RESUMEN
A commonly observed survival strategy in protozoan parasites is the sequential expression of clonally variant-surface antigens to avoid elimination by the host's immune response. In malaria-causing P. falciparum, the immunovariant erythrocyte-membrane protein-1 (PfEMP1) adhesin family, encoded by var genes, is responsible for both antigenic variation and cytoadherence of infected erythrocytes to the microvasculature. Until recently, the biological function of these variant genes was believed to be restricted to intraerythrocytic developmental stages. With the advent of new technologies, var gene expression has been confirmed in transmission and pre-erythrocytic stages. Here, we discuss how repurposing of var gene expression beyond chronic blood-stage infection may be critical for successful transmission.
Asunto(s)
Antígenos de Protozoos , Malaria Falciparum , Plasmodium falciparum , Humanos , Variación Antigénica , Antígenos de Protozoos/genética , Eritrocitos/parasitología , Genes Protozoarios , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Proteínas Protozoarias/genéticaRESUMEN
Malaria-causing Plasmodium parasites undergo multiple phenotypic transitions as they cycle between diverse niches in the mammalian and mosquito hosts. Recent applications of single-cell technologies to Plasmodium have enabled the systematic investigation of the distinct stages across the life cycle. Most single-cell data have focused on the parasite exclusively, but a few studies have started to profile both parasite and host cells to shed light on the heterogeneity of cell states that underpin host-parasite interactions. In this opinion article, we highlight how atlasing initiatives are starting to be used to infer functional interactions between parasite and host and could be a powerful tool in drug discovery and vaccine development.
Asunto(s)
Culicidae , Malaria , Plasmodium , Animales , Culicidae/parasitología , Interacciones Huésped-Parásitos , Humanos , Estadios del Ciclo de Vida , Malaria/parasitología , Malaria/prevención & control , MamíferosRESUMEN
Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years and reactivate to cause recurrent blood-stage infection. Although they are an important target for malaria eradication, little is known about the molecular features of replicative and non-replicative intracellular liver-stage parasites and their host cell dependence. Here, we leverage a bioengineered human microliver platform to culture patient-derived P. vivax parasites for transcriptional profiling. Coupling enrichment strategies with bulk and single-cell analyses, we capture both parasite and host transcripts in individual hepatocytes throughout the course of infection. We define host- and state-dependent transcriptional signatures and identify unappreciated populations of replicative and non-replicative parasites that share features with sexual transmissive forms. We find that infection suppresses the transcription of key hepatocyte function genes and elicits an anti-parasite innate immune response. Our work provides a foundation for understanding host-parasite interactions and reveals insights into the biology of P. vivax dormancy and transmission.
Asunto(s)
Malaria Vivax , Malaria , Hepatocitos/parasitología , Humanos , Hígado/parasitología , Malaria/parasitología , Malaria Vivax/parasitología , Plasmodium vivax/genéticaRESUMEN
Progress towards a protective vaccine against malaria remains slow. To date, only limited protection has been routinely achieved following immunisation with either whole-parasite (sporozoite) or subunit-based vaccines. One major roadblock to vaccine progress, and to pre-erythrocytic parasite biology in general, is the continued reliance on manual salivary gland dissection for sporozoite isolation from infected mosquitoes. Here, we report development of a multi-step method, based on batch processing of homogenised whole mosquitoes, slurry, and density-gradient filtration, which combined with free-flow electrophoresis rapidly produces a pure, infective sporozoite inoculum. Human-infective Plasmodium falciparum and rodent-infective Plasmodium berghei sporozoites produced in this way are two- to threefold more infective than salivary gland dissection sporozoites in in vitro hepatocyte infection assays. In an in vivo rodent malaria model, the same P. berghei sporozoites confer sterile protection from mosquito-bite challenge when immunisation is delivered intravenously or 60-70% protection when delivered intramuscularly. By improving purity, infectivity, and immunogenicity, this method represents a key advancement in capacity to produce research-grade sporozoites, which should impact delivery of a whole-parasite based malaria vaccine at scale in the future.
Asunto(s)
Culicidae/parasitología , Malaria/prevención & control , Plasmodium berghei/patogenicidad , Plasmodium falciparum/patogenicidad , Esporozoítos/patogenicidad , Animales , Modelos Animales de Enfermedad , Drosophila , Células Hep G2 , Humanos , Inmunización , Masculino , Ratas , Esporozoítos/crecimiento & desarrolloRESUMEN
Malaria parasites have a complex life cycle featuring diverse developmental strategies, each uniquely adapted to navigate specific host environments. Here we use single-cell transcriptomics to illuminate gene usage across the transmission cycle of the most virulent agent of human malaria - Plasmodium falciparum. We reveal developmental trajectories associated with the colonization of the mosquito midgut and salivary glands and elucidate the transcriptional signatures of each transmissible stage. Additionally, we identify both conserved and non-conserved gene usage between human and rodent parasites, which point to both essential mechanisms in malaria transmission and species-specific adaptations potentially linked to host tropism. Together, the data presented here, which are made freely available via an interactive website, provide a fine-grained atlas that enables intensive investigation of the P. falciparum transcriptional journey. As well as providing insights into gene function across the transmission cycle, the atlas opens the door for identification of drug and vaccine targets to stop malaria transmission and thereby prevent disease.
Asunto(s)
Anopheles/parasitología , Estadios del Ciclo de Vida/genética , Malaria Falciparum/transmisión , Mosquitos Vectores/parasitología , Plasmodium falciparum/genética , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Femenino , Interacciones Huésped-Parásitos/genética , Humanos , Estadios del Ciclo de Vida/efectos de los fármacos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Masculino , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/patogenicidad , RNA-Seq , Análisis de la Célula Individual , Especificidad de la Especie , Transcriptoma/efectos de los fármacosRESUMEN
The causative agent of malaria, Plasmodium, replicates inside a membrane-bound parasitophorous vacuole (PV), which shields this intracellular parasite from the cytosol of the host cell 1 . One common threat for intracellular pathogens is the homeostatic process of autophagy, through which cells capture unwanted intracellular material for lysosomal degradation 2 . During the liver stage of a malaria infection, Plasmodium parasites are targeted by the autophagy machinery of the host cell, and the PV membrane (PVM) becomes decorated with several autophagy markers, including LC3 (microtubule-associated protein 1 light chain 3) 3,4 . Here we show that Plasmodium berghei parasites infecting hepatic cells rely on the PVM transmembrane protein UIS3 to avoid elimination by host-cell-mediated autophagy. We found that UIS3 binds host LC3 through a non-canonical interaction with a specialized surface on LC3 where host proteins with essential functions during autophagy also bind. UIS3 acts as a bona fide autophagy inhibitor by competing with host LC3-interacting proteins for LC3 binding. Our work identifies UIS3, one of the most promising candidates for a genetically attenuated vaccine against malaria 5 , as a unique and potent mediator of autophagy evasion in Plasmodium. We propose that the protein-protein interaction between UIS3 and host LC3 represents a target for antimalarial drug development.
Asunto(s)
Autofagia/fisiología , Hepatocitos/patología , Malaria/patología , Malaria/parasitología , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Plasmodium berghei/genética , Animales , Autofagosomas/metabolismo , Línea Celular , Células HEK293 , Células Hep G2 , Hepatocitos/parasitología , Hepatocitos/ultraestructura , Interacciones Huésped-Patógeno , Humanos , Malaria/fisiopatología , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Plasmodium berghei/metabolismo , Plasmodium berghei/patogenicidad , Unión Proteica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Vacuolas/metabolismoRESUMEN
During invasion, Plasmodium, the causative agent of malaria, wraps itself in a parasitophorous vacuole membrane (PVM), which constitutes a critical interface between the parasite and its host cell. Within hepatocytes, each Plasmodium sporozoite generates thousands of new parasites, creating high demand for lipids to support this replication and enlarge the PVM. Here, a global analysis of the total lipid repertoire of Plasmodium-infected hepatocytes reveals an enrichment of neutral lipids and the major membrane phospholipid, phosphatidylcholine (PC). While infection is unaffected in mice deficient in key enzymes involved in neutral lipid synthesis and lipolysis, ablation of rate-limiting enzymes in hepatic PC biosynthetic pathways significantly decreases parasite numbers. Host PC is taken up by both P. berghei and P. falciparum and is necessary for correct localization of parasite proteins to the PVM, which is essential for parasite survival. Thus, Plasmodium relies on the abundance of these lipids within hepatocytes to support infection.
Asunto(s)
Hígado/parasitología , Malaria/metabolismo , Fosfatidilcolinas/biosíntesis , Plasmodium berghei/metabolismo , Plasmodium falciparum/metabolismo , Animales , Línea Celular , Supervivencia Celular , Femenino , Interacciones Huésped-Parásitos , Humanos , Metabolismo de los Lípidos , Hígado/metabolismo , Malaria/parasitología , Ratones , Ratones Endogámicos C57BL , Plasmodium berghei/crecimiento & desarrollo , Plasmodium falciparum/crecimiento & desarrollo , Esporozoítos/crecimiento & desarrollo , Esporozoítos/metabolismoRESUMEN
Esse artigo tem por objetivo discutir desafios e potencialidades da construção de um programa de assistência familiar em um Hospital-Dia Psiquiátrico. Para tanto, apresentamos algumas práticas voltadas ao cuidado e a inclusão da família no tratamento ao portador de doença mental que vêm sendo desenvolvidas nesse contexto, tendo como base as contribuições do movimento construcionista social em Psicologia. A partir disso, discutimos como a adoção de posturas construcionistas têm permitido à equipe a revisão de conceitos e posicionamentos, favorecendo reflexões acerca das implicações do uso dos discursos da doença mental e do profissional como especialista no cuidado à família. (AU)
The aim of this article is to discuss challenges and potentialities of the construction of a family assistance program in a day care Psychiatric hospital. Thus, we present some practices that have been developed in this context order to take care and to include the family of the mental health patient in his treatment. These practices are based on the the adoption of a social constructionist stance allows the health team to review its concepts and positionings, thus bringing reflections on the implications of the mental health discourse and of the professional as an expert for the family health assistance. (AU)
RESUMEN
Plasmodium sporozoites, the causative agent of malaria, are injected into their vertebrate host through the bite of an infected Anopheles mosquito, homing to the liver where they invade hepatocytes to proliferate and develop into merozoites that, upon reaching the bloodstream, give rise to the clinical phase of infection. To investigate how host cell signal transduction pathways affect hepatocyte infection, we used RNAi to systematically test the entire kinome and associated genes in human Huh7 hepatoma cells for their potential roles during infection by P. berghei sporozoites. The three-phase screen covered 727 genes, which were tested with a total of 2,307 individual siRNAs using an automated microscopy assay to quantify infection rates and qRT-PCR to assess silencing levels. Five protein kinases thereby emerged as top hits, all of which caused significant reductions in infection when silenced by RNAi. Follow-up validation experiments on one of these hits, PKCsigma (PKCzeta), confirmed the physiological relevance of our findings by reproducing the inhibitory effect on P. berghei infection in adult mice treated systemically with liposome-formulated PKCsigma-targeting siRNAs. Additional cell-based analyses using a pseudo-substrate inhibitor of PKCsigma added further RNAi-independent support, indicating a role for host PKCsigma on the invasion of hepatocytes by sporozoites. This study represents the first comprehensive, functional genomics-driven identification of novel host factors involved in Plasmodium sporozoite infection.
Asunto(s)
Genoma Humano , Malaria , Fosfotransferasas/genética , Plasmodium berghei/patogenicidad , Proteína Quinasa C , ARN Interferente Pequeño/farmacología , Animales , Línea Celular , Silenciador del Gen , Hepatocitos/enzimología , Hepatocitos/parasitología , Humanos , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , EsporozoítosRESUMEN
Leukocyte locomotion is a polarized process with diverse regulatory assemblies segregating along an anterior-posterior axis that defines two regions within the cell, the leading edge and the uropod. However, the mechanisms that generate T cell asymmetry downstream of chemokine receptors are ill defined. In this study we show that the atypical protein kinases C (aPKCs), PKCiota and PKCzeta, are required for an early symmetry breaking step. Once the polarity is established, aPKCs also drive uropod formation. These effects depend on the interaction between Par6 and aPKCs. Finally, failure to transduce aPKC-dependent signals reduces T cell motility and their ability to scan dendritic cells. Altogether, our findings suggest that lymphocyte motor activity is regulated by a signaling cascade that relays chemokinetic input to aPKCs.
Asunto(s)
Polaridad Celular , Proteína Quinasa C/metabolismo , Linfocitos T/enzimología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Activación Enzimática , Humanos , Receptores CCR7/inmunología , Transducción de Señal/inmunología , Linfocitos T/citologíaRESUMEN
The molecular mechanisms responsible for the sustained basal motility of T cells within lymph nodes (LNs) remain elusive. To study T cell motility in a LN environment, we have developed a new experimental system based on slices of LNs that allows the assessment of T cell trafficking after adoptive transfer or direct addition of T cells to the slice. Using this experimental system, we show that T cell motility is highly sensitive to pertussis toxin and strongly depends on CCR7 and its ligands. Our results also demonstrate that, despite its established role in myeloid cell locomotion, phosphoinositide 3-kinase (PI3K) activity does not contribute to the exploratory behavior of the T lymphocytes within LN slices. Likewise, although PI3K activation is detectable in chemokine-treated T cells, PI3K plays only a minor role in T cell polarization and migration in vitro. Collectively, our results suggest that the common amplification system that, in other cells, facilitates large phosphatidylinositol 3,4,5-trisphosphate increases at the plasma membrane is absent in T cells. We conclude that T cell motility within LNs is not an intrinsic property of T lymphocytes but is driven in a PI3K-independent manner by the lymphoid chemokine-rich environment.
Asunto(s)
Movimiento Celular/inmunología , Ganglios Linfáticos/inmunología , Receptores CCR7/metabolismo , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Inmunohistoquímica , Ligandos , Ganglios Linfáticos/citología , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Toxina del Pertussis/toxicidad , Fosfatidilinositol 3-Quinasas/metabolismo , Linfocitos T/efectos de los fármacos , TransfecciónRESUMEN
Dendritic cells (DCs) possess a number of unique features that distinguish them from other APCs. One such feature is their ability to trigger Ag-independent responses in T cells. Previous studies have focused on mature DCs, but the prevalence of this phenomenon in the resting-state immature DCs has never been considered. In this study, we show that, in the absence of Ag, human immature DCs trigger multiple responses in autologous primary CD4+ T cells, namely, increased motility, small Ca2+ transients, and up-regulation of CD69. These responses are particularly marked in CD4+ memory T cells. By using several experimental approaches, we found that DC-specific ICAM-3-grabbing nonintegrin plays no role in the induction of T cell responses, whereas ICAM-1/LFA-1 interactions are required. In addition, DC-produced chemokines contribute to the Ag-independent T cell stimulatory ability of DCs, because pertussis toxin-treated T cells exhibit diminished responses to immature DCs. More particularly, CCL17 and CCL22, which are constitutively produced by immature DCs, mediate both T cell polarization and attraction. Thus, immature DCs owe part of their outstanding Ag-independent T cell stimulatory ability to chemokines and ICAM-1, but not DC-specific ICAM-3-grabbing nonintegrin.