RESUMEN
Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antimaláricos/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Esporozoítos/inmunología , Adolescente , Adulto , Animales , Línea Celular , Línea Celular Tumoral , Epítopos/inmunología , Femenino , Células HEK293 , Hepatocitos/inmunología , Hepatocitos/parasitología , Humanos , Hígado/inmunología , Hígado/parasitología , Malaria/inmunología , Malaria/parasitología , Vacunas contra la Malaria/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Adulto JovenRESUMEN
An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.
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Plasmodium falciparum , Esporozoítos , Animales , Humanos , Ratones , Culicidae/parasitología , Malaria/parasitología , Malaria/prevención & control , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/química , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Esporozoítos/crecimiento & desarrollo , Esporozoítos/patogenicidad , Hepatocitos/parasitología , Hígado/parasitología , Proteína 1 de Superficie de Merozoito , Eritrocitos/parasitología , Técnicas In VitroRESUMEN
Malaria infection involves an obligatory, yet clinically silent liver stage1,2. Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis3, but the effects of hepatocyte zonation on parasite development at the molecular level remain unknown. Here we combine single-cell RNA sequencing4 and single-molecule transcript imaging5 to characterize the host and parasite temporal expression programmes in a zonally controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, including potentially co-adaptive programmes related to iron and fatty acid metabolism. We find that parasites develop more rapidly in the pericentral lobule zones and identify a subpopulation of periportally biased hepatocytes that harbour abortive infections, reduced levels of Plasmodium transcripts and parasitophorous vacuole breakdown. These 'abortive hepatocytes', which appear predominantly with high parasite inoculum, upregulate immune recruitment and key signalling programmes. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights the heterogeneous behaviour of both the parasite and the host hepatocyte.
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Regulación de la Expresión Génica , Hepatocitos , Hígado , Malaria , Parásitos , Plasmodium berghei , Análisis de la Célula Individual , Animales , Hepatocitos/citología , Hepatocitos/inmunología , Hepatocitos/metabolismo , Hepatocitos/parasitología , Hígado/anatomía & histología , Hígado/citología , Hígado/inmunología , Hígado/parasitología , Malaria/genética , Malaria/inmunología , Malaria/parasitología , Parásitos/genética , Parásitos/inmunología , Parásitos/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/inmunología , Plasmodium berghei/metabolismo , Imagen Individual de Molécula , Análisis de Secuencia de ARN , Hierro/metabolismo , Ácidos Grasos/metabolismo , Transcripción Genética , Genes Protozoarios/genética , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/inmunologíaRESUMEN
Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking.
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Hepatocitos , Hígado , Malaria Falciparum , Plasmodium falciparum , Proteínas Protozoarias , Esporozoítos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Animales , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Esporozoítos/metabolismo , Esporozoítos/crecimiento & desarrollo , Ratones , Hígado/parasitología , Hígado/metabolismo , Humanos , Hepatocitos/parasitología , Hepatocitos/metabolismo , Malaria Falciparum/parasitologíaRESUMEN
An essential step in the life cycle of malaria parasites is their egress from hepatocytes, which enables the transition from the asymptomatic liver stage to the pathogenic blood stage of infection. To exit the liver, Plasmodium parasites first disrupt the parasitophorous vacuole membrane that surrounds them during their intracellular replication. Subsequently, parasite-filled structures called merosomes emerge from the infected cell. Shrouded by host plasma membrane, like in a Trojan horse, parasites enter the vasculature undetected by the host immune system and travel to the lung where merosomes rupture, parasites are released, and the blood infection stage begins. This complex, multi-step process must be carefully orchestrated by the parasite and requires extensive manipulation of the infected host cell. This review aims to outline the known signaling pathways that trigger exit, highlight Plasmodium proteins that contribute to the release of liver-stage merozoites, and summarize the accompanying changes to the hepatic host cell.
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Malaria , Parásitos , Plasmodium , Animales , Humanos , Parásitos/metabolismo , Hígado/parasitología , Hepatocitos/parasitología , Plasmodium/metabolismo , Malaria/parasitología , Eritrocitos/parasitología , Proteínas Protozoarias/metabolismoRESUMEN
The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites.
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Proteínas Relacionadas con la Autofagia , Autofagia , Hepatocitos , Hígado , Proteínas Asociadas a Microtúbulos , Plasmodium berghei , ATPasas de Translocación de Protón Vacuolares , Vacuolas , Vacuolas/metabolismo , Vacuolas/parasitología , Plasmodium berghei/genética , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/metabolismo , Plasmodium berghei/enzimología , Animales , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Hígado/parasitología , Ratones , Hepatocitos/parasitología , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Malaria/parasitología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , HumanosRESUMEN
Plasmodium falciparum (Pf) is a major cause of human malaria and is transmitted by infected Anopheles mosquitoes. The initial asymptomatic infection is characterized by parasite invasion of hepatocytes, followed by massive replication generating schizonts with blood-infective merozoites. Hepatocytes can be categorized by their zonal location and metabolic functions within a liver lobule. To understand specific host conditions that affect infectivity, we studied Pf parasite liver stage development in relation to the metabolic heterogeneity of fresh human hepatocytes. We found selective preference of different Pf strains for a minority of hepatocytes, which are characterized by the particular presence of glutamine synthetase (hGS). Schizont growth is significantly enhanced by hGS uptake early in development, showcasing a novel import system. In conclusion, Pf development is strongly determined by the differential metabolic status in hepatocyte subtypes. These findings underscore the importance of detailed understanding of hepatocyte host-Pf interactions and may delineate novel pathways for intervention strategies.
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Glutamato-Amoníaco Ligasa/metabolismo , Hepatocitos/parasitología , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Transporte Biológico/fisiología , Proliferación Celular/fisiología , Glucosa/metabolismo , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Humanos , Hígado/parasitología , Hígado/patologíaRESUMEN
In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8+ T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.
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Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Hígado/inmunología , Malaria/inmunología , Animales , Linfocitos T CD8-positivos/parasitología , Culicidae , Células Dendríticas/inmunología , Células Dendríticas/parasitología , Hepatocitos/inmunología , Hepatocitos/parasitología , Hígado/parasitología , Hepatopatías/inmunología , Hepatopatías/parasitología , Vacunas contra la Malaria/inmunología , Ratones , Plasmodium berghei/inmunología , Esporozoítos/inmunología , Esporozoítos/parasitología , Vacunación/métodosRESUMEN
Schistosomiasis is a serious public health problem, and previous studies found that liver function and hepatic cells are damaged. To evaluate the serum parameters of liver function and fibrosis in schistosomiasis patients infected with Schistosoma japonicum (Schistosoma J.) and analyze the correlations between liver function and serum fibrosis markers in patients infected with Schistosoma J., this retrospective study enrolled 133 patients. The study population was divided into four groups: healthy people control group (n = 20), chronic schistosomiasis without liver cirrhosis (CS) group (n = 21), schistosomiasis cirrhosis without hypoalbuminemia (SC-HA) group (n = 68), and schistosomiasis cirrhosis with hypoalbuminemia (SC +HA) group (n = 24). Clinical and laboratory data were collected for analysis. In the multiple comparison of abnormal rates of aspartate aminotransferase (AST) and total bilirubin (TBIL), the abnormal rate of the SC +HA group was significantly higher than that of the other three groups (P < 0.05), and the abnormal rate of γ-GT in the SC +HA group was significantly higher than that in the control group (P < 0.05). Multiple comparison results of serum levels of fibrosis markers showed that the SC group had a significantly higher level of indexes than other groups (P < 0.05). The levels of TGF-ß1 in the CS group, SC-HA group and SC +HA group were significantly higher than those in the control group (P < 0.001). Our study demonstrated that the liver function and hepatic cells were damaged with the progression of liver disease in patients infected with Schistosoma J., and they played an important role in the occurrence and development of liver fibrosis.
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Hepatocitos , Cirrosis Hepática , Schistosoma japonicum , Esquistosomiasis Japónica , Humanos , Cirrosis Hepática/patología , Cirrosis Hepática/parasitología , Esquistosomiasis Japónica/complicaciones , Esquistosomiasis Japónica/patología , Masculino , Femenino , Persona de Mediana Edad , Animales , Adulto , Estudios Retrospectivos , Hepatocitos/patología , Hepatocitos/parasitología , Biomarcadores/sangre , Anciano , Hígado/patología , Hígado/parasitología , Pruebas de Función HepáticaRESUMEN
Ivermectin, a broad-spectrum anti-parasitic drug, has been proposed as a novel vector control tool to reduce malaria transmission by mass drug administration. Ivermectin and some metabolites have mosquito-lethal effect, reducing Anopheles mosquito survival. Ivermectin inhibits liver stage development in a rodent malaria model, but no inhibition was observed in a primate malaria model or in a human malaria challenge trial. In the liver, cytochrome P450 3A4 and 3A5 enzymes metabolize ivermectin, which may impact drug efficacy. Thus, understanding ivermectin metabolism and assessing this impact on Plasmodium liver stage development is critical. Using primary human hepatocytes (PHHs), we characterized ivermectin metabolism and evaluated the efficacy of ivermectin and its primary metabolites M1 (3â³-O-demethyl ivermectin) and M3 (4-hydroxymethyl ivermectin) against Plasmodium falciparum liver stages. Two different modes of ivermectin exposure were evaluated: prophylactic mode (days 0-3 post-infection) and curative mode (days 3-5 post-infection). We used two different PHH donors and modes to determine the inhibitory concentration (IC50) of ivermectin, M1, M3, and the known anti-malarial drug pyrimethamine, with IC50 values ranging from 1.391 to 14.44, 9.95-23.71, 4.767-8.384, and 0.9073-5.416 µM, respectively. In our PHH model, ivermectin and metabolites M1 and M3 demonstrated inhibitory activity against P. falciparum liver stages in curative treatment mode (days 3-5) and marginal activity in prophylactic treatment mode (days 0-3). Ivermectin had improved efficacy when co-administered with ketoconazole, a specific inhibitor of cytochrome P450 3A4 enzyme. Further studies should be performed to examine ivermectin liver stage efficacy when co-administered with CYP3A4 inhibitors and anti-malarial drugs to understand the pharmacokinetic and pharmacodynamic drug-drug interactions that enhance efficacy against human malaria parasites in vitro.
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Hepatocitos , Ivermectina , Plasmodium falciparum , Ivermectina/farmacología , Hepatocitos/parasitología , Hepatocitos/efectos de los fármacos , Humanos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Citocromo P-450 CYP3A/metabolismo , Antimaláricos/farmacología , Hígado/parasitología , Hígado/efectos de los fármacos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Animales , Células Cultivadas , Anopheles/parasitología , Anopheles/efectos de los fármacosRESUMEN
OBJECTIVES: Alveolar echinococcosis (AE) represents one of the deadliest helminthic infections, characterized by an insidious onset and high lethality. METHODS: This study utilized the Gene Expression Omnibus (GEO) database, applied Weighted Correlation Network Analysis (WGCNA) and Differential Expression Analysis (DEA), and employed the Matthews Correlation Coefficient (MCC) to identify CCL17 and CCL19 as key genes in AE. Immunohistochemistry and immunofluorescence co-localization techniques were used to examine the expression of CCL17 and CCL19 in liver tissue lesions of AE patients. Additionally, a mouse model of multilocular echinococcus larvae infection was developed to study the temporal expression patterns of these genes, along with liver fibrosis and inflammatory responses. RESULTS: The in vitro model simulating echinococcal larva infection mirrored the hepatic microenvironment post-infection with multilocular echinococcal tapeworms. Quantitative RT-PCR analysis showed that liver fibrosis occurred in AE patients, with proximal activation and increased expression of CCL17 and CCL19 over time post-infection. Notably, expression peaked during the late stages of infection. Similarly, F4/80, a macrophage marker, exhibited corresponding trends in expression. Upon stimulation of normal hepatocytes by vesicular larvae in cellular experiments, there was a significant increase in CCL17 and CCL19 expression at 12 h post-infection, mirroring the upregulation observed with F4/80. CONCLUSION: CCL17 and CCL19 facilitate macrophage aggregation via the chemokine pathway and their increased expression correlates with the progression of infection, suggesting their potential as biomarkers for AE progression.
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Biomarcadores , Quimiocina CCL17 , Quimiocina CCL19 , Progresión de la Enfermedad , Animales , Humanos , Ratones , Biomarcadores/metabolismo , Quimiocina CCL19/metabolismo , Quimiocina CCL17/metabolismo , Quimiocina CCL17/genética , Equinococosis/metabolismo , Cirrosis Hepática/parasitología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Modelos Animales de Enfermedad , Hígado/parasitología , Hígado/metabolismo , Hígado/patología , Equinococosis Hepática/metabolismo , Equinococosis Hepática/parasitología , Femenino , Masculino , Hepatocitos/metabolismo , Hepatocitos/parasitologíaRESUMEN
BACKGROUND: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood. METHODS: To gain a better understanding of the protein-protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. RESULTS: A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. CONCLUSION: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.
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Hepatocitos , Plasmodium falciparum , Proteínas Protozoarias , Esporozoítos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Hepatocitos/parasitología , Humanos , Esporozoítos/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Interacciones Huésped-Patógeno , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Interacciones Huésped-Parásitos , Unión ProteicaRESUMEN
Vaccination strategies in mice inducing high numbers of memory CD8+ T cells specific to a single epitope are able to provide sterilizing protection against infection with Plasmodium sporozoites. We have recently found that Plasmodium-specific CD8+ T cells cluster around sporozoite-infected hepatocytes but whether such clusters are important in elimination of the parasite remains incompletely understood. Here, we used our previously generated data in which we employed intravital microscopy to longitudinally image 32 green fluorescent protein (GFP)-expressing Plasmodium yoelii parasites in livers of mice that had received activated Plasmodium-specific CD8+ T cells after sporozoite infection. We found significant heterogeneity in the dynamics of the normalized GFP signal from the parasites (termed 'vitality index' or VI) that was weakly correlated with the number of T cells near the parasite. We also found that a simple model assuming mass-action, additive killing by T cells well describes the VI dynamics for most parasites and predicts a highly variable killing efficacy by individual T cells. Given our estimated median per capita kill rate of k = 0.031/h we predict that a single T cell is typically incapable of killing a parasite within the 48 h lifespan of the liver stage in mice. Stochastic simulations of T cell clustering and killing of the liver stage also suggested that: (i) three or more T cells per infected hepatocyte are required to ensure sterilizing protection; (ii) both variability in killing efficacy of individual T cells and resistance to killing by individual parasites may contribute to the observed variability in VI decline, and (iii) the stable VI of some clustered parasites cannot be explained by measurement noise. Taken together, our analysis for the first time provides estimates of efficiency at which individual CD8+ T cells eliminate intracellular parasitic infection in vivo.
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Malaria , Plasmodium yoelii , Ratones , Animales , Linfocitos T CD8-positivos , Hígado/parasitología , Hepatocitos/parasitología , Esporozoítos , Plasmodium berghei/metabolismoRESUMEN
Parasites of the genus Plasmodium, the etiological agent of malaria, are transmitted through the bite of anopheline mosquitoes, which deposit sporozoites into the host skin. Sporozoites migrate through the dermis, enter the bloodstream, and rapidly traffic to the liver. They cross the liver sinusoidal barrier and traverse several hepatocytes before switching to productive invasion of a final one for replication inside a parasitophorous vacuole. Cell traversal and productive invasion are functionally independent processes that require proteins secreted from specialized secretory organelles known as micronemes. In this review, we summarize the current understanding of how sporozoites traverse through cells and productively invade hepatocytes, and discuss the role of environmental sensing in switching from a migratory to an invasive state. We propose that timely controlled secretion of distinct microneme subsets could play a key role in successful migration and infection of hepatocytes. A better understanding of these essential biological features of the Plasmodium sporozoite may contribute to the development of new strategies to fight against the very first and asymptomatic stage of malaria.
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Hepatocitos/parasitología , Malaria/parasitología , Plasmodium/fisiología , Esporozoítos/fisiología , Animales , Humanos , Hígado/parasitología , Plasmodium/genética , Plasmodium/crecimiento & desarrollo , Esporozoítos/genética , Esporozoítos/crecimiento & desarrolloRESUMEN
Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury.
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Hepatocitos , Evasión Inmune , Interleucina-10/inmunología , Trypanosoma congolense , Tripanosomiasis Africana , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Hepatocitos/inmunología , Hepatocitos/parasitología , Hepatocitos/patología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Activación de Linfocitos , Ratones , Monocitos/inmunología , Monocitos/patología , Linfocitos T/inmunología , Linfocitos T/patología , Trypanosoma congolense/inmunología , Trypanosoma congolense/patogenicidad , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/patologíaRESUMEN
Recently, a population of non-recirculating, tissue-resident memory CD8+ T cells has been identified; cells that seems to act as key sentinels for invading microorganisms with enhanced effector functions. In malaria, the liver represents the first site for parasite development before a definite infection is established in circulating red blood cells. Here, we discuss the evidence obtained from animal models on several diseases and hypothesize that liver-resident memory CD8+ T cells (hepatic TRM) play a critical role in providing protective liver-stage immunity against Plasmodium malaria parasites. Although observations in human malaria trials are limited to peripheral blood, we propose recommendations for the translation of some of these findings to human malaria research.
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Linfocitos T CD8-positivos/inmunología , Hepatocitos/inmunología , Hígado/inmunología , Vacunas contra la Malaria/inmunología , Malaria/inmunología , Plasmodium falciparum/fisiología , Linfocitos T Reguladores/inmunología , Animales , Hepatocitos/parasitología , Humanos , Memoria Inmunológica , Interferón gamma/metabolismo , Estadios del Ciclo de Vida , Activación de Linfocitos , Malaria/prevención & control , VacunaciónRESUMEN
The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterised by a long-lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3-phosphate (PI3P)-labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P-labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3-labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites.
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Autofagia , Hepatocitos/parasitología , Fosfatos de Fosfatidilinositol/metabolismo , Plasmodium berghei/metabolismo , Plasmodium berghei/parasitología , Esporozoítos/metabolismo , Vacuolas/parasitología , Animales , Línea Celular , Femenino , Células HeLa , Interacciones Huésped-Parásitos , Humanos , Malaria/parasitología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Organismos Modificados GenéticamenteRESUMEN
Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co-factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2-deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single-membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2-deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.
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Apicoplastos/metabolismo , Transportadores de Ácido Fólico/genética , Transportadores de Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Malaria/parasitología , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Animales , Anopheles/parasitología , Hepatocitos/parasitología , Estadios del Ciclo de Vida , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Mosquitos Vectores , Oocistos/citología , Oocistos/genética , Oocistos/metabolismo , Organismos Modificados Genéticamente , Plasmodium berghei/citología , Proteínas Protozoarias/metabolismo , Esporozoítos/metabolismoRESUMEN
BACKGROUND: The zoonotic simian parasite Plasmodium cynomolgi develops into replicating schizonts and dormant hypnozoites during the infection of hepatocytes and is used as a model organism to study relapsing malaria. The transcriptional profiling of P. cynomolgi liver stages was previously reported and revealed many important biological features of the parasite but left out the host response to malaria infection. METHODS: Previously published RNA sequencing data were used to quantify the expression of host genes in rhesus macaque hepatocytes infected with P. cynomolgi in comparison to either cells from uninfected samples or uninfected bystander cells. RESULTS: Although the dataset could not be used to resolve the transcriptional profile of hypnozoite-infected hepatocytes, it provided a snapshot of the host response to liver stage schizonts at 9-10 day post-infection and identified specific host pathways that are modulated during the exo-erythrocytic stage of P. cynomolgi. CONCLUSIONS: This study constitutes a valuable resource characterizing the hepatocyte response to P. cynomolgi infection and provides a framework to build on future research that aims at understanding hepatocyte-parasite interactions during relapsing malaria infection.
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Malaria , Parásitos , Plasmodium cynomolgi , Animales , Plasmodium cynomolgi/genética , Macaca mulatta/parasitología , Hepatocitos/parasitología , Malaria/parasitología , Hígado/parasitologíaRESUMEN
Upon entering its mammalian host, the malaria parasite productively invades two distinct cell types, that is, hepatocytes and erythrocytes during which several adhesins/invasins are thought to be involved. Many surface-located proteins containing thrombospondin Type I repeat (TSR) which help establish host-parasite molecular crosstalk have been shown to be essential for mammalian infection. Previous reports indicated that antibodies produced against Plasmodium falciparum secreted protein with altered thrombospondin repeat (SPATR) block hepatocyte invasion by sporozoites but no genetic evidence of its contribution to invasion has been reported. After failing to generate Spatr knockout in Plasmodium berghei blood stages, a conditional mutagenesis system was employed. Here, we show that SPATR plays an essential role during parasite's blood stages. Mutant salivary gland sporozoites exhibit normal motility, hepatocyte invasion, liver stage development and rupture of the parasitophorous vacuole membrane resulting in merosome formation. But these mutant hepatic merozoites failed to establish a blood stage infection in vivo. We provide direct evidence that SPATR is not required for hepatocyte invasion but plays an essential role during the blood stages of P. berghei.