RESUMEN
The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here, we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to have evolved from a putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans.
Asunto(s)
Apicoplastos , Transportadores de Ácidos Monocarboxílicos , Parásitos , Toxoplasma , Animales , Ratones , Apicoplastos/metabolismo , Ácidos Grasos/metabolismo , Compuestos Orgánicos/metabolismo , Parásitos/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismoRESUMEN
Apicomplexan parasites are unicellular eukaryotes responsible for major human diseases such as malaria and toxoplasmosis, which cause massive social and economic burden. Toxoplasmosis, caused by Toxoplasma gondii, is a global chronic infectious disease affecting ~1/3 of the world population and is a major threat for any immunocompromised patient. To date, there is no efficient vaccine against these parasites and existing treatments are threatened by rapid emergence of parasite resistance. Throughout their life cycle, Apicomplexa require large amount of nutrients, especially lipids for propagation and survival. Understanding lipid acquisition is key to decipher host-parasite metabolic interactions. Parasite membrane biogenesis relies on a combination of (a) host lipid scavenging, (b) de novo lipid synthesis in the parasite, and (c) fluxes of lipids between host and parasite and within. We recently uncovered that parasite need to store the host-scavenged lipids to avoid their toxic accumulation and to mobilize them for division. How can parasites orchestrate the many lipids fluxes essential for survival? Here, we developed metabolomics approaches coupled to stable isotope labelling to track, monitor, and quantify fatty acid and lipids fluxes between the parasite, its human host cell, and its extracellular environment to unravel the complex lipid fluxes in any physiological environment the parasite could meet.
Asunto(s)
Parásitos , Toxoplasma , Toxoplasmosis , Animales , Humanos , Parásitos/metabolismo , Plastidios/metabolismo , Ácidos Grasos/metabolismo , Toxoplasma/metabolismo , Toxoplasmosis/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
The honey bee trypanosomatid parasite, Lotmaria passim, contains two genes that encode the flagellar calcium binding protein (FCaBP) through tandem duplication in its genome. FCaBPs localize in the flagellum and entire body membrane of L. passim through specific N-terminal sorting sequences. This finding suggests that this is an example of protein subcellular relocalization resulting from gene duplication, altering the intracellular localization of FCaBP. However, this phenomenon may not have occurred in Leishmania, as one or both of the duplicated genes have become pseudogenes. Multiple copies of the FCaBP gene are present in several Trypanosoma species and Leptomonas pyrrhocoris, indicating rapid evolution of this gene in trypanosomatid parasites. The N-terminal flagellar sorting sequence of L. passim FCaBP1 is in close proximity to the BBSome complex, while that of Trypanosoma brucei FCaBP does not direct GFP to the flagellum in L. passim. Deletion of the two FCaBP genes in L. passim affected growth and impaired flagellar morphogenesis and motility, but it did not impact host infection. Therefore, FCaBP represents a duplicated gene with a rapid evolutionary history that is essential for flagellar structure and function in a trypanosomatid parasite.
Asunto(s)
Leishmania , Parásitos , Abejas/genética , Animales , Proteínas de Unión al Calcio/genética , Parásitos/metabolismo , Flagelos/genética , Flagelos/metabolismo , Cilios/metabolismoRESUMEN
Plasmodium vivax, the most widespread human malaria parasite, and P. knowlesi, an emerging Plasmodium that infects humans, are the phylogenetically closest malarial species that infect humans, which may induce cross-species reactivity across most co-endemic areas in Southeast Asia. The thrombospondin-related anonymous protein (TRAP) family is indispensable for motility and host cell invasion in the growth and development of Plasmodium parasites. The merozoite-specific TRAP (MTRAP), expressed in blood-stage merozoites, is supposed to be essential for human erythrocyte invasion. We aimed to characterize MTRAPs in blood-stage P. vivax and P. knowlesi parasites and ascertain their cross-species immunoreactivity. Recombinant P. vivax and P. knowlesi MTRAPs of full-length ectodomains were expressed in a mammalian expression system. The MTRAP-specific immunoglobulin G, obtained from immune animals, was used in an immunofluorescence assay for subcellular localization and invasion inhibitory activity in blood-stage parasites was determined. The cross-species humoral immune responses were analyzed in the sera of patients with P. vivax or P. knowlesi infections. The MTRAPs of P. vivax (PvMTRAP) and P. knowlesi (PkMTRAP) were localized on the rhoptry body of merozoites in blood-stage parasites. Both anti-PvMTRAP and anti-PkMTRAP antibodies inhibited erythrocyte invasion of blood-stage P. knowlesi parasites. The humoral immune response to PvMTRAP showed high immunogenicity, longevity, and cross-species immunoreactivity with P. knowlesi. MTRAPs are promising candidates for development of vaccines and therapeutics against vivax and knowlesi malaria.
Asunto(s)
Malaria Vivax , Malaria , Parásitos , Plasmodium , Animales , Humanos , Plasmodium vivax/genética , Parásitos/metabolismo , Merozoítos , Trombospondinas/metabolismo , Plasmodium/metabolismo , Malaria/parasitología , Malaria Vivax/parasitología , Proteínas Protozoarias/metabolismo , Mamíferos/metabolismoRESUMEN
While the malaria parasite Plasmodium falciparum has low average genome-wide diversity levels, likely due to its recent introduction from a gorilla-infecting ancestor (approximately 10,000 to 50,000 years ago), some genes display extremely high diversity levels. In particular, certain proteins expressed on the surface of human red blood cell-infecting merozoites (merozoite surface proteins (MSPs)) possess exactly 2 deeply diverged lineages that have seemingly not recombined. While of considerable interest, the evolutionary origin of this phenomenon remains unknown. In this study, we analysed the genetic diversity of 2 of the most variable MSPs, DBLMSP and DBLMSP2, which are paralogs (descended from an ancestral duplication). Despite thousands of available Illumina WGS datasets from malaria-endemic countries, diversity in these genes has been hard to characterise as reads containing highly diverged alleles completely fail to align to the reference genome. To solve this, we developed a pipeline leveraging genome graphs, enabling us to genotype them at high accuracy and completeness. Using our newly- resolved sequences, we found that both genes exhibit 2 deeply diverged lineages in a specific protein domain (DBL) and that one of the 2 lineages is shared across the genes. We identified clear evidence of nonallelic gene conversion between the 2 genes as the likely mechanism behind sharing, leading us to propose that gene conversion between diverged paralogs, and not recombination suppression, can generate this surprising genealogy; a model that is furthermore consistent with high diversity levels in these 2 genes despite the strong historical P. falciparum transmission bottleneck.
Asunto(s)
Hominidae , Malaria Falciparum , Malaria , Parásitos , Animales , Humanos , Plasmodium falciparum/metabolismo , Parásitos/metabolismo , Conversión Génica , Antígenos de Superficie , Malaria/parasitología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Variación GenéticaRESUMEN
Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells. IMPORTANCE: Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.
Asunto(s)
Malaria , Parásitos , Plasmodium , Toxoplasma , Animales , Plasmodium falciparum/metabolismo , Tomografía con Microscopio Electrónico , Proteínas Protozoarias/metabolismo , Parásitos/metabolismo , Interacciones Huésped-Parásitos , Toxoplasma/metabolismoRESUMEN
Typical two-cysteine peroxiredoxins (2-Cys-PRXs) are H2O2-metabolizing enzymes whose activity relies on two cysteine residues. Protists of the family Trypanosomatidae invariably express one cytosolic 2-Cys-PRX (cPRX1). However, the Leishmaniinae sub-family features an additional isoform (cPRX2), almost identical to cPRX1, except for the lack of an elongated C-terminus with a Tyr-Phe (YF) motif. Previously, cytosolic PRXs were considered vital components of the trypanosomatid antioxidant machinery. Here, we shed new light on the properties, functions and relevance of cPRXs from the human pathogen Leishmania infantum. We show first that LicPRX1 is sensitive to inactivation by hyperoxidation, mirroring other YF-containing PRXs participating in redox signaling. Using genetic fusion constructs with roGFP2, we establish that LicPRX1 and LicPRX2 can act as sensors for H2O2 and oxidize protein thiols with implications for signal transduction. Third, we show that while disrupting the LicPRX-encoding genes increases susceptibility of L. infantum promastigotes to external H2O2in vitro, both enzymes are dispensable for the parasites to endure the macrophage respiratory burst, differentiate into amastigotes and initiate in vivo infections. This study introduces a novel perspective on the functions of trypanosomatid cPRXs, exposing their dual roles as both peroxidases and redox sensors. Furthermore, the discovery that Leishmania can adapt to the absence of both enzymes has significant implications for our understanding of Leishmania infections and their treatment. Importantly, it questions the conventional notion that the oxidative response of macrophages during phagocytosis is a major barrier to infection and the suitability of cPRXs as drug targets for leishmaniasis.
Asunto(s)
Leishmania , Leishmaniasis , Parásitos , Animales , Humanos , Peroxirredoxinas/metabolismo , Cisteína/metabolismo , Peróxido de Hidrógeno/metabolismo , Parásitos/metabolismo , Oxidación-ReducciónRESUMEN
Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we functionally characterized a putative Trypanosoma PEX1 orthologue by bioinformatic and experimental approaches and show that it is a true PEX1 orthologue. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to the bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggest that these parasites possess a highly efficient quality control mechanism that exports the import receptors from glycosomes to the cytosol in the absence of a functional TbPEX1-TbPEX6 complex.
Asunto(s)
Parásitos , Proteínas de Saccharomyces cerevisiae , Trypanosoma , Animales , Parásitos/metabolismo , Saccharomyces cerevisiae/metabolismo , Peroxisomas/genética , Peroxisomas/metabolismo , Microcuerpos , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Nematode infections are common in both humans and livestock, with major adverse planetary health and economic impacts. Wuchereria bancrofti is a parasitic nematode that causes lymphatic filariasis, a neglected tropical disease that can lead to severe disability and deformity worldwide. For the long-term survival of the bancroftian parasites in the host, a complex immune invasion strategy is involved through immunomodulation. Therefore, immunomodulation can serve as a site of research and innovation for molecular targets. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine crucial to the host antimicrobial alarm system and stress response. Interestingly, the nematode parasite W. bancrofti also produces two homologs of MIF (Wba-MIF1 and 2). Using a mass spectrometry-based phosphoproteomics approach, we report new findings on the immunomodulatory effect and signaling mechanism of Wba-MIF2 in macrophage cells. Accordingly, we observed 1201 phosphorylated sites on 467 proteins. Out of the 1201 phosphorylated sites, 1075, 117, and 9 were found on serine (S), threonine (T), and tyrosine (Y) residues, respectively. Our bioinformatics analysis led to identification of major pathways, including spliceosomes, T cell receptor signaling pathway, Th17 differentiation pathway, interleukin-17 signaling pathway, and insulin signaling pathway upon Wba-MIF2 treatment. Wba-MIF2 treatment also enriched CDK4, CDK1, and DNAPK kinases. The comparison of the signaling pathway of Wba-MIF2 with that of human-MIF suggests both share similar signaling pathways. These findings collectively offer new insights into the role and mechanism of Wba-MIF2 as an immunomodulator and inform future diagnostics and drug discovery research for W. bancrofti.
Asunto(s)
Antiinfecciosos , Filariasis Linfática , Factores Inhibidores de la Migración de Macrófagos , Parásitos , Animales , Humanos , Wuchereria bancrofti/metabolismo , Parásitos/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Filariasis Linfática/parasitologíaRESUMEN
Helminth parasites modulate the host immune system to ensure a long-lasting asymptomatic form of infection generally, mediated by the secretion of immunomodulatory molecules and one such molecule is a homologue of human host cytokine, Macrophage migratory Inhibitory Factor (hMIF). In this study, we sought to understand the role of homologue of hMIF from the lymphatic filarial parasite, Wuchereria bancrofti (Wba-MIF2), in the immunomodulation of the Streptozotocin (STZ)-induced Type1 Diabetes Mellitus (T1DM) animal model. Full-length recombinant Wba-MIF2 was expressed and found to have both oxidoreductase and tautomerase activities. Wba-MIF2 recombinant protein was treated to STZ induced T1DM animals, and after 5 weeks pro-inflammatory (IL-1, IL-2, IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and gene expressions were determined in sera samples and spleen respectively. Pro-inflammatory and anti-inflammatory cytokine levels were significantly (p<0.05) up-regulated and down-regulated respectively, in the STZ-T1DM animals, as compared to treated groups. Histopathology showed macrophage infiltration and greater damage of islets of beta cells in the pancreatic tissue of STZ-T1DM animals, than Wba-MIF2 treated STZ-T1DM animals. The present study clearly showed the potential of Wba-MIF2 as an immunomodulatory molecule, which could modulate the host immune system in the STZ-T1DM mice model from a pro-inflammatory to anti-inflammatory milieu.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Filarioidea , Factores Inhibidores de la Migración de Macrófagos , Parásitos , Humanos , Animales , Ratones , Wuchereria bancrofti , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Parásitos/metabolismo , Estreptozocina , Factores Inmunológicos , Diabetes Mellitus Experimental/genética , Antiinflamatorios , Oxidorreductasas IntramolecularesRESUMEN
Intracellular cargo transport is a ubiquitous cellular process in all eukaryotes. In many cell types, membrane bound cargo is associated with molecular motors which transport cargo along microtubule and actin tracks. In Toxoplasma gondii (T. gondii), an obligate intracellular parasite in the phylum Apicomplexa, organization of the endomembrane pathway depends on actin and an unconventional myosin motor, myosin F (MyoF). Loss of MyoF and actin disrupts vesicle transport, organelle positioning, and division of the apicoplast, a nonphotosynthetic plastid organelle. How this actomyosin system contributes to these cellular functions is still unclear. Using live-cell imaging, we observed that MyoF-EmeraldFP (MyoF-EmFP) displayed a dynamic and filamentous-like organization in the parasite cytosol, reminiscent of cytosolic actin filament dynamics. MyoF was not associated with the Golgi, apicoplast or dense granule surfaces, suggesting that it does not function using the canonical cargo transport mechanism. Instead, we found that loss of MyoF resulted in a dramatic rearrangement of the actin cytoskeleton in interphase parasites accompanied by significantly reduced actin dynamics. However, actin organization during parasite replication and motility was unaffected by the loss of MyoF. These findings revealed that MyoF is an actin organizing protein in Toxoplasma and facilitates cargo movement using an unconventional transport mechanism.
Asunto(s)
Parásitos , Toxoplasma , Animales , Actinas/metabolismo , Toxoplasma/metabolismo , Miosinas/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto de Actina/metabolismo , Parásitos/metabolismoRESUMEN
Parasitic diseases are still a major public health problem especially among individuals of low socioeconomic status in underdeveloped countries. In recent years it has been demonstrated that parasites can release extracellular vesicles that participate in the host-parasite communication, immune evasion, and in governing processes associated with host infection. Extracellular vesicles are membrane-bound structures released into the extracellular space that can carry several types of biomolecules, including proteins, lipids, nucleic acids, and metabolites, which directly impact the target cells. Extracellular vesicles have attracted wide attention due to their relevance in host-parasite communication and for their potential value in applications such as in the diagnostic biomarker discovery. This review of the literature aimed to join the current knowledge on the role of extracellular vesicles in host-parasite interaction and summarize its molecular content, providing information for the acquisition of new tools that can be used in the diagnosis of parasitic diseases. These findings shed light to the potential of extracellular vesicle cargo derived from protozoan parasites as novel diagnostic tools.
Asunto(s)
Vesículas Extracelulares , Enfermedades Parasitarias , Animales , Humanos , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Interacciones Huésped-Parásitos , Parásitos/metabolismo , Enfermedades Parasitarias/diagnóstico , Enfermedades Parasitarias/parasitologíaRESUMEN
The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using an assay for LPC hydrolysis in P. falciparum-infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, constitute the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ, loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy.
Asunto(s)
Malaria Falciparum , Parásitos , Animales , Humanos , Plasmodium falciparum , Lisofosfatidilcolinas/metabolismo , Lisofosfolipasa/genética , Lisofosfolipasa/metabolismo , Malaria Falciparum/parasitología , Eritrocitos/metabolismo , Parásitos/metabolismo , Ácidos Grasos/metabolismo , Lipasa/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
The microbiota community is composed of bacteria, fungi, viruses, and protists that exert symbiotic effects within the human body. Unlike microbiota, parasites are characteristically reliant on their hosts to thrive and flourish, producing toxic metabolites that agitate microbiota and disturb homeostasis. The proper management of parasitic infections addresses several important challenges related to low socioeconomic status and emergent resistance. Therefore, understanding the microbiota's role in interactions with hosts and parasites is crucial for managing parasite diseases with fewer economic and adverse effects associated with pharmaceutical interventions. The current review was divided into three sections. Section 1 focused on the mutual microbiota-host interaction through the purinergic P2X7 receptor (P2X7R) and secretory immunoglobulin A (SIgA). The P2X7R is an abundant intestinal cation channel that is crucial in mucosal immunity, facilitated by SIgA-mediated protection in both innate and adaptive immunity. This study demonstrated that microbiota continually "teach and train" host immunity to attain homeostasis via SIgA production (in T cell-independent and T cell-dependent pathways) and the purinergic receptor P2X7R. In addition, we discussed the potential of manipulating SIgA and P2X7R in immune therapies targeting parasitic infections. Section 2 exhibited parasite-microbiota (microbe-microbe) interactions wherein each can indirectly affect one another through physical and immunogenic alterations and directly via predation, bactericidal protein production, and overlapping of nutrient resources. Thus, microbe-microbe interactions appeared to be multifaceted and species-dependent. Section 3 showed the relationship between microbiota and specific parasites, and the promising role of probiotics. In this section, the review discussed examples of tissue, blood, gastrointestinal, genitourinary, and respiratory parasitic diseases, while highlighting the associated dysbiosis. Furthermore, Section 3 acknowledged the importance of "strain-dependent" biotherapy to boost beneficial microbiota, modulate immunity, and exert anti-parasitic effects.
Asunto(s)
Microbiota , Parásitos , Enfermedades Parasitarias , Animales , Humanos , Parásitos/metabolismo , Receptores Purinérgicos P2X7 , Inmunoglobulina A Secretora/metabolismoRESUMEN
The cytoskeletal protein actin plays a critical role in the pathogenicity of the intracellular parasite, Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there are conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of jasplakinolide-stabilized and native (i.e. unstabilized) filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune the dynamic properties of actin filaments without disrupting their conserved structure.
Asunto(s)
Parásitos , Toxoplasma , Animales , Actinas/metabolismo , Toxoplasma/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Parásitos/metabolismoRESUMEN
Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.
Asunto(s)
Citoesqueleto de Actina , Actinas , Leishmania major , Parásitos , Profilinas , Animales , Humanos , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , Leishmania major/citología , Leishmania major/metabolismo , Parásitos/citología , Parásitos/metabolismo , Profilinas/química , Profilinas/metabolismo , Unión Proteica , Dominios ProteicosRESUMEN
Recently, a S168T variant in the acetylcholine receptor subunit ACR-8 was associated with levamisole resistance in the parasitic helminth Haemonchus contortus. Here, we used the Xenopus laevis oocyte expression system and two-electrode voltage-clamp electrophysiology to measure the functional impact of this S168T variant on the H. contortus levamisole-sensitive acetylcholine receptor, L-AChR-1.1. Expression of the ACR-8 S168T variant significantly reduced the current amplitude elicited by levamisole compared to acetylcholine, with levamisole changing from a full to partial agonist on the recombinant L-AChR. Functional validation of the S168T mutation on modulating levamisole activity at the receptor level highlights its critical importance as both a mechanism and a marker of levamisole resistance.
Asunto(s)
Antihelmínticos , Haemonchus , Parásitos , Animales , Levamisol/farmacología , Haemonchus/genética , Haemonchus/metabolismo , Antinematodos/farmacología , Receptores Colinérgicos/genética , Parásitos/metabolismo , Resistencia a Medicamentos/genética , Antihelmínticos/farmacología , Antihelmínticos/metabolismoRESUMEN
The tactics used by animal pathogens to combat host immunity are largely unclear. Here, we report the depiction of the virulence-required effector Tge1 deployed by the entomopathogen Metarhizium robertsii to suppress Drosophila antifungal immunity. Tge1 can target both GNBP3 and GNBP-like 3 (GL3), and the latter can bind to ß-glucans like GNBP3, whereas the glucan binding by both receptors can be attenuated by Tge1. As opposed to the surveillance GNBP3, GL3 is inducible in Drosophila depending on the Toll pathway via a positive feedback loop mechanism. Losses of GNBP3 and GL3 genes result in the deregulations of protease cascade, Spätzle maturation, and antimicrobial gene expressions in Drosophila upon fungal challenges. Fly survival assays confirm that GL3 plays a more essential role than GNBP3 in combating fungal infections. In addition to evidencing the gene-for-gene interactions between fungi and insects, our data advance insights into Drosophila antifungal immunity.
Asunto(s)
Proteínas de Drosophila , Parásitos , beta-Glucanos , Animales , Drosophila/metabolismo , Antifúngicos/farmacología , beta-Glucanos/farmacología , beta-Glucanos/metabolismo , Parásitos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Portadoras/metabolismoRESUMEN
Apicomplexans are ubiquitous intracellular parasites of animals. These parasites use a programmed sequence of secretory events to find, invade, and then re-engineer their host cells to enable parasite growth and proliferation. The secretory organelles micronemes and rhoptries mediate the first steps of invasion. Both secrete their contents through the apical complex which provides an apical opening in the parasite's elaborate inner membrane complex (IMC) - an extensive subpellicular system of flattened membrane cisternae and proteinaceous meshwork that otherwise limits access of the cytoplasm to the plasma membrane for material exchange with the cell exterior. After invasion, a second secretion programme drives host cell remodelling and occurs from dense granules. The site(s) of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii, small subapical annular structures that are embedded in the IMC have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown. Here, we determined that integral membrane proteins of the plasma membrane occur specifically at these apical annular sites, that these proteins include SNARE proteins, and that the apical annuli are sites of vesicle fusion and exocytosis. Specifically, we show that dense granules require these structures for the secretion of their cargo proteins. When secretion is perturbed at the apical annuli, parasite growth is strongly impaired. The apical annuli, therefore, represent a second type of IMC-embedded structure to the apical complex that is specialised for protein secretion, and reveal that in Toxoplasma there is a physical separation of the processes of pre- and post-invasion secretion that mediate host-parasite interactions.
Asunto(s)
Parásitos , Toxoplasma , Animales , Toxoplasma/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Orgánulos/metabolismo , Parásitos/metabolismo , Membrana Celular/metabolismoRESUMEN
A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. Additionally, Δ-PolK parasites lost their capability to utilize prenols for protein prenylation. Experiments using culture medium supplemented with whole/delipidated human plasma in transgenic parasites revealed that human plasma has components that can diminish the effectiveness of fosmidomycin. Mass spectrometry tests indicated that both bovine supplements used in culture and human plasma contain GGOH. These findings suggest that the FOH/GGOH salvage pathway might offer an alternate source of isoprenoids for malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme related to isoprenoid metabolism.