF by preventing the development of a constitutively active acetylcholine sensitive inward rectifier potassium current (IKACh). Moreover, blocking this abnormal current in the old heart reduces AF inducibility. Our studies demonstrate that in the aging heart, IKACh is constitutively active in a PKCϵ-dependent manner, contributing to the perpetuation of AF.
RESUMEN
The emergence and spread of Plasmodium falciparum parasites resistant to front-line antimalarial artemisinin-combination therapies (ACT) threatens to erase the considerable gains against the disease of the last decade. Here, we develop a large-scale phenotypic screening pipeline and use it to carry out a large-scale forward-genetic phenotype screen in P. falciparum to identify genes allowing parasites to survive febrile temperatures. Screening identifies more than 200 P. falciparum mutants with differential responses to increased temperature. These mutants are more likely to be sensitive to artemisinin derivatives as well as to heightened oxidative stress. Major processes critical for P. falciparum tolerance to febrile temperatures and artemisinin include highly essential, conserved pathways associated with protein-folding, heat shock and proteasome-mediated degradation, and unexpectedly, isoprenoid biosynthesis, which originated from the ancestral genome of the parasite's algal endosymbiont-derived plastid, the apicoplast. Apicoplast-targeted genes in general are upregulated in response to heat shock, as are other Plasmodium genes with orthologs in plant and algal genomes. Plasmodium falciparum parasites appear to exploit their innate febrile-response mechanisms to mediate resistance to artemisinin. Both responses depend on endosymbiont-derived genes in the parasite's genome, suggesting a link to the evolutionary origins of Plasmodium parasites in free-living ancestors.
Asunto(s)
Apicoplastos/metabolismo , Artemisininas/farmacología , Resistencia a Medicamentos , Fiebre/parasitología , Malaria Falciparum/parasitología , Parásitos/fisiología , Animales , Apicoplastos/efectos de los fármacos , Resistencia a Medicamentos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Respuesta al Choque Térmico/efectos de los fármacos , Mutación/genética , Parásitos/efectos de los fármacos , Fenotipo , Plasmodium falciparum/genética , Transducción de Señal/efectos de los fármacos , Temperatura , Terpenos/metabolismo , Transcripción Genética/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Resistance to antimalarial drugs, and in particular to the artemisinin derivatives and their partner drugs, threatens recent progress toward regional malaria elimination and eventual global malaria eradication. Population-level studies utilizing whole-genome sequencing approaches have facilitated the identification of regions of the parasite genome associated with both clinical and in vitro drug-resistance phenotypes. However, the biological relevance of genes identified in these analyses and the establishment of a causal relationship between genotype and phenotype requires functional characterization. Here we examined data from population genomic and transcriptomic studies in the context of data generated from recent functional studies, using a new population genetic approach designed to identify potential favored mutations within the region of a selective sweep (iSAFE). We identified several genes functioning in pathways now known to be associated with artemisinin resistance that were supported in early population genomic studies, as well as potential new drug targets/pathways for further validation and consideration for treatment of artemisinin-resistant Plasmodium falciparum. In addition, we establish the utility of iSAFE in identifying positively-selected mutations in population genomic studies, potentially accelerating the time to functional validation of candidate genes.
Asunto(s)
Antimaláricos , Artemisininas , Malaria Falciparum , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Artemisininas/farmacología , Artemisininas/uso terapéutico , Genómica , Humanos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/genéticaRESUMEN
Mosquito transmission of the deadly malaria parasite Plasmodium falciparum is mediated by mature sexual forms (gametocytes). Circulating in the vertebrate host, relatively few intraerythrocytic gametocytes are picked up during a bloodmeal to continue sexual development in the mosquito vector. Human-to-vector transmission thus represents an infection bottleneck in the parasite's life cycle for therapeutic interventions to prevent malaria. Even though recent progress has been made in the identification of genetic factors linked to gametocytogenesis, a plethora of genes essential for sexual-stage development are yet to be unraveled. In this review, we revisit P. falciparum transmission biology by discussing targetable features of gametocytes and provide a perspective on a forward-genetic approach for identification of novel transmission-blocking candidates in the future.
RESUMEN
Genome-scale mutagenesis screens for genes essential for apicomplexan parasite survival have been completed in three species: Plasmodium falciparum, the major human malaria parasite, Plasmodium berghei, a model rodent malaria parasite, and the more distantly related Toxoplasma gondii, the causative agent of toxoplasmosis. These three species share 2606 single-copy orthologs, 1500 of which have essentiality data in all three screens. In this review, we explore the overlap between these datasets to define the core essential genes of the phylum Apicomplexa. We further discuss the implications of these groundbreaking studies for understanding apicomplexan parasite biology, and we identify promising areas of focus for developing new pan-apicomplexan parasite interventions.
Asunto(s)
Apicomplexa , Genes Esenciales , Genoma de Protozoos , Apicomplexa/genética , Genes Esenciales/genética , Genoma de Protozoos/genéticaRESUMEN
The human malaria parasite Plasmodium falciparum thrives in radically different host environments in mosquitoes and humans, with only a limited set of transcription factors. The nature of regulatory elements or their target genes in the P. falciparum genome remains elusive. Here, we found that this eukaryotic parasite uses an efficient way to maximally use genetic and epigenetic regulation to form regulatory units (RUs) during blood infections. Genes located in the same RU tend to have the same pattern of expression over time and are associated with open chromatin along regulatory elements. To precisely define and quantify these RUs, a novel hidden Markov model was developed to capture the regulatory structure in a genome-wide fashion by integrating expression and epigenetic evidence. We successfully identified thousands of RUs and cross-validated with previous findings. We found more genes involved in red blood cell (RBC) invasion located in the same RU as the PfAP2-I (AP2-I) transcription factor, demonstrating that AP2-I is responsible for regulating RBC invasion. Our study has provided a regulatory mechanism for a compact eukaryotic genome and offers new insights into the in vivo transcriptional regulation of the P. falciparum intraerythrocytic stage.
Asunto(s)
Regulación de la Expresión Génica/genética , Malaria Falciparum/genética , Plasmodium falciparum/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Cromatina/genética , Cromosomas/genética , Epigénesis Genética/genética , Eritrocitos , Genoma Humano , Humanos , Malaria Falciparum/parasitología , Malaria Falciparum/patología , Plasmodium falciparum/patogenicidadRESUMEN
Background: Basic and clinical scientific research at the University of South Florida (USF) have intersected to support a multi-faceted approach around a common focus on rare iron-related diseases. We proposed a modified version of the National Center for Biotechnology Information's (NCBI) Hackathon-model to take full advantage of local expertise in building "Iron Hack", a rare disease-focused hackathon. As the collaborative, problem-solving nature of hackathons tends to attract participants of highly-diverse backgrounds, organizers facilitated a symposium on rare iron-related diseases, specifically porphyrias and Friedreich's ataxia, pitched at general audiences. Methods: The hackathon was structured to begin each day with presentations by expert clinicians, genetic counselors, researchers focused on molecular and cellular biology, public health/global health, genetics/genomics, computational biology, bioinformatics, biomolecular science, bioengineering, and computer science, as well as guest speakers from the American Porphyria Foundation (APF) and Friedreich's Ataxia Research Alliance (FARA) to inform participants as to the human impact of these diseases. Results: As a result of this hackathon, we developed resources that are relevant not only to these specific disease-models, but also to other rare diseases and general bioinformatics problems. Within two and a half days, "Iron Hack" participants successfully built collaborative projects to visualize data, build databases, improve rare disease diagnosis, and study rare-disease inheritance. Conclusions: The purpose of this manuscript is to demonstrate the utility of a hackathon model to generate prototypes of generalizable tools for a given disease and train clinicians and data scientists to interact more effectively.
Asunto(s)
Ataxia de Friedreich , Porfirias , Bases de Datos Factuales , Humanos , Hierro , Enfermedades Raras , Estados UnidosRESUMEN
Among the many anthropogenic changes that impact humans and wildlife, one of the most pervasive but least understood is light pollution. Although detrimental physiological and behavioural effects resulting from exposure to light at night are widely appreciated, the impacts of light pollution on infectious disease risk have not been studied. Here, we demonstrate that artificial light at night (ALAN) extends the infectious-to-vector period of the house sparrow (Passer domesticus), an urban-dwelling avian reservoir host of West Nile virus (WNV). Sparrows exposed to ALAN maintained transmissible viral titres for 2 days longer than controls but did not experience greater WNV-induced mortality during this window. Transcriptionally, ALAN altered the expression of gene regulatory networks including key hubs (OASL, PLBD1 and TRAP1) and effector genes known to affect WNV dissemination (SOCS). Despite mounting anti-viral immune responses earlier, transcriptomic signatures indicated that ALAN-exposed individuals probably experienced pathogen-induced damage and immunopathology, potentially due to evasion of immune effectors. A simple mathematical modelling exercise indicated that ALAN-induced increases of host infectious-to-vector period could increase WNV outbreak potential by approximately 41%. ALAN probably affects other host and vector traits relevant to transmission, and additional research is needed to advise the management of zoonotic diseases in light-polluted areas.
Asunto(s)
Enfermedades de las Aves/virología , Reservorios de Enfermedades/veterinaria , Luz/efectos adversos , Gorriones , Fiebre del Nilo Occidental/veterinaria , Virus del Nilo Occidental/fisiología , Animales , Reservorios de Enfermedades/virología , Florida , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/efectos de la radiaciónRESUMEN
With relatively few known specific transcription factors to control the abundance of specific mRNAs, Plasmodium parasites may rely more on the regulation of transcript stability and turnover to provide sufficient gene regulation. Plasmodium transmission stages impose translational repression on specific transcripts in part to accomplish this. However, few proteins are known to participate in this process, and those that are characterized primarily affect female gametocytes. We have identified and characterized Plasmodium yoelii (Py) CCR4-1, a putative deadenylase, which plays a role in the development and activation of male gametocytes, regulates the abundance of specific mRNAs in gametocytes, and ultimately increases the efficiency of host-to-vector transmission. We find that when pyccr4-1 is deleted or its protein made catalytically inactive, there is a loss in the initial coordination of male gametocyte maturation and a reduction of parasite infectivity of the mosquito. Expression of only the N-terminal CAF1 domain of the essential CAF1 deadenylase leads to a similar phenotype. Comparative RNA-seq revealed that PyCCR4-1 affects transcripts important for transmission-related functions that are associated with male or female gametocytes, some of which directly associate with the immunoprecipitated complex. Finally, circular RT-PCR of one of the bound, dysregulated transcripts showed that deletion of the pyccr4-1 gene does not result in gross changes to its UTR or poly(A) tail length. We conclude that the two putative deadenylases of the CAF1/CCR4/NOT complex play critical and intertwined roles in gametocyte maturation and transmission.
Asunto(s)
Plasmodium falciparum/genética , Receptores CCR4/metabolismo , Animales , Culicidae/metabolismo , Exorribonucleasas , Gametogénesis/fisiología , Regulación de la Expresión Génica , Proteínas de Homeodominio , Masculino , Ratones , Mosquitos Vectores , Plasmodium/genética , Plasmodium falciparum/metabolismo , Proteínas , ARN Mensajero/genética , Proteínas Represoras , Ribonucleasas , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Life cycles of spirorchiids that infect the vascular system of turtles are poorly understood. Few life cycles of these blood flukes have been elucidated and all intermediate hosts reported are gastropods (Mollusca), regardless of whether the definitive host is a freshwater or a marine turtle. During a recent survey of blood fluke larvae in polychaetes on the coast of South Carolina, USA, spirorchiid-like cercariae were found to infect the polychaetes Amphitrite ornata (Terebellidae) and Enoplobranchus sanguineus (Polycirridae). Cercariae were large, furcate, with a ventral acetabulum, but no eyespots were observed. Partial sequences of D1-D2 domains of the large ribosomal subunit, the internal transcribed spacer 2, and the mitochondrial cytochrome oxidase 1 genes allowed the identification of sporocysts and cercariae as belonging to two unidentified Neospirorchis species reported from the green turtle, Chelonia mydas, in Florida: Neospirorchis sp. (Neogen 13) in A. ornata and Neospirorchis sp. (Neogen 14) in E. sanguineus. Phylogenetic analysis suggests that infection of annelids by blood flukes evolved separately in aporocotylids and spirorchiids. Our results support the contention that the Spirorchiidae is not a valid family and suggest that Neospirorchis is a monophyletic clade within the paraphyletic Spirorchiidae. Since specificity of spirorchiids for their intermediate hosts is broader than it was thus far assumed, surveys of annelids in turtle habitats are necessary to further our understanding of the life history of these pathogenic parasites.
Asunto(s)
Oocistos/aislamiento & purificación , Poliquetos/parasitología , Trematodos/aislamiento & purificación , Tortugas/parasitología , Animales , Interacciones Huésped-ParásitosRESUMEN
Severe malaria is caused by the apicomplexan parasite Plasmodium falciparum. Despite decades of research, the distinct biology of these parasites has made it challenging to establish high-throughput genetic approaches to identify and prioritize therapeutic targets. Using transposon mutagenesis of P. falciparum in an approach that exploited its AT-rich genome, we generated more than 38,000 mutants, saturating the genome and defining mutability and fitness costs for over 87% of genes. Of 5399 genes, our study defined 2680 genes as essential for optimal growth of asexual blood stages in vitro. These essential genes are associated with drug resistance, represent leading vaccine candidates, and include approximately 1000 Plasmodium-conserved genes of unknown function. We validated this approach by testing proteasome pathways for individual mutants associated with artemisinin sensitivity.
Asunto(s)
Genes Protozoarios , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Reproducción Asexuada/genética , Animales , Antimaláricos/farmacología , Artemisininas/farmacología , Secuencia Conservada , Resistencia a Medicamentos/genética , Eritrocitos/parasitología , Genes Esenciales , Aptitud Genética , Humanos , Vacunas contra la Malaria/genética , Mutagénesis , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
Aporocotylidae comprises a diverse family of fish blood flukes, with adults found in blood or body cavity of marine, brackish, or freshwater fish. Aporocotylids are unique among the Digenea with many developing in polychaetes. The life cycle has been elucidated for only a few species that develop in polychaetes from marine/brackish environments and none for western Atlantic aporocotylids. The basis for this study was observations of blood fluke larvae in annelids from South Carolina, USA in 1982 prior to possible usage of molecular tools to specifically identify parasite larvae. Recent description of aporocotylid species and genotyping tools prompted revisiting original collection sites to examine polychaetes and fish as potential hosts. Polycirrid Enoplobranchus sanguineus and terebellids Amphitrite ornata, and Terebella lapidaria revealed infections with aporocotylid larvae. Adult blood flukes were also collected from fish commonly encountered in the same habitat: spotted seatrout (Cynoscion nebulosus), red drum (Sciaenops ocellatus), black drum (Pogonias cromis), and Atlantic croaker (Micropogonias undulatus). Sporocysts containing cercariae were found in individuals of each annelid species. Adult Cardicola parvus were found in spotted seatrout and Atlantic croaker, C. laruei in spotted seatrout, C. currani in red drum, and C. palmeri in black drum. Genotype analysis of ITS-2 and lsrDNA of all forms confirmed conspecific infections by C. parvus in E. sanguineus and A. ornata and C. laruei in T. lapidaria. This is the first description of complete life cycles of aporocotylids in the Western Atlantic and first evidence of cryptic infections of Cy. nebulosus with C. parvus.
Asunto(s)
Poliquetos/parasitología , Trematodos/fisiología , Infecciones por Trematodos/parasitología , Trucha/parasitología , Animales , Océano Atlántico , Cercarias/aislamiento & purificación , Genotipo , Estadios del Ciclo de Vida/fisiología , Oocistos/aislamiento & purificación , Perciformes/parasitología , Trematodos/genéticaRESUMEN
Malaria remains one of the most devastating parasitic diseases worldwide, with 90% of the malaria deaths in Africa in 2013 attributable to Plasmodium falciparum. The clinical symptoms of malaria include cycles of fever, corresponding to parasite rupture from red blood cells every 48 h. Parasite pathways involved in the parasite's ability to survive the host fever response, and indeed, the functions of ~40% of P. falciparum genes as a whole, are still largely unknown. Here, we evaluated the potential of scalable forward-genetic screening methods to identify genes involved in the host fever response. We performed a phenotypic screen for genes linked to the parasite response to febrile temperatures by utilizing a selection of single-disruption P. falciparum mutants generated via random piggyBac transposon mutagenesis in a previous study. We identified several mutants presenting significant phenotypes in febrile response screens compared to the wild type, indicating possible roles for the disrupted genes in this process. We present these initial studies as proof that forward genetics can be used to gain insight into critical factors associated with parasite biology. IMPORTANCE Though the P. falciparum genome sequence has been available for many years, ~40% of its genes do not have informative annotations, as they show no detectable homology to those of studied organisms. More still have not been evaluated via genetic methods. Scalable forward-genetic approaches that allow interrogation of gene function without any pre-existing knowledge are needed to hasten understanding of parasite biology, which will expedite the identification of drug targets and the development of future interventions in the face of spreading resistance to existing frontline drugs. In this work, we describe a new approach to pursue forward-genetic phenotypic screens for P. falciparum to identify factors associated with virulence. Future large-scale phenotypic screens developed to probe other such interesting phenomena, when considered in parallel, will prove a powerful tool for functional annotation of the P. falciparum genome, where so much remains undiscovered.