RESUMEN
Sandflies are known vectors of leishmaniasis. In the Old World, sandflies are also vectors of viruses while little is known about the capacity of New World insects to transmit viruses to humans. Here, we relate the identification of RNA sequences with homology to rhabdovirus nucleocapsids (NcPs) genes, initially in the Lutzomyia longipalpis LL5 cell lineage, named NcP1.1 and NcP2. The Rhabdoviridae family never retrotranscribes its RNA genome to DNA. The sequences here described were identified in cDNA and DNA from LL-5 cells and in adult insects indicating that they are transcribed endogenous viral elements (EVEs). The presence of NcP1.1 and NcP2 in the L. longipalpis genome was confirmed in silico. In addition to showing the genomic location of NcP1.1 and NcP2, we identified another rhabdoviral insertion named NcP1.2. Analysis of small RNA molecules derived from these sequences showed that NcP1.1 and NcP1.2 present a profile consistent with elements targeted by primary piRNAs, while NcP2 was restricted to the degradation profile. The presence of NcP1.1 and NcP2 was investigated in sandfly populations from South America and the Old World. These EVEs are shared by different sandfly populations in South America while none of the Old World species studied presented the insertions.
Asunto(s)
Leishmaniasis , Psychodidae , Rhabdoviridae , Humanos , Animales , América del Sur , ARN , ADN , BrasilRESUMEN
BACKGROUND: The leishmaniases are important neglected diseases caused by Leishmania spp. which are transmitted by sand flies, Lutzomyia longipalpis being the main vector of visceral leishmaniasis in the Americas. The methodologies for leishmaniasis control are not efficient, causing 1.5 million reported cases annually worldwide, therefore showing the need for development of novel strategies and interventions to control transmission of the disease. The bacterium Wolbachia pipientis is being used to control viruses transmitted by mosquitoes, such as dengue and Zika, and its introduction in disease vectors has been effective against parasites such as Plasmodium. Here we show the first successful establishment of Wolbachia into two different embryonic cell lines from L. longipalpis, LL-5 and Lulo, and analysed its effects on the sand fly innate immune system, followed by in vitro Leishmania infantum interaction. RESULTS: Our results show that LL-5 cells respond to wMel and wMelPop-CLA strains within the first 72 h post-infection, through the expression of antimicrobial peptides and inducible nitric oxide synthase resulting in a decrease of Wolbachia detection in the early stages of infection. In subsequent passages, the wMel strain was not able to infect any of the sand fly cell lines while the wMelPop-CLA strain was able to stably infect Lulo cells and LL-5 at lower levels. In Wolbachia stably infected cells, the expression of immune-related genes involved with downregulation of the IMD, Toll and Jak-Stat innate immune pathways was significantly decreased, in comparison with the uninfected control, suggesting immune activation upon Wolbachia transinfection. Furthermore, Wolbachia transinfection did not promote a negative effect on parasite load in those cells. CONCLUSIONS: Initial strong immune responses of LL5 cells might explain the inefficiency of stable infections in these cells while we found that Lulo cells are more permissive to infection with Wolbachia causing an effect on the cell immune system, but not against in vitro L. infantum interaction. This establishes Lulo cells as a good system for the adaptation of Wolbachia in L. longipalpis.
Asunto(s)
Expresión Génica , Inmunidad Innata , Factores Inmunológicos/biosíntesis , Leishmania infantum/crecimiento & desarrollo , Interacciones Microbianas , Psychodidae/inmunología , Wolbachia/inmunología , Animales , Línea Celular , Carga de Parásitos , Psychodidae/microbiología , Wolbachia/crecimiento & desarrolloRESUMEN
Innate immunity is an ancient and conserved defense system that provides an early effective response against invaders. Many immune genes of Anopheles mosquitoes have been implicated in defense against a variety of pathogens, including plasmodia. Nevertheless, only recent work identified some immune genes of Anopheles aquasalis mosquitoes upon P. vivax infection. Among these was a GATA transcription factor gene, which is described here. This is an ortholog of GATA factor Serpent genes described in Drosophila melanogaster and Anopheles gambiae. Gene expression analyses showed an increase of GATA-Serpent mRNA in P. vivax-infected A. aquasalis and functional RNAi experiments identified this transcription factor as an important immune gene of A. aquasalis against both bacteria and P. vivax. Besides, we were able to identify an effect of GATA-Serpent knockdown on A. aquasalis hemocyte proliferation and differentiation. These findings expand our understanding of the poorly studied A. aquasalis-P. vivax interactions and uncover GATA-Serpent as a key player of the mosquito innate immune response.
Asunto(s)
Anopheles/inmunología , Bacterias/inmunología , Factores de Transcripción GATA/metabolismo , Inmunidad Innata , Plasmodium/inmunología , Animales , Anopheles/genética , Diferenciación Celular , Proliferación Celular , Femenino , Factores de Transcripción GATA/genética , Perfilación de la Expresión Génica , Silenciador del Gen , Hemocitos/inmunología , Hemocitos/fisiologíaRESUMEN
Phlebotomine sand flies are the subject of much research because of the role of their females as the only proven natural vectors of Leishmania species, the parasitic protozoans that are the causative agents of the neglected tropical disease leishmaniasis. Activity in this field was highlighted by the eighth International Symposium on Phlebotomine Sand flies (ISOPS) held in September 2014, which prompted this review focusing on vector control. Topics reviewed include: Taxonomy and phylogenetics, Vector competence, Genetics, genomics and transcriptomics, Eco-epidemiology, and Vector control. Research on sand flies as leishmaniasis vectors has revealed a diverse array of zoonotic and anthroponotic transmission cycles, mostly in subtropical and tropical regions of Africa, Asia and Latin America, but also in Mediterranean Europe. The challenge is to progress beyond descriptive eco-epidemiology, in order to separate vectors of biomedical importance from the sand fly species that are competent vectors but lack the vectorial capacity to cause much human disease. Transmission modelling is required to identify the vectors that are a public health priority, the ones that must be controlled as part of the integrated control of leishmaniasis. Effective modelling of transmission will require the use of entomological indices more precise than those usually reported in the leishmaniasis literature.
Asunto(s)
Transmisión de Enfermedad Infecciosa/prevención & control , Entomología/tendencias , Control de Insectos/métodos , Control de Insectos/tendencias , Leishmaniasis/epidemiología , Leishmaniasis/prevención & control , Psychodidae/fisiología , África/epidemiología , Animales , Asia/epidemiología , Europa (Continente)/epidemiología , Humanos , Insectos Vectores , América Latina/epidemiología , Clima TropicalRESUMEN
In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Anopheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.
Asunto(s)
Anopheles/parasitología , Insectos Vectores/parasitología , Malaria/transmisión , Plasmodium/clasificación , Animales , Anopheles/clasificación , Anopheles/genética , Anopheles/inmunología , Anopheles/ultraestructura , Modelos Animales de Enfermedad , Insectos Vectores/clasificación , Insectos Vectores/genética , Insectos Vectores/inmunología , Insectos Vectores/ultraestructura , Malaria/inmunología , Control de Mosquitos , Carga de Parásitos , Bosque LluviosoRESUMEN
Leishmaniasis is a serious problem that affects mostly poor countries. Various species of Leishmania are the agents of the disease, which take different clinical manifestations. The parasite is transmitted by sandflies, predominantly from the Phlebotomus genus in the Old World and Lutzomyia in the New World. During development in the gut, Leishmania must survive various challenges, which include avoiding being expelled with blood remnants after digestion. It is believed that attachment to the gut epithelium is a necessary step for vector infection, and molecules from parasites and sand flies have been implicated in this attachment. In previous work, monoclonal antibodies were produced against Leishmania. Among these an antibody was obtained against Leishmania braziliensis flagella, which blocked the attachment of Leishmania panamensis flagella to Phlebotomus papatasi guts. The protein recognized by this antibody was identified and named FLAG1, and the complete FLAG1 gene sequence was obtained. This protein was later independently identified as a small, myristoylated protein and called SMP1, so from now on it will be denominated FLAG1/SMP1. The FLAG1/SMP1 gene is expressed in all developmental stages of the parasite, but has higher expression in promastigotes. The anti-FLAG1/SMP1 antibody recognized the flagellum of all Leishmania species tested and generated the expected band by western blots. This antibody was used in attachment and infection blocking experiments. Using the New World vector Lutzomyia longipalpis and Leishmania infantum chagasi, no inhibition of attachment ex vivo or infection in vivo was seen. On the other hand, when the Old World vectors P. papatasi and Leishmania major were used, a significant decrease of both attachment and infection were seen in the presence of the antibody. We propose that FLAG1/SMP1 is involved in the attachment/infection of Leishmania in the strict vector P. papatasi and not the permissive vector L. longipalpis.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Leishmania/fisiología , Proteínas Protozoarias/metabolismo , Psychodidae/parasitología , Secuencia de Aminoácidos , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente , Interacciones Huésped-Parásitos , Leishmania/genética , Leishmania/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas Protozoarias/genéticaRESUMEN
In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.
Asunto(s)
Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Amoxicilina/administración & dosificación , Antibacterianos/administración & dosificación , Medicamentos Herbarios Chinos/administración & dosificación , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Omeprazol/análogos & derivados , Úlcera Péptica/tratamiento farmacológico , Antiulcerosos/administración & dosificación , Claritromicina/administración & dosificación , Método Doble Ciego , Quimioterapia Combinada , Estudios de Seguimiento , Infecciones por Helicobacter/patología , Lansoprazol , Omeprazol/administración & dosificación , Estudios Prospectivos , Úlcera Péptica/microbiología , Úlcera Péptica/patología , Recurrencia , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Malaria affects millions of people worldwide and hundreds of thousands of people each year in Brazil. The mosquito Anopheles aquasalis is an important vector of Plasmodium vivax, the main human malaria parasite in the Americas. Reactive oxygen species (ROS) have been shown to have a role in insect innate immune responses as a potent pathogen-killing agent. We investigated the mechanisms of free radicals modulation after A. aquasalis infection with P. vivax. ROS metabolism was evaluated in the vector by studying expression and activity of three key detoxification enzymes, one catalase and two superoxide dismutases (SOD3A and SOD3B). Also, the involvement of free radicals in the mosquito immunity was measured by silencing the catalase gene followed by infection of A. aquasalis with P. vivax. Catalase, SOD3A and SOD3B expression in whole A. aquasalis were at the same levels of controls at 24 h and upregulated 36 h after ingestion of blood containing P. vivax. However, in the insect isolated midgut, the mRNA for these enzymes was not regulated by P. vivax infection, while catalase activity was reduced 24 h after the infectious meal. RNAi-mediated silencing of catalase reduced enzyme activity in the midgut, resulted in increased P. vivax infection and prevalence, and decreased bacterial load in the mosquito midgut. Our findings suggest that the interactions between A. aquasalis and P. vivax do not follow the model of ROS-induced parasite killing. It appears that P. vivax manipulates the mosquito detoxification system in order to allow its own development. This can be an indirect effect of fewer competitive bacteria present in the mosquito midgut caused by the increase of ROS after catalase silencing. These findings provide novel information on unique aspects of the main malaria parasite in the Americas interaction with one of its natural vectors.
Asunto(s)
Anopheles/metabolismo , Anopheles/parasitología , Plasmodium vivax/fisiología , Especies Reactivas de Oxígeno/metabolismo , Secuencia de Aminoácidos , Animales , Anopheles/genética , Catalasa/genética , Catalasa/metabolismo , Susceptibilidad a Enfermedades , Activación Enzimática , Femenino , Silenciador del Gen , Humanos , Masculino , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: Phlebotomine insects harbor bacterial, viral and parasitic pathogens that can cause diseases of public health importance. Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the New World. Insects can mount a powerful innate immune response to pathogens. Defensin peptides take part in this response and are known to be active against Gram-positive and Gram-negative bacteria, and some parasites. We studied the expression of a defensin gene from Lutzomyia longipalpis to understand its role in sand fly immune response. METHODS: We identified, sequenced and evaluated the expression of a L. longipalpis defensin gene by semi-quantitative RT-PCR. The gene sequence was compared to other vectors defensins and expression was determined along developmental stages and after exposure of adult female L. longipalpis to bacteria and Leishmania. RESULTS: Phylogenetic analysis showed that the L. longipalpis defensin is closely related to a defensin from the Old World sand fly Phlebotomus duboscqi. Expression was high in late L4 larvae and pupae in comparison to early larval stages and newly emerged flies. Defensin expression was modulated by oral infection with bacteria. The Gram-positive Micrococcus luteus induced early high defensin expression, whilst the Gram-negative entomopathogenic Serratia marcescens induced a later response. Bacterial injection also induced defensin expression in adult insects. Female sand flies infected orally with Leishmania mexicana showed no significant difference in defensin expression compared to blood fed insects apart from a lower defensin expression 5 days post Leishmania infection. When Leishmania was introduced into the hemolymph by injection there was no induction of defensin expression until 72 h later. CONCLUSIONS: Our results suggest that L. longipalpis modulates defensin expression upon bacterial and Leishmania infection, with patterns of expression that are distinct among bacterial species and routes of infection.
Asunto(s)
Bacterias/inmunología , Defensinas/biosíntesis , Leishmania mexicana/inmunología , Micrococcus luteus/inmunología , Psychodidae/microbiología , Psychodidae/parasitología , Serratia marcescens/inmunología , Animales , Análisis por Conglomerados , Defensinas/inmunología , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Filogenia , Psychodidae/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on gram-negative and gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Leishmania infantum/inmunología , Leishmania mexicana/inmunología , Leishmaniasis Visceral/inmunología , Psychodidae , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/inmunología , Femenino , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/parasitología , Genes de Insecto/inmunología , Inmunidad Innata/inmunología , Leishmania infantum/crecimiento & desarrollo , Leishmania mexicana/crecimiento & desarrollo , Filogenia , Psychodidae/genética , Psychodidae/inmunología , Psychodidae/parasitologíaRESUMEN
BACKGROUND: Parasites of the Leishmania genus alternate between the flagellated extracellular promastigote stage and intracellular amastigotes. Here we report the characterization of a Leishmania isolate, obtained from a cutaneous leishmaniasis patient, which presents peculiar morphological features. METHODS: The parasite was cultured in vitro and characterized morphologically using optical and electron microscopy. Identification was performed based on monoclonal antibodies and internal ribosomal spacer typing. In vitro macrophage cultures, murine experimental models and sand fly infections were used to evaluate infectivity in vitro and in vivo. RESULTS: The isolate was identified as Leishmania (Viannia) braziliensis. In the atypical promastigotes grown in culture, a short flagellum surrounded or interrupted by a protuberance of disorganized material was observed. A normal axoneme was present close to the basal body but without elongation much further outside the flagellar pocket. A disorganized swelling at the precocious end of the axoneme coincided with the lack of a paraflagellar rod structure. The isolate was able to infect macrophages in vitro, induce lesions in BALB/c mice and infect Lutzomyia longipalpis. CONCLUSIONS: Notwithstanding the lack of an extracellular flagellum, this isolate infects macrophages in vitro and produces lesions when inoculated into mice. Moreover, it is able to colonize phlebotomine sand flies. Considering the importance attributed to the flagellum in the successful infection and survival of Leishmania in the insect midgut and in the invasion of macrophages, these findings may bring new light into the infectious mechanisms of L. (V.) braziliensis.
Asunto(s)
Leishmania braziliensis/aislamiento & purificación , Leishmaniasis Cutánea/parasitología , Animales , Anticuerpos Monoclonales , Antiprotozoarios/administración & dosificación , Secuencia de Bases , ADN Protozoario/química , ADN Protozoario/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Femenino , Flagelos , Humanos , Leishmania braziliensis/genética , Leishmania braziliensis/ultraestructura , Leishmaniasis Cutánea/tratamiento farmacológico , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación , Psychodidae/parasitología , Análisis de Secuencia de ADN , Resultado del TratamientoRESUMEN
Malaria affects 300 million people worldwide every year and 450,000 in Brazil. In coastal areas of Brazil, the main malaria vector is Anopheles aquasalis, and Plasmodium vivax is responsible for the majority of malaria cases in the Americas. Insects possess a powerful immune system to combat infections. Three pathways control the insect immune response: Toll, IMD, and JAK-STAT. Here we analyze the immune role of the A. aquasalis JAK-STAT pathway after P. vivax infection. Three genes, the transcription factor Signal Transducers and Activators of Transcription (STAT), the regulatory Protein Inhibitors of Activated STAT (PIAS) and the Nitric Oxide Synthase enzyme (NOS) were characterized. Expression of STAT and PIAS was higher in males than females and in eggs and first instar larvae when compared to larvae and pupae. RNA levels for STAT and PIAS increased 24 and 36 hours (h) after P. vivax challenge. NOS transcription increased 36 h post infection (hpi) while this protein was already detected in some midgut epithelial cells 24 hpi. Imunocytochemistry experiments using specific antibodies showed that in non-infected insects STAT and PIAS were found mostly in the fat body, while in infected mosquitoes the proteins were found in other body tissues. The knockdown of STAT by RNAi increased the number of oocysts in the midgut of A. aquasalis. This is the first clear evidence for the involvement of a specific immune pathway in the interaction of the Brazilian malaria vector A. aquasalis with P. vivax, delineating a potential target for the future development of disease controlling strategies.
Asunto(s)
Anopheles/inmunología , Anopheles/parasitología , Óxido Nítrico Sintasa/biosíntesis , Plasmodium vivax/inmunología , Plasmodium vivax/aislamiento & purificación , Proteínas Inhibidoras de STAT Activados/biosíntesis , Factores de Transcripción STAT/biosíntesis , Animales , Brasil , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/inmunología , Proteínas Inhibidoras de STAT Activados/inmunología , Factores de Transcripción STAT/inmunología , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Leishmania parasites have been reported to interfere and even subvert their host immune responses to enhance their chances of survival and proliferation. Experimental Leishmania infection in mice has been widely used in the identification of specific parasite virulence factors involved in the interaction with the host immune system. Cysteine-proteinase B (CPB) is an important virulence factor in parasites from the Leishmania (Leishmania) mexicana complex: it inhibits lymphocytes Th1 and/or promotes Th2 responses either through proteolytic activity or through epitopes derived from its COOH-terminal extension. In the present study we analyzed the effects of Leishmania (Leishmania) amazonensis CPB COOH-terminal extension-derived peptides on cell cultures from murine strains with distinct levels of susceptibility to infection: BALB/c, highly susceptible, and CBA, mildly resistant. RESULTS: Predicted epitopes, obtained by in silico mapping, displayed the ability to induce cell proliferation and expression of cytokines related to Th1 and Th2 responses. Furthermore, we applied in silico simulations to investigate how the MHC/epitopes interactions could be related to the immunomodulatory effects on cytokines, finding evidence that specific interaction patterns can be related to in vitro activities. CONCLUSIONS: Based on our results, we consider that some peptides from the CPB COOH-terminal extension may influence host immune responses in the murine infection, thus helping Leishmania survival.
Asunto(s)
Proteasas de Cisteína/inmunología , Epítopos/inmunología , Leishmania mexicana/inmunología , Leishmania mexicana/patogenicidad , Leishmaniasis/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteasas de Cisteína/genética , Citocinas/biosíntesis , Epítopos/genética , Epítopos/metabolismo , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Femenino , Antígenos H-2/inmunología , Antígenos H-2/metabolismo , Leishmaniasis/parasitología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Óxido Nítrico/biosíntesis , Unión Proteica/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Malaria affects 300 million people worldwide every year and is endemic in 22 countries in the Americas where transmission occurs mainly in the Amazon Region. Most malaria cases in the Americas are caused by Plasmodium vivax, a parasite that is almost impossible to cultivate in vitro, and Anopheles aquasalis is an important malaria vector. Understanding the interactions between this vector and its parasite will provide important information for development of disease control strategies. To this end, we performed mRNA subtraction experiments using A. aquasalis 2 and 24 hours after feeding on blood and blood from malaria patients infected with P. vivax to identify changes in the mosquito vector gene induction that could be important during the initial steps of infection. A total of 2,138 clones of differentially expressed genes were sequenced and 496 high quality unique sequences were obtained. Annotation revealed 36% of sequences unrelated to genes in any database, suggesting that they were specific to A. aquasalis. A high number of sequences (59%) with no matches in any databases were found 24 h after infection. Genes related to embryogenesis were down-regulated in insects infected by P. vivax. Only a handful of genes related to immune responses were detected in our subtraction experiment. This apparent weak immune response of A. aquasalis to P. vivax infection could be related to the susceptibility of this vector to this important human malaria parasite. Analysis of some genes by real time PCR corroborated and expanded the subtraction results. Taken together, these data provide important new information about this poorly studied American malaria vector by revealing differences between the responses of A. aquasalis to P. vivax infection, in relation to better studied mosquito-Plasmodium pairs. These differences may be important for the development of malaria transmission-blocking strategies in the Americas.
Asunto(s)
Anopheles/parasitología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Plasmodium vivax/metabolismo , Actinas/genética , Secuencia de Aminoácidos , Animales , Etiquetas de Secuencia Expresada , Femenino , Biblioteca de Genes , Masculino , Modelos Genéticos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Sand fly and Leishmania are one of the best studied vector-parasite models. Much is known about the development of these parasites within the sand fly, and how transmission to a suitable vertebrate host takes place. Various molecules secreted by the vector assist the establishment of the infection in a vertebrate, and changes to the vector are promoted by the parasites in order to facilitate or enhance transmission. Despite a generally accepted view that sand flies and Leishmania are also one of the oldest vector-pathogen pairs known, such long history has not been translated into a harmonic relationship. Leishmania are faced with many barriers to the establishment of a successful infection within the sand fly vector, and specific associations have been developed which are thought to represent aspects of a co-evolution between the parasite and its vectors. In this review, we highlight the journey taken by Leishmania during its development within the vector, and describe the issues associated with the natural barriers encountered by the parasite. Recent data revealed sexual replication of Leishmania within the sand fly, but it is yet unknown if such reproduction affects disease outcome. New approaches targeting sand fly molecules to prevent parasite transmission are being sought, and various techniques related to genetic manipulation of sand flies are being utilized.
RESUMEN
Leishmaniasis is an important worldwide public health problem. Visceral leishmaniasis caused by Leishmania infantum chagasi is mainly transmitted by Lutzomyia longipalpis in the Americas. Leishmania development within the sand fly vector is mostly restricted to the midgut. Thus, a comparative analysis of blood-fed versus infected midguts may provide an invaluable insight into various aspects of sand fly immunity, physiology of blood digestion, and, more importantly, of Leishmania development. To that end, we have engaged in a study to identify expressed sequenced tags (ESTs) from L. longipalpis cDNA libraries produced from midguts dissected at different times post blood meal and also after artificial infection with L. i. chagasi. A total of 2,520 ESTs were obtained and, according to the quality of the sequencing data obtained, assembled into 378 clusters and 1,526 individual sequences or singletons totalizing 1,904 sequences. Several sequences associated with defense, apoptosis, RNAi, and digestion processes were annotated. The data presented here increases current knowledge on the New World sand fly transcriptome, contributing to the understanding of various aspects of the molecular physiology of L. longipalpis, and mechanisms underlying the relationship of this sand fly species with L. i. chagasi.
Asunto(s)
Etiquetas de Secuencia Expresada , Insectos Vectores/genética , Leishmania , Leishmaniasis Visceral/transmisión , Psychodidae/genética , Animales , Perfilación de la Expresión Génica , Insectos Vectores/parasitología , Psychodidae/parasitología , Análisis de Secuencia de ADNRESUMEN
Cysteine proteinases have been implicated in many aspects of protozoan parasite pathogenesis. These hydrolases are normally found as zymogens, and some classes in trypanosomatids possess a long C-terminal extension (CTE), for which no function has been assigned. In this paper we hypothesize that the CTE domain of Lpcys2, the abundant lysosomal cysteine proteinase of Leishmania pifanoi amastigotes, is involved in host cell infection. Confirming previous reports that this peptide is highly immunogenic in Trypanosoma cruzi, we detected antibodies against CTE in sera of leishmaniasis patients. We produced a polyclonal antibody specific to Lpcys2 CTE and determined that this antibody was capable of recognizing both L. pifanoi and Leishmania amazonensis cysteine proteinases. Using this antibody, we detected a predominant localization of Lpcys2 CTE in the lysosome and flagellar pocket of cultured axenic amastigotes of both parasite species; however, its location was shifted towards the surface of the parasites during macrophage infection. We examined the role of Lpcys2 CTE in macrophage infection and found a significant reduction in the percentage of infected cells when macrophages were infected with L. pifanoi and L. amazonensis in the presence of anti-CTE antibody. This study suggests a role for leishmanial cysteine proteinases CTE at early stages of infection.
Asunto(s)
Cisteína Endopeptidasas , Interacciones Huésped-Parásitos , Leishmania/enzimología , Leishmania/patogenicidad , Leishmaniasis/parasitología , Macrófagos Peritoneales/parasitología , Animales , Anticuerpos Antiprotozoarios/sangre , Células Cultivadas , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , Humanos , Leishmania/clasificación , Leishmania/ultraestructura , Leishmaniasis/inmunología , Lisosomas/enzimología , Lisosomas/ultraestructura , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de TransmisiónRESUMEN
Leishmaniasis, caused by Leishmania parasites, is an important public health problem worldwide. Leishmania, like other trypanosomatids, present unique biological features as compared to higher eukaryotes that can be exploited with the intent of finding new chemotherapeutical/vaccine candidates. Mechanisms of cellular sorting in Leishmania can be viewed as such potential targets. We have previously demonstrated a role for the pro-domain of a Leishmania cysteine proteinase in lysosomal targeting. In this paper, we show that this signal is not recognized by mammalian cells and is recognized by yeast; we also discuss here the implications of these findings related to evolution and further characterization of the Leishmania trafficking machinery.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Leishmania , Proteínas/metabolismo , Proteínas Protozoarias/metabolismo , Saccharomyces/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Cisteína Endopeptidasas/genética , Transporte de Proteínas , Proteínas/genética , Proteínas Protozoarias/genéticaRESUMEN
Amastigotes of Leishmania species belonging to the Leishmania mexicana complex exhibit large lysosomes, called megasomes, which are rich in cysteine proteinases. Various aspects of the host-parasite interaction, the differentiation process as well as intracellular survival, have been attributed to these proteinases. The in vitro differentiation from promastigote to amastigote forms of Leishmania amazonensis was evaluated using the analysis of the expression of cysteine proteinase (Lpcys2) by Northern blot, Western blot and enzymatic activity. As promastigotes transformed to amastigotes, there was an increase in Lpcys2 RNA transcription, as well as an increase in Lpcys2 production and activity. Moreover, the processing rate of the cysteine proteinase precursor forms was also increased in amastigote forms. These results are in agreement with our previous study in which megasome development was demonstrated by morphometric and immunochemical analysis.