RESUMO
As part of on-going efforts to control hookworm infection, the "human hookworm vaccine initiative" has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm.
Assuntos
Anticorpos Anti-Helmínticos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Eritrócitos/efeitos dos fármacos , Infecções por Uncinaria/prevenção & controle , Necator americanus/enzimologia , Nippostrongylus/crescimento & desenvolvimento , Infecções por Strongylida/prevenção & controle , Ancylostomatoidea/efeitos dos fármacos , Ancylostomatoidea/crescimento & desenvolvimento , Animais , Antígenos de Helmintos/imunologia , Ácido Aspártico Endopeptidases/imunologia , Eritrócitos/parasitologia , Feminino , Infecções por Uncinaria/parasitologia , Estágios do Ciclo de Vida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nippostrongylus/efeitos dos fármacos , Infecções por Strongylida/parasitologiaRESUMO
Hookworms infect more than 700 million people worldwide and cause more morbidity than most other human parasitic infections. Nippostrongylus brasiliensis (the rat hookworm) has been used as an experimental model for human hookworm because of its similar life cycle and ease of maintenance in laboratory rodents. Adult N. brasiliensis, like the human hookworm, lives in the intestine of the host and releases excretory/secretory products (ESP), which represent the major host-parasite interface. We performed a comparative proteomic analysis of infective larval (L3) and adult worm stages of N. brasiliensis to gain insights into the molecular bases of host-parasite relationships and determine whether N. brasiliensis could indeed serve as an appropriate model for studying human hookworm infections. Proteomic data were matched to a transcriptomic database assembled from 245,874,892 Illumina reads from different developmental stages (eggs, L3, L4, and adult) of N. brasiliensis yieldingâ¼18,426 unigenes with 39,063 possible isoform transcripts. From this analysis, 313 proteins were identified from ESPs by LC-MS/MS-52 in the L3 and 261 in the adult worm. Most of the proteins identified in the study were stage-specific (only 13 proteins were shared by both stages); in particular, two families of proteins-astacin metalloproteases and CAP-domain containing SCP/TAPS-were highly represented in both L3 and adult ESP. These protein families are present in most nematode groups, and where studied, appear to play roles in larval migration and evasion of the host's immune response. Phylogenetic analyses of defined protein families and global gene similarity analyses showed that N. brasiliensis has a greater degree of conservation with human hookworm than other model nematodes examined. These findings validate the use of N. brasiliensis as a suitable parasite for the study of human hookworm infections in a tractable animal model.
Assuntos
Ancylostomatoidea/crescimento & desenvolvimento , Trato Gastrointestinal/parasitologia , Proteínas de Helminto/metabolismo , Estágios do Ciclo de Vida , Proteoma/análise , Ancylostomatoidea/metabolismo , Animais , Sequência de Bases , Sequência Conservada , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Filogenia , Proteoma/metabolismo , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNARESUMO
Complement may be important for immunity to infection with parasitic helminths, by promoting the recruitment of leukocytes to infected tissues and by modulating the function of cytotoxic effector leukocytes. However, the importance of complement in vivo during helminth infection is poorly understood. In this study, mice lacking classical (C1q-deficient), alternative (factor B-deficient) or all pathways of complement activation (C3-deficient) were used to assess the role of complement in immunity to the nematode Nippostrongylus brasiliensis. Double-mutant complement-deficient/IL-5 transgenic (Tg) mice were used to determine if complement is required for the strong eosinophil-dependent resistance to this parasite. Complement activation on larvae (C3 deposition), extracellular eosinophil peroxidase activity, larval aggregation and eosinophil recruitment to the skin 30 min post-injection (p.i.) of larvae were reduced in factor B-deficient mice. Inhibition of the C5a receptor with the antagonist PMX53 impaired eosinophil and neutrophil recruitment to the skin. C3 deposition on larvae was minimal by 150 min p.i. and at this time cell adherence, larval aggregation, eosinophil recruitment and degranulation were complement-independent. Factor B and C3 deficiency were associated with higher lung larval burdens in primary infections. Complement-deficient/IL-5 Tg mice were highly resistant to N. brasiliensis, suggesting that eosinophils can limit infection in a complement-independent manner. Potent secondary immunity was similarly complement-independent. In conclusion, although the alternative pathway is important for parasite recognition and leukocyte recruitment early in N. brasiliensis infections, the parasite soon becomes resistant to complement and other factors can compensate to promote eosinophil-dependent immunity.
Assuntos
Proteínas do Sistema Complemento/imunologia , Eosinófilos/imunologia , Imunidade Celular/imunologia , Imunidade Inata/imunologia , Nippostrongylus/imunologia , Animais , Basófilos/citologia , Basófilos/imunologia , Basófilos/parasitologia , Adesão Celular , Degranulação Celular/imunologia , Movimento Celular , Complemento C3/imunologia , Eosinófilos/citologia , Eosinófilos/parasitologia , Eosinófilos/fisiologia , Feminino , Fertilidade , Intestinos/imunologia , Intestinos/parasitologia , Larva/citologia , Pulmão/imunologia , Pulmão/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/imunologia , Nippostrongylus/citologia , Pele/imunologia , Pele/parasitologia , Infecções por Strongylida/imunologiaRESUMO
Eosinophils are an important feature of immune responses to infections with many of the tissue-invasive helminth parasites. The cytokine IL-5 and a high-affinity double GATA-binding site within the GATA-1 promoter are critical for eosinophilopoiesis. In this study, we believe we demonstrate for the first time that defects in eosinophilopoiesis are associated with impaired resistance to Nippostrongylus brasiliensis. Primary and secondary infections were established in wildtype (WT), IL-5(-/-) and DeltadblGATA mice. Resistance to secondary infections was impaired in IL-5(-/-) and DeltadblGATA mice, with significantly more larvae able to reach the lungs 2 days p.i. Pulmonary inflammation was minimal in all strains in the first 2 days of both primary and secondary infections, suggesting that eosinophil-dependent resistance occurred before larvae reached this site. Intestinal worm burdens and/or parasite egg production in primary infections were greater in animals with defective eosinophilopoiesis. While larvae did reach the gut by day 3 of secondary infections of WT and IL-5(-/-) mice, worms were expelled by day 7, even in the complete absence of eosinophils in tissues of the small intestine. This and our previous studies indicate that N. brasiliensis are likely to be exquisitely sensitive to attack by eosinophils soon after entry into the skin. Eosinophils in the gut may make a modest contribution to resistance on first exposure to the parasite, but are not required for expulsion in either primary or secondary infections. In order to mount an effective immune response it may be vital for the host to identify and attack the parasite before it implements immune evasion strategies and migrates to other anatomical sites. These observations may be of particular significance for the development of successful vaccines against hookworms and other nematodes.
Assuntos
Eosinófilos/imunologia , Interleucina-5/imunologia , Enteropatias Parasitárias/imunologia , Nippostrongylus/imunologia , Infecções por Strongylida/imunologia , Animais , Eosinófilos/parasitologia , Feminino , Interleucina-5/genética , Enteropatias Parasitárias/genética , Pulmão/imunologia , Pulmão/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos CBA , Nippostrongylus/genética , Contagem de Ovos de Parasitas , Pele/imunologia , Pele/parasitologia , Estatística como Assunto , Infecções por Strongylida/genética , Infecções por Strongylida/parasitologiaRESUMO
Complement activation and C3 deposition on the surface of parasitic helminths may be important for recruitment of leukocytes and for damage to the target organism via cell-mediated mechanisms. Inhibition of complement activation would therefore be advantageous to parasites, minimizing damage and enhancing migration through tissues. The aim of this study was to determine ex vivo if complement activation by, and leukocyte adherence to, the nematode Nippostrongylus brasiliensis change as the parasite matures and migrates through the murine host. Pathways of activation of complement and the mechanism of adherence of leukocytes were also defined using sera from mice genetically deficient in either C1q, factor B, C1q and factor B, C3, or C4. Substantive deposition of C3 and adherence of eosinophil-rich leukocytes were seen with infective-stage (L3) but not with lung-stage (L4) larvae. Adult intestinal worms had low to intermediate levels of both C3 and leukocyte binding. For L3 and adult worms, complement deposition was principally dependent on the alternative pathway. For lung-stage larvae, the small amount of C3 detected was dependent to similar degrees on both the lectin and alternative pathways. The classical pathway was not involved for any of the life stages of the parasite. These results suggest that in primary infections, the infective stage of N. brasiliensis is vulnerable to complement-dependent attack by leukocytes. However, within the first 24 h of infection, N. brasiliensis acquires the ability to largely avoid complement-dependent immune responses.