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
BACKGROUND: Eukaryotic genome assembly remains a challenge in part due to the prevalence of complex DNA repeats. This is a particularly acute problem for holocentric nematodes because of the large number of satellite DNA sequences found throughout their genomes. These have been recalcitrant to most genome sequencing methods. At the same time, many nematodes are parasites and some represent a serious threat to human health. There is a pressing need for better molecular characterization of animal and plant parasitic nematodes. The advent of long-read DNA sequencing methods offers the promise of resolving complex genomes. RESULTS: Using Nippostrongylus brasiliensis as a test case, applying improved base-calling algorithms and assembly methods, we demonstrate the feasibility of de novo genome assembly matching current community standards using only MinION long reads. In doing so, we uncovered an unexpected diversity of very long and complex DNA sequences repeated throughout the N. brasiliensis genome, including massive tandem repeats of tRNA genes. CONCLUSION: Base-calling and assembly methods have improved sufficiently that de novo genome assembly of large complex genomes is possible using only long reads. The method has the added advantage of preserving haplotypic variants and so has the potential to be used in population analyses.
Assuntos
Genoma Helmíntico/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nippostrongylus/genética , Análise de Sequência de DNA/métodos , Animais , Sequência de Bases/genética , Feminino , Nippostrongylus/isolamento & purificação , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-DawleyRESUMO
Nippostrongylus brasiliensis, a nematode parasite of rodents, has a parasitic life cycle that is an extremely useful model for the study of human hookworm infection, particularly in regards to the induced immune response. The current reference genome for this parasite is highly fragmented with minimal annotation, but new advances in long-read sequencing suggest that a more complete and annotated assembly should be an achievable goal. We de-novo assembled a single contig mitochondrial genome from N. brasiliensis using MinION R9 nanopore data. The assembly was error-corrected using existing Illumina HiSeq reads, and annotated in full (i.e. gene boundary definitions without substantial gaps) by comparing with annotated genomes from similar parasite relatives. The mitochondrial genome has also been annotated with a preliminary electrical consensus sequence, using raw signal data generated from a Nanopore R9 flow cell.
RESUMO
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
Of all the microbial infections relevant to mammals the relationship between parasitic worms and what constitutes and regulates a host protective immune response is perhaps the most complex and evolved. Nippostrongylus brasiliensis is a tissue migrating parasitic roundworm of rodents that exemplifies many of the salient features of parasitic worm infection, including parasite development through sequential larval stages as it migrates through specific tissue sites. Immune competent hosts respond to infection by N. brasiliensis with a rapid and selective development of a profound Th2 immune response that appears able to confer life long protective immunity against reinfection. This review details how the lung can be the site of migrating nematode immune killing and the gut a site of rapid immune mediated clearance of worms. Furthermore it appears that N. brasiliensis induced responses in the lung are sufficient for conferring immunity in lung and gut while infection of the gut only confers immunity in the gut. This review also covers the role of IL-4, STAT6, and the innate cytokines IL-25, IL-33, and thymic stromal lymphopoietin in the generation of CD4-mediated immunity against N. brasiliensis reinfection and discusses what cytokines might be involved in mediated killing or expulsion of helminth parasites.
RESUMO
Hookworms represent a major infectious burden globally, especially in developing countries. The murine hookworm Nippostrongylus brasiliensis is normally cleared in a manner dependent on IL-13, IL4-R and STAT6 signalling. Here we have used STAT6-deficient animals to model a non-resistant population and describe 2 novel STAT6-independent processes for the clearance of N. brasiliensis. During primary infection STAT6-/- animals are able to clear gut-dwelling N. brasiliensis by a mechanism involving the trapping and degradation of worms in the gut mucosa. Here, a previously undescribed STAT6-independent up-regulation of Relm-ß was observed which correlated with the mucosal trapping and degradation of worms. Previous studies have indicated that during secondary infection STAT6 deficient animals fail to expel adult worms and remain susceptible to re-infection and long-term colonization of the gut. We report here that an initial partially protective response occurs early upon re-infection in the absence of STAT6, and that a late-phase protective secondary response arises in the gut of STAT6-deficient mice leading to the clearance of the majority of N. brasiliensis, through their trapping and death in the mucosal layer of the lower region of the small intestine. These findings show that there are a number of redundant effector pathways which act to reduce worm burden in the gut which can be activated by mechanisms that do not work through the dominant STAT6 signalling pathway and may be useful as targets for future vaccination strategies against resistant hookworm strains.
Assuntos
Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Nippostrongylus/imunologia , Fator de Transcrição STAT6/deficiência , Fator de Transcrição STAT6/imunologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia , Animais , Citometria de Fluxo , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nippostrongylus/genética , RNA de Helmintos/química , RNA de Helmintos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT6/genética , Transdução de Sinais , Organismos Livres de Patógenos EspecíficosRESUMO
Allergic asthma is responsible for widespread morbidity and mortality and its incidence has increased dramatically in industrialized countries over the past two decades. Here, we describe a new murine model of allergic asthma utilizing a novel allergen with intrinsic enzymatic activity similar to that reported for many environmental allergens. The allergen, NES, is excreted and secreted from the nematode Nippostrongylus brasiliensis, and can readily be isolated from in vitro parasite cultures. When NES is administered intranasally to presensitized mice, allergic airway disease develops, including airway hyper-responsiveness, airway eosinophilia, IgE antibody production and Th2 cytokine production. This disease is characteristic of atopic asthma and can be induced within 11 days, thus providing a platform for the rapid analysis of allergic disease and high throughput testing of immunomodulatory factors.
Assuntos
Antígenos de Helmintos/farmacologia , Asma/etiologia , Hipersensibilidade/etiologia , Nippostrongylus/patogenicidade , Infecções por Strongylida/complicações , Alérgenos/administração & dosagem , Alérgenos/farmacologia , Animais , Antígenos de Helmintos/imunologia , Asma/imunologia , Asma/parasitologia , Citocinas/biossíntese , Modelos Animais de Doenças , Eosinofilia/induzido quimicamente , Eosinofilia/imunologia , Imunoglobulina E/imunologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Sistema Respiratório/imunologia , Sistema Respiratório/patologia , Células Th2/imunologiaRESUMO
Animal models of Nippostrongylus brasiliensis and Heligmosomoides polygyrus infection are powerful tools for the investigation of the basic biology of immune responses and protective immunity. In particular, they model the induction and maintenance of Th2 type immune responses and exhibit all the requisite hallmarks of CD4 T cell-dependent IgE production, eosinophilia, mastocytosis, and mucus production. This chapter describes simple, cost-effective techniques for using and maintaining these easy-to-work-with parasites in the context of a modern laboratory.