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1.
Annu Rev Microbiol ; 70: 63-81, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27359216

RESUMO

Toxoplasma gondii is a widespread parasite of warm-blooded vertebrates that also causes opportunistic infections in humans. Rodents are a natural host for asexually replicating forms, whereas cats serve as the definitive host for sexual development. The laboratory mouse provides a model to study pathogenesis. Strains of T. gondii are globally diverse, with more than 16 distinct haplogroups clustered into 6 major clades. Forward genetic analysis of genetic crosses between different lineages has been used to define the molecular basis of acute virulence in the mouse. These studies have identified a family of secretory serine/threonine rhoptry kinases that target innate immune pathways to protect intracellular parasites from destruction. Rhoptry kinases target immunity-related GTPases, a family of immune effectors that is expanded in rodents. Similar forward genetic studies may be useful to define the basis of pathogenesis in other hosts, including humans, where infections of different strains present with variable clinical severity.


Assuntos
Mapeamento Cromossômico , Toxoplasma/genética , Toxoplasma/patogenicidade , Toxoplasmose Animal/parasitologia , Toxoplasmose/parasitologia , Animais , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Virulência
2.
PLoS Genet ; 11(8): e1005434, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26291965

RESUMO

Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg) in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL) analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5 and ROP18 are conserved virulence factors in genetically diverse strains from North and South America, suggesting they evolved to resist innate immune defenses in ancestral T. gondii strains, and they have subsequently diversified under positive selection.


Assuntos
Proteínas de Protozoários/genética , Doenças dos Roedores/parasitologia , Toxoplasma/genética , Toxoplasmose Animal/parasitologia , Sequência de Aminoácidos , Animais , Animais não Endogâmicos , Variações do Número de Cópias de DNA , Dados de Sequência Molecular , Filogenia , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/química , Locos de Características Quantitativas , América do Sul , Toxoplasma/patogenicidade , Virulência/genética , Fatores de Virulência/genética
3.
Eukaryot Cell ; 14(2): 140-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25480939

RESUMO

Quantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasite Toxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDR(r) and type 10 VAND-SNF(r). The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of the SNR1 gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies in T. gondii.


Assuntos
Adenosina/análogos & derivados , Sistemas de Transporte de Aminoácidos/genética , Antiprotozoários/toxicidade , Resistência a Medicamentos/genética , Ligação Genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Adenosina/toxicidade , Marcadores Genéticos , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Seleção Genética , Toxoplasma/efeitos dos fármacos
4.
BMC Genomics ; 15: 350, 2014 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-24885521

RESUMO

BACKGROUND: Considerable work has been carried out to understand the biology of tachyzoites and bradyzoites of Toxoplasma gondii in large part due to in vitro culture methods for these stages. However, culturing methods for stages that normally develop in the gut of the definitive felid host, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite's life cycle. Here, we begin to unravel the molecular aspects of enteric stages by providing new data on merozoite stage gene expression. RESULTS: To profile gene expression differences in enteric stages we harvested merozoites from the intestine of infected cats and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by comparing it to previously published data for the oocyst, tachyzoite, and bradyzoite stages. Principal component analysis highlighted the unique profile of merozoites, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen one (SAG1) and many dense granule proteins do not label merozoites: our microarray data confirms that these genes were not expressed at this stage. Also, the expression for many rhoptry and microneme proteins was drastically reduced while the expression for many surface antigens was increased at the merozoite stage. Gene Ontology and KEGG analysis revealed that genes involved in transcription/translation and many metabolic pathways were upregulated at the merozoite stage, highlighting unique growth requirements of this stage. To functionally test these predictions, we demonstrated that an upstream promoter region of a merozoite specific gene was sufficient to control expression in merozoites in vivo. CONCLUSIONS: Merozoites are the first developmental stage in the coccidian cycle that takes place within the gut of the definitive host. The data presented here describe the global gene expression profile of the merozoite stage and the creation of transgenic parasite strains that show stage-specific expression of reporter genes in the cat intestine. These data and reagents will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate enteric development.


Assuntos
Merozoítos/metabolismo , Toxoplasma/genética , Animais , Antígenos de Protozoários/genética , Gatos , Análise por Conglomerados , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , Regiões Promotoras Genéticas , Proteínas de Protozoários/genética , RNA Mensageiro/metabolismo , Toxoplasma/metabolismo , Toxoplasmose Animal/metabolismo , Toxoplasmose Animal/parasitologia , Toxoplasmose Animal/patologia
5.
PLoS Pathog ; 8(11): e1002992, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23144612

RESUMO

Secretory polymorphic serine/threonine kinases control pathogenesis of Toxoplasma gondii in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-γ mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 in vitro, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δrop5 and Δrop18 mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3(-/-) mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-γ dependent, indicting it plays a general role in governing virulence factors that block immunity.


Assuntos
Macrófagos Peritoneais/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Toxoplasma/imunologia , Toxoplasma/patogenicidade , Toxoplasmose/imunologia , Animais , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Deleção de Genes , Interferon gama/genética , Interferon gama/imunologia , Macrófagos Peritoneais/microbiologia , Camundongos , Camundongos Knockout , Fosforilação/genética , Fosforilação/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas de Protozoários , Toxoplasma/genética , Toxoplasmose/genética
6.
Proc Natl Acad Sci U S A ; 108(23): 9631-6, 2011 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-21586633

RESUMO

The population structure of Toxoplasma gondii includes three highly prevalent clonal lineages referred to as types I, II, and III, which differ greatly in virulence in the mouse model. Previous studies have implicated a family of serine/threonine protein kinases found in rhoptries (ROPs) as important in mediating virulence differences between strain types. Here, we explored the genetic basis of differences in virulence between the highly virulent type I lineage and moderately virulent type II based on successful genetic cross between these lineages. Genome-wide association revealed that a single quantitative trait locus controls the dramatic difference in lethality between these strain types. Neither ROP16 nor ROP18, previously implicated in virulence of T. gondii, was found to contribute to differences between types I and II. Instead, the major virulence locus contained a tandem cluster of polymorphic alleles of ROP5, which showed similar protein expression between strains. ROP5 contains a conserved serine/threonine protein kinase domain that includes only part of the catalytic triad, and hence, all members are considered to be pseudokinases. Genetic disruption of the entire ROP5 locus in the type I lineage led to complete attenuation of acute virulence, and complementation with ROP5 restored lethality to WT levels. These findings reveal that a locus of polymorphic pseudokinases plays an important role in pathogenesis of toxoplasmosis in the mouse model.


Assuntos
Família Multigênica/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Toxoplasma/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Gatos , Mapeamento Cromossômico , Feminino , Perfilação da Expressão Gênica , Teste de Complementação Genética , Variação Genética , Genoma de Protozoário/genética , Camundongos , Dados de Sequência Molecular , Mutação , Filogenia , Proteínas Serina-Treonina Quinases/genética , Proteínas de Protozoários/classificação , Locos de Características Quantitativas/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Toxoplasma/classificação , Toxoplasma/patogenicidade , Toxoplasmose Animal/parasitologia , Virulência/genética
7.
PLoS Pathog ; 7(12): e1002392, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22144892

RESUMO

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes--a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.


Assuntos
Apicomplexa/genética , Plastídeos/genética , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA de Protozoário/genética , Apicomplexa/metabolismo , Apicomplexa/patogenicidade , Ciclo Celular/fisiologia , Plastídeos/metabolismo , Transporte Proteico/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/biossíntese , RNA de Protozoário/biossíntese
8.
Mol Microbiol ; 79(1): 192-204, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21166903

RESUMO

Intracellular microbes have evolved efficient strategies for transitioning from one cell to another in a process termed intercellular transmission. Here we show that host cell transmission of the obligate intracellular parasite Toxoplasma gondii is closely tied to specific cell cycle distributions, with egress and reinvasion occurring most proficiently by parasites in the G1 phase. We also reveal that Toxoplasma undergoes marked changes in mRNA expression when transitioning from the extracellular environment to its intracellular niche. These mRNA level changes reflect a modal switch from expression of proteins involved in invasion, motility and signal transduction in extracellular parasites to expression of metabolic and DNA replication proteins in intracellular parasites. Host cell binding and signalling associated with the discharge of parasite secretory proteins was not sufficient to induce this switch in gene expression, suggesting that the regulatory mechanisms responsible are tied to the establishment of the intracellular environment. The genes whose expression increased after parasite invasion belong to a progressive cascade known to underlie the parasite division cycle indicating that the unique relationship between the G1 phase and invasion effectively synchronizes short-term population growth. This work provides new insight into how this highly successful parasite competently transits from cell to cell.


Assuntos
Ciclo Celular , Fibroblastos/fisiologia , Fibroblastos/parasitologia , Regulação da Expressão Gênica , Toxoplasma/genética , Toxoplasma/patogenicidade , Células Cultivadas , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita , Humanos , Transdução de Sinais , Toxoplasma/crescimento & desenvolvimento
9.
Eukaryot Cell ; 8(12): 1828-36, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19801420

RESUMO

Toxoplasma gondii has an unusual population structure consisting of three clonal lineages that predominate in North America and Europe. This simple pattern has encouraged the use of only a few laboratory isolates that are representative of each lineage. Principle among these is the type I RH strain, originally isolated from a child with encephalitis some 70 years ago. Comparison of different passages of the RH strain that have been propagated differently over the intervening time period revealed that the commonly used clonal line called RH-ERP was not representative of natural isolates of the type I lineage. Notably, RH-ERP formed much larger plaques than other type 1 strains, including a separate, earlier derived isolate of the RH strain. The RH-ERP variant also showed enhanced extracellular survival, faster growth, and decreased differentiation compared to the prototype type I strain GT1. Comparison of gene expression differences in the RH-ERP line revealed that several ABC transporters were upregulated, which may provide a growth advantage in vitro. These findings illustrate that dramatic phenotypic changes can arise in laboratory strains, emphasizing the need for comparison with recent clinical isolates.


Assuntos
Regulação da Expressão Gênica , Toxoplasma/citologia , Toxoplasma/genética , Animais , Diferenciação Celular/genética , Sobrevivência Celular , Células Clonais , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Perfilação da Expressão Gênica , Genótipo , Humanos , Masculino , Camundongos , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/patogenicidade , Toxoplasmose Animal/genética , Toxoplasmose Animal/parasitologia , Virulência/genética
10.
mSphere ; 5(4)2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669460

RESUMO

Immunity to Toxoplasma gondii at early stages of infection in C57BL/6 mice depends on gamma interferon (IFN-γ) production by NK cells, while at later stages it is primarily mediated by CD8 T cells. We decided to explore the requirement for CD4 T cells during T. gondii infection in Batf3-/- mice, which lack CD8α+ dendritic cells (DCs) that are necessary for cross-presentation of cell-associated antigens to CD8 T cells. We show that in this immunodeficient background on a BALB/c background, CD4 T cells become important effector cells and are able to protect Batf3-/- mice from infection with the avirulent strain RHΔku80Δrop5 Independently of the initial NK cell activation, CD4 T cells in wild-type and Batf3-/- mice were the major source of IFN-γ. Importantly, memory CD4 T cells were sufficient to provide protective immunity following transfer into Batf3-/- mice and secondary challenge with the virulent RHΔku80 strain. Collectively, these results show that under situations where CD8 cell responses are impaired, CD4 T cells provide an important alternative immune response to T. gondiiIMPORTANCEToxoplasma gondii is a widespread parasite of animals that causes zoonotic infections in humans. Although healthy individuals generally control the infection with only moderate symptoms, it causes serious illness in newborns and those with compromised immune systems such as HIV-infected AIDS patients. Because rodents are natural hosts for T. gondii, laboratory mice provide an excellent model for studying immune responses. Here, we used a combination of an attenuated mutant strain of the parasite that effectively vaccinates mice, with a defect in a transcriptional factor that impairs a critical subset of dendritic cells, to studying the immune response to infection. The findings reveal that in BALB/c mice, CD4 memory T cells play a dominant role in producing IFN-γ needed to control chronic infection. Hence, BALB/c mice may provide a more appropriate model for declining immunity seen in HIV-AIDS patients where loss of CD4 cells is associated with emergence of opportunistic infections.


Assuntos
Imunidade Adaptativa , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD4-Positivos/imunologia , Proteínas Repressoras/genética , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Feminino , Regulação da Expressão Gênica , Memória Imunológica , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Organismos Livres de Patógenos Específicos , Toxoplasma/genética , Vacinação
11.
Mol Microbiol ; 68(6): 1502-18, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18433450

RESUMO

Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis-elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii, we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis-acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6-8 bp resolution and these minimal cis-elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis-elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a 'poised' chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.


Assuntos
Proteínas de Protozoários/genética , Elementos de Resposta , Toxoplasma/genética , Transcrição Gênica , Acetilação , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Histonas/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Mutagênese Sítio-Dirigida , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência , Toxoplasma/metabolismo , Toxoplasmose/parasitologia
12.
PLoS Pathog ; 2(10): e105, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17069459

RESUMO

Toxoplasma is a significant opportunistic pathogen in AIDS, and bradyzoite differentiation is the critical step in the pathogenesis of chronic infection. Bradyzoite development has an apparent tropism for cells and tissues of the central nervous system, suggesting the need for a specific molecular environment in the host cell, but it is unknown whether this environment is parasite directed or the result of molecular features specific to the host cell itself. We have determined that a trisubstituted pyrrole acts directly on human and murine host cells to slow tachyzoite replication and induce bradyzoite-specific gene expression in type II and III strain parasites but not type I strains. New mRNA synthesis in the host cell was required and indicates that novel host transcripts encode signals that were able to induce parasite development. We have applied multivariate microarray analyses to identify and correlate host gene expression with specific parasite phenotypes. Human cell division autoantigen-1 (CDA1) was identified in this analysis, and small interfering RNA knockdown of this gene demonstrated that CDA1 expression causes the inhibition of parasite replication that leads subsequently to the induction of bradyzoite differentiation. Overexpression of CDA1 alone was able to slow parasite growth and induce the expression of bradyzoite-specific proteins, and thus these results demonstrate that changes in host cell transcription can directly influence the molecular environment to enable bradyzoite development. Investigation of host biochemical pathways with respect to variation in strain type response will help provide an understanding of the link(s) between the molecular environment in the host cell and parasite development.


Assuntos
Autoantígenos/metabolismo , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Toxoplasma/crescimento & desenvolvimento , Animais , Autoantígenos/genética , Células Cultivadas , DNA/genética , DNA/metabolismo , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Interações Hospedeiro-Parasita/genética , Humanos , Masculino , Análise Multivariada , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Pirróis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Toxoplasma/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
13.
Biol Open ; 6(5): 698-705, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28347989

RESUMO

Recent years have seen significant developments in the ability to continuously propagate organoids derived from intestinal crypts. These advancements have been applied to mouse and human samples providing models for gastrointestinal tissue development and disease. We adapt these methods for the propagation of intestinal organoids (enteroids) from various large farm and small companion (LF/SC) animals, including cat, dog, cow, horse, pig, sheep and chicken. We show that LF/SC enteroids propagate and expand in L-WRN conditioned media containing signaling factors Wnt3a, R-spondin-3, and Noggin (WRN). Multiple successful isolations were achieved for each species, and the growth of LF/SC enteroids was maintained to high passage number. LF/SC enteroids expressed crypt stem cell marker LGR5 and low levels of mesenchymal marker VIM. Labeling with EdU also showed distinct regions of cell proliferation within the enteroids marking crypt-like regions. The ability to grow and maintain LF/SC enteroid cell lines provides additional models for the study of gastrointestinal developmental biology as well as platforms for the study of host-pathogen interactions between intestinal cells and zoonotic enteric pathogens of medical importance.

14.
J Vis Exp ; (124)2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28671645

RESUMO

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Resistência a Medicamentos/genética , Genes de Protozoários , Locos de Características Quantitativas/genética , Toxoplasma/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Antiprotozoários/farmacologia , Quebras de DNA de Cadeia Dupla , Endonucleases/genética , Genoma de Protozoário/genética , RNA Guia de Cinetoplastídeos/genética , Toxoplasma/efeitos dos fármacos
15.
BMC Biol ; 3: 26, 2005 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-16324218

RESUMO

BACKGROUND: Toxoplasma gondii gives rise to toxoplasmosis, among the most prevalent parasitic diseases of animals and man. Transformation of the tachzyoite stage into the latent bradyzoite-cyst form underlies chronic disease and leads to a lifetime risk of recrudescence in individuals whose immune system becomes compromised. Given the importance of tissue cyst formation, there has been intensive focus on the development of methods to study bradyzoite differentiation, although the molecular basis for the developmental switch is still largely unknown. RESULTS: We have used serial analysis of gene expression (SAGE) to define the Toxoplasma gondii transcriptome of the intermediate-host life cycle that leads to the formation of the bradyzoite/tissue cyst. A broad view of gene expression is provided by >4-fold coverage from nine distinct libraries (approximately 300,000 SAGE tags) representing key developmental transitions in primary parasite populations and in laboratory strains representing the three canonical genotypes. SAGE tags, and their corresponding mRNAs, were analyzed with respect to abundance, uniqueness, and antisense/sense polarity and chromosome distribution and developmental specificity. CONCLUSION: This study demonstrates that phenotypic transitions during parasite development were marked by unique stage-specific mRNAs that accounted for 18% of the total SAGE tags and varied from 1-5% of the tags in each developmental stage. We have also found that Toxoplasma mRNA pools have a unique parasite-specific composition with 1 in 5 transcripts encoding Apicomplexa-specific genes functioning in parasite invasion and transmission. Developmentally co-regulated genes were dispersed across all Toxoplasma chromosomes, as were tags representing each abundance class, and a variety of biochemical pathways indicating that trans-acting mechanisms likely control gene expression in this parasite. We observed distinct similarities in the specificity and expression levels of mRNAs in primary populations (Day-6 post-sporozoite infection) that occur prior to the onset of bradyzoite development that were uniquely shared with the virulent Type I-RH laboratory strain suggesting that development of RH may be arrested. By contrast, strains from Type II-Me49B7 and Type III-VEGmsj contain SAGE tags corresponding to bradyzoite genes, which suggests that priming of developmental expression likely plays a role in the greater capacity of these strains to complete bradyzoite development.


Assuntos
Toxoplasma/genética , Transcrição Gênica , Animais , Sequência de Bases , Primers do DNA , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica , Genes de Protozoários , Poli A/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , RNA de Protozoário/genética , Toxoplasma/crescimento & desenvolvimento
16.
Trends Parasitol ; 32(7): 542-553, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27184069

RESUMO

Toxoplasma gondii is a classic model for studying obligate intracellular microorganisms as various genetic manipulation tools have been developed in T. gondii over the past 20 years. Here we summarize the major strategies for T. gondii genetic manipulation including genetic crosses, insertional mutagenesis, chemical mutagenesis, homologous gene replacement, conditional knockdown techniques, and the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. We evaluate the advantages and limitations of each of these tools in a historical perspective. We also discuss additional applications of modified CRISPR-Cas9 systems for use in T. gondii, such as regulation of gene expression, labeling of specific genomic loci, and epigenetic modifications. These approaches have the potential to revolutionize the analysis of T. gondii biology and help us to better develop new drugs and vaccines.


Assuntos
Técnicas Genéticas/tendências , Parasitologia/tendências , Pesquisa/tendências , Toxoplasma/genética , Regulação da Expressão Gênica , Marcadores Genéticos , Técnicas Genéticas/normas
17.
Nat Commun ; 7: 10147, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26738725

RESUMO

Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity.


Assuntos
Genoma de Protozoário , Toxoplasma/genética , Toxoplasma/patogenicidade , Sequência Conservada , DNA de Protozoário/genética , Regulação da Expressão Gênica/fisiologia , Filogenia , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sintenia , Virulência
18.
Mol Biochem Parasitol ; 142(1): 56-65, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15878790

RESUMO

The apicomplexa parasite Toxoplasma gondii expresses two distinct proliferating cell nuclear antigens (PCNA) that exhibit distinct patterns of subcellular localization during tachyzoite growth. In all cell cycle phases, TgPCNA1 is concentrated in the nucleus, while TgPCNA2 is only concentrated in the nucleus during S-phase and uniformly distributed throughout the cell during mitosis and early G1-phase. TgPCNA1-GFP and native TgPCNA2 display a punctate staining pattern that is consistent with assembly into replication foci during S-phase; however, TgPCNA2 disassociates from replication foci before TgPCNA1-GFP. Consistent with the distinct pattern of TgPCNA2 cellular localization, homotypic TgPCNA2 interactions were primarily observed by yeast two-hybrid or co-immunoprecipitation analysis. Transgenic parasites in which the TgPCNA2 gene was disrupted displayed a slower growth rate in vitro; however, no difference in DNA polymerase activity, response to chemical mutagens, or recombinational frequency was observed in these mutant clones demonstrating that TgPCNA2 is non-essential in the tachyzoite developmental stage. Heterologous expression of TgPCNA1, but not TgPCNA2, was able to complement a POL30 cold-sensitive yeast strain suggesting that this isoform may serve as a major replisomal factor in T. gondii and is consistent with the failure to disrupt this gene in tachyzoites.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Antígeno Nuclear de Célula em Proliferação , Proteínas de Protozoários , Toxoplasma/crescimento & desenvolvimento , Animais , Ciclo Celular , Células Cultivadas , Deleção de Genes , Teste de Complementação Genética , Humanos , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo
19.
Cell Rep ; 6(5): 928-37, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-24582962

RESUMO

microRNAs were recently found to be regulators of the host response to infection by apicomplexan parasites. In this study, we identified two immunomodulatory microRNAs, miR-146a and miR-155, that were coinduced in the brains of mice challenged with Toxoplasma in a strain-specific manner. These microRNAs define a characteristic fingerprint for infection by type II strains, which are the most prevalent cause of human toxoplasmosis in Europe and North America. Using forward genetics, we showed that strain-specific differences in miR-146a modulation were in part mediated by the rhoptry kinase, ROP16. Remarkably, we found that miR-146a deficiency led to better control of parasite burden in the gut and most likely of early parasite dissemination in the brain tissue, resulting in the long-term survival of mice.


Assuntos
Encéfalo/parasitologia , MicroRNAs/genética , Toxoplasma/fisiologia , Toxoplasmose/genética , Animais , Fracionamento Celular , Feminino , Fibroblastos/parasitologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais , Análise Serial de Tecidos , Transfecção
20.
PLoS One ; 5(8): e12354, 2010 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-20865045

RESUMO

BACKGROUND: Apicomplexan parasites replicate by varied and unusual processes where the typically eukaryotic expansion of cellular components and chromosome cycle are coordinated with the biosynthesis of parasite-specific structures essential for transmission. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the global cell cycle transcriptome of the tachyzoite stage of Toxoplasma gondii. In dividing tachyzoites, more than a third of the mRNAs exhibit significant cyclical profiles whose timing correlates with biosynthetic events that unfold during daughter parasite formation. These 2,833 mRNAs have a bimodal organization with peak expression occurring in one of two transcriptional waves that are bounded by the transition into S phase and cell cycle exit following cytokinesis. The G1-subtranscriptome is enriched for genes required for basal biosynthetic and metabolic functions, similar to most eukaryotes, while the S/M-subtranscriptome is characterized by the uniquely apicomplexan requirements of parasite maturation, development of specialized organelles, and egress of infectious daughter cells. Two dozen AP2 transcription factors form a series through the tachyzoite cycle with successive sharp peaks of protein expression in the same timeframes as their mRNA patterns, indicating that the mechanisms responsible for the timing of protein delivery might be mediated by AP2 domains with different promoter recognition specificities. CONCLUSION/SIGNIFICANCE: Underlying each of the major events in apicomplexan cell cycles, and many more subordinate actions, are dynamic changes in parasite gene expression. The mechanisms responsible for cyclical gene expression timing are likely crucial to the efficiency of parasite replication and may provide new avenues for interfering with parasite growth.


Assuntos
Ciclo Celular , Perfilação da Expressão Gênica , Toxoplasma/citologia , Toxoplasma/genética , Células Cultivadas , Fibroblastos/parasitologia , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/parasitologia
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