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1.
PLoS Negl Trop Dis ; 13(8): e0007691, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31469835

RESUMO

Lung disease is regularly reported in human filarial infections but the molecular pathogenesis of pulmonary filariasis is poorly understood. We used Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity responsible for pleural inflammation, to model responses to human filarial infections and probe the mechanisms. Wild-type and Th2-deficient mice (ΔdblGata1 and Il-4receptor(r)a-/-/IL-5-/-) were infected with L. sigmodontis. Survival and growth of adult filariae and prevalence and density of microfilariae were evaluated. Cells and cytokines in the pleural cavity and bronchoalveolar space were characterized by imaging, flow cytometry and ELISA. Inflammatory pathways were evaluated by transcriptomic microarrays and lungs were isolated and analyzed for histopathological signatures. 40% of WT mice were amicrofilaremic whereas almost all mutant mice display blood microfilaremia. Microfilariae induced pleural, bronchoalveolar and lung-tissue inflammation associated with an increase in bronchoalveolar eosinophils and perivascular macrophages, production of mucus, visceral pleura alterations and fibrosis. Inflammation and pathology were decreased in Th2-deficient mice. An IL-4R-dependent increase of CD169 was observed on pleural and bronchoalveolar macrophages in microfilaremic mice. CD169+ tissue-resident macrophages were identified in the lungs with specific localizations. Strikingly, CD169+ macrophages increased significantly in the perivascular area in microfilaremic mice. These data describe lung inflammation and pathology in chronic filariasis and emphasize the role of Th2 responses according to the presence of microfilariae. It is also the first report implicating CD169+ lung macrophages in response to a Nematode infection.

2.
Sci Transl Med ; 11(491)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068442

RESUMO

Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets Wolbachia, a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of Wolbachia in the in vivo Litomosoides sigmodontis filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify Wolbachia elimination in Brugia pahangi filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in L. sigmodontis, Brugia malayi, and Onchocerca ochengi in vivo preclinical models of filarial disease and in vitro selectivity against Loa loa (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.

3.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30953430

RESUMO

The Wolbachia endosymbionts encompass a large group of intracellular bacteria of biomedical and veterinary relevance, closely related to Anaplasma, Ehrlichia, and Rickettsia. This genus of Gram-negative members of the Alphaproteobacteria does not infect vertebrates but is instead restricted to ecdysozoan species, including terrestrial arthropods and a family of parasitic filarial nematodes, the Onchocercidae. The Wolbachia profoundly impact not only the ecology and evolution but also the reproductive biology of their hosts, through a wide range of symbiotic interactions. Because they are essential to the survival and reproduction of their filarial nematode hosts, they represent an attractive target to fight filariasis. Their abilities to spread through insect populations and to affect vector competence through pathogen protection have made Wolbachia a staple for controlling vector-borne diseases. Estimated to be present in up to 66% of insect species, the Wolbachia are probably the most abundant endosymbionts on earth. Their success resides in their unique capacity to infect and manipulate the host germ line to favor their vertical transmission through the maternal lineage. Because the Wolbachia resist genetic manipulation and growth in axenic culture, our understanding of their biology is still in its infancy. Despite these limitations, the "-omics" revolution combined with the use of well-established and emerging experimental host models is accelerating our comprehension of the host phenotypes caused by Wolbachia, and the identification of Wolbachia effectors is ongoing.


Assuntos
Interações entre Hospedeiro e Microrganismos/fisiologia , Wolbachia/fisiologia , Animais , Artrópodes/microbiologia , Filarioidea/microbiologia , Insetos/microbiologia , Simbiose , Wolbachia/classificação , Wolbachia/genética , Wolbachia/patogenicidade
4.
PLoS Negl Trop Dis ; 13(3): e0007218, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30893296

RESUMO

The reproductive parasites Wolbachia are the most common endosymbionts on earth, present in a plethora of arthropod species. They have been introduced into mosquitos to successfully prevent the spread of vector-borne diseases, yet the strategies of host cell subversion underlying their obligate intracellular lifestyle remain to be explored in depth in order to gain insights into the mechanisms of pathogen-blocking. Like some other intracellular bacteria, Wolbachia reside in a host-derived vacuole in order to replicate and escape the immune surveillance. Using here the pathogen-blocking Wolbachia strain from Drosophila melanogaster, introduced into two different Drosophila cell lines, we show that Wolbachia subvert the endoplasmic reticulum to acquire their vacuolar membrane and colonize the host cell at high density. Wolbachia redistribute the endoplasmic reticulum, and time lapse experiments reveal tight coupled dynamics suggesting important signalling events or nutrient uptake. Wolbachia infection however does not affect the tubular or cisternal morphologies. A fraction of endoplasmic reticulum becomes clustered, allowing the endosymbionts to reside in between the endoplasmic reticulum and the Golgi apparatus, possibly modulating the traffic between these two organelles. Gene expression analyses and immunostaining studies suggest that Wolbachia achieve persistent infections at very high titers without triggering endoplasmic reticulum stress or enhanced ERAD-driven proteolysis, suggesting that amino acid salvage is achieved through modulation of other signalling pathways.


Assuntos
Drosophila melanogaster/microbiologia , Retículo Endoplasmático/microbiologia , Membranas Intracelulares/microbiologia , Estresse Fisiológico/fisiologia , Simbiose/fisiologia , Wolbachia/fisiologia , Animais , Linhagem Celular , Drosophila melanogaster/citologia , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/microbiologia , Interações Hospedeiro-Patógeno , Membranas Intracelulares/metabolismo , Estresse Fisiológico/genética , Simbiose/genética , Vacúolos/microbiologia , Wolbachia/patogenicidade
5.
PLoS Pathog ; 14(10): e1007364, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30321239

RESUMO

Wolbachia are maternally inherited endosymbiotic bacteria, widespread among arthropods thanks to host reproductive manipulations that increase their prevalence into host populations. The most commonly observed manipulation is cytoplasmic incompatibility (CI). CI leads to embryonic death in crosses between i) infected males and uninfected females and ii) individuals infected with incompatible Wolbachia strains. CI can be conceptualized as a toxin-antidote system where a toxin deposited by Wolbachia in the sperm would induce embryonic death unless countered by an antidote produced by Wolbachia present in the eggs. In Drosophila melanogaster, transgenic expression of Wolbachia effector cidB revealed its function of CI-inducing toxin. Moreover in Culex pipiens, the diversity of cidB variants present in wPip strains accounts for the diversity in crossing-types. We conducted cytological analyses to determine the CI mechanisms that lead to embryonic death in C. pipiens, and assess whether diversity in crossing-types could be based on variations in these mechanisms. We revealed that paternal chromatin condensation and segregation defects during the first embryonic division are always responsible for embryonic death. The strongest observed defects lead to an exclusion of the paternal chromatin from the first zygotic division, resulting in haploid embryos unable to hatch. The proportion of unhatched haploid embryos, developing with only maternal chromatin, which reflects the frequency of strong defects can be considered as a proxy of CI intensity at the cellular level. We thus studied the putative effect of variations in crossing types and cidB diversification on CI defects intensity. Incompatible crosses involving distinct wPip strains revealed that CI defects intensity depends on the Wolbachia strains hosted by the males and is linked to the diversity of cidB genes harbored in their genomes. These results support that, additionally to its implication in C. pipiens crossing type variability, cidB diversification also influences the strength of CI embryonic defects.


Assuntos
Animais Geneticamente Modificados/microbiologia , Proteínas de Bactérias/metabolismo , Culex/microbiologia , Citoplasma/microbiologia , Drosophila melanogaster/microbiologia , Polimorfismo Genético , Wolbachia/fisiologia , Animais , Animais Geneticamente Modificados/genética , Proteínas de Bactérias/genética , Culex/genética , Citoplasma/patologia , Drosophila melanogaster/genética , Feminino , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/patologia , Especificidade de Hospedeiro , Masculino , Fenótipo , Simbiose
7.
Dev Cell ; 45(2): 198-211.e3, 2018 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-29689195

RESUMO

Although symbiotic interactions are ubiquitous in the living world, examples of developmental symbioses are still scarce. We show here the crucial role of Wolbachia in the oogenesis of filarial nematodes, a class of parasites of biomedical and veterinary relevance. We applied newly developed techniques to demonstrate the earliest requirements of Wolbachia in the parasite germline preceding the production of faulty embryos in Wolbachia-depleted nematodes. We show that Wolbachia stimulate germline proliferation in a cell-autonomous manner, and not through nucleotide supplementation as previously hypothesized. We also found Wolbachia to maintain the quiescence of a pool of germline stem cells to ensure a constant delivery of about 1,400 eggs per day for many years. The loss of quiescence upon Wolbachia depletion as well as the disorganization of the distal germline suggest that Wolbachia are required to execute the proper germline stem cell developmental program in order to produce viable eggs and embryos.


Assuntos
Brugia Malayi/crescimento & desenvolvimento , Filariose/patologia , Células Germinativas/citologia , Proteínas de Helminto/metabolismo , Células-Tronco/fisiologia , Simbiose , Wolbachia/fisiologia , Animais , Brugia Malayi/microbiologia , Proliferação de Células , Feminino , Filariose/metabolismo , Filariose/parasitologia , Células Germinativas/microbiologia , Células Germinativas/fisiologia , Proteínas de Helminto/genética , Masculino , Células-Tronco/citologia , Células-Tronco/microbiologia
8.
Development ; 143(1): 160-73, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26586219

RESUMO

C. elegans embryonic elongation is a morphogenetic event driven by actomyosin contractility and muscle-induced tension transmitted through hemidesmosomes. A role for the microtubule cytoskeleton has also been proposed, but its contribution remains poorly characterized. Here, we investigate the organization of the non-centrosomal microtubule arrays present in the epidermis and assess their function in elongation. We show that the microtubule regulators γ-tubulin and NOCA-1 are recruited to hemidesmosomes and adherens junctions early in elongation. Several parallel approaches suggest that microtubule nucleation occurs from these sites. Disrupting the epidermal microtubule array by overexpressing the microtubule-severing protein Spastin or by inhibiting the C. elegans ninein homolog NOCA-1 in the epidermis mildly affected elongation. However, microtubules were essential for elongation when hemidesmosomes or the activity of the Rho kinase LET-502/ROCK were partially compromised. Imaging of junctional components and genetic analyses suggest that epidermal microtubules function together with Rho kinase to promote the transport of E-cadherin to adherens junctions and myotactin to hemidesmosomes. Our results indicate that the role of LET-502 in junctional remodeling is likely to be independent of its established function as a myosin II activator, but requires a microtubule-dependent pathway involving the syntaxin SYX-5. Hence, we propose that non-centrosomal microtubules organized by epidermal junctions contribute to elongation by transporting junction remodeling factors, rather than having a mechanical role.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Células Epidérmicas , Microtúbulos/metabolismo , Quinases Associadas a rho/metabolismo , Actomiosina/metabolismo , Junções Aderentes/metabolismo , Animais , Caderinas/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas do Citoesqueleto , Citoesqueleto/metabolismo , Epiderme/metabolismo , Hemidesmossomos/metabolismo , Morfogênese/fisiologia , Proteínas Musculares/metabolismo , Miosina Tipo II/metabolismo , Proteínas Nucleares , Transporte Proteico/genética , Proteínas Qa-SNARE/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Tubulina (Proteína)/metabolismo
9.
G3 (Bethesda) ; 4(11): 2241-5, 2014 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-25236732

RESUMO

During spermiogenesis, histones are massively replaced with protamines. A previous report showed that Drosophila males homozygous for a genomic deletion covering several genes including the protamine-like genes Mst35Ba/b are surprisingly fertile. Here, we have precisely deleted the Mst35B locus by homologous recombination, and we confirm the dispensability of Mst35Ba/b for fertility.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Infertilidade Masculina/genética , Protaminas/genética , Espermatozoides/citologia , Sequência de Aminoácidos , Animais , Drosophila/fisiologia , Deleção de Genes , Masculino , Dados de Sequência Molecular
10.
PLoS Negl Trop Dis ; 8(8): e3096, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25165813

RESUMO

While bacterial symbionts influence a variety of host cellular responses throughout development, there are no documented instances in which symbionts influence early embryogenesis. Here we demonstrate that Wolbachia, an obligate endosymbiont of the parasitic filarial nematodes, is required for proper anterior-posterior polarity establishment in the filarial nematode B. malayi. Characterization of pre- and post-fertilization events in B. malayi reveals that, unlike C. elegans, the centrosomes are maternally derived and produce a cortical-based microtubule organizing center prior to fertilization. We establish that Wolbachia rely on these cortical microtubules and dynein to concentrate at the posterior cortex. Wolbachia also rely on PAR-1 and PAR-3 polarity cues for normal concentration at the posterior cortex. Finally, we demonstrate that Wolbachia depletion results in distinct anterior-posterior polarity defects. These results provide a striking example of endosymbiont-host co-evolution operating on the core initial developmental event of axis determination.


Assuntos
Evolução Biológica , Brugia Malayi/microbiologia , Polaridade Celular/fisiologia , Simbiose/fisiologia , Wolbachia/fisiologia , Animais , Brugia Malayi/genética , Brugia Malayi/fisiologia , Centrossomo/fisiologia , Dineínas/fisiologia , Centro Organizador dos Microtúbulos/fisiologia , Oócitos/fisiologia
11.
Parasit Vectors ; 7: 140, 2014 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-24685011

RESUMO

BACKGROUND: Wolbachia endosymbionts are a proven target for control of human disease caused by filarial nematodes. However, little is known about the occurrence of Wolbachia in taxa closely related to the superfamily Filarioidea. Our study addressed the status of Wolbachia presence in members of the superfamily Dracunculoidea by screening the human parasite Dracunculus medinensis and related species from wildlife for Wolbachia. FINDINGS: D. medinensis, D. lutrae and D. insignis specimens were all negative for Wolbachia colonization by PCR screening for the Wolbachia ftsZ, 16S rRNA and Wolbachia surface protein (wsp) sequences. The quality and purity of the DNA preparations was confirmed by amplification of nematode 18S rRNA and cytochrome c oxidase subunit I sequences. Furthermore, Wolbachia endobacteria were not detected by whole mount fluorescence staining, or by immunohistochemistry using a Wolbachia-specific antiserum. In contrast, positive control Brugia malayi worms were shown to harbour Wolbachia by PCR, fluorescence staining and immunohistochemistry. CONCLUSIONS: Three examined species of Dracunculus showed no evidence of Wolbachia endobacteria. This supports that members of the superfamily Dracunculoidea are free of Wolbachia. Within the order Spirurida, these endosymbionts appear restricted to the Filarioidea.


Assuntos
Dracunculíase/veterinária , Dracunculus/microbiologia , Wolbachia/isolamento & purificação , Animais , Animais Selvagens , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Dracunculíase/epidemiologia , Dracunculíase/parasitologia , Feminino , Gana/epidemiologia , Humanos , Vison/parasitologia , Ontário/epidemiologia , Lontras/parasitologia , Especificidade da Espécie
12.
Proc Natl Acad Sci U S A ; 110(19): 7748-53, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23610429

RESUMO

Lateral gene transfer events between bacteria and animals highlight an avenue for evolutionary genomic loss/gain of function. Herein, we report functional lateral gene transfer in animal parasitic nematodes. Members of the Nematoda are heme auxotrophs, lacking the ability to synthesize heme; however, the human filarial parasite Brugia malayi has acquired a bacterial gene encoding ferrochelatase (BmFeCH), the terminal step in heme biosynthesis. BmFeCH, encoded by a 9-exon gene, is a mitochondrial-targeted, functional ferrochelatase based on enzyme assays, complementation, and inhibitor studies. Homologs have been identified in several filariae and a nonfilarial nematode. RNAi and ex vivo inhibitor experiments indicate that BmFeCH is essential for viability, validating it as a potential target for filariasis control.


Assuntos
Brugia Malayi/enzimologia , Ferroquelatase/genética , Transferência Genética Horizontal , Animais , Animais Geneticamente Modificados , Teorema de Bayes , Brugia Malayi/genética , Caenorhabditis elegans/genética , Clonagem Molecular , Escherichia coli/metabolismo , Éxons , Feminino , Teste de Complementação Genética , Genoma , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Masculino , Microscopia Confocal , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Filogenia , Interferência de RNA
13.
Biol Open ; 1(6): 536-47, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23213446

RESUMO

Parasitic filarial nematodes that belong to the Onchocercidae family live in mutualism with Wolbachia endosymbionts. We developed whole-mount techniques to follow the segregation patterns of Wolbachia through the somatic and germline lineages of four filarial species. These studies reveal multiple evolutionarily conserved mechanisms that are required for Wolbachia localization to the germline. During the initial embryonic divisions, Wolbachia segregate asymmetrically such that they concentrate in the posteriorly localized P(2) blastomere, a precursor to the adult germline and hypodermal lineages. Surprisingly, in the next division they are excluded from the germline precursor lineage. Rather, they preferentially segregate to the C blastomere, a source of posterior hypodermal cells. Localization to the germline is accomplished by a distinct mechanism in which Wolbachia invade first the somatic gonadal cells close to the ovarian distal tip cell, the nematode stem cell niche, from the hypodermis. This tropism is associated with a cortical F-actin disruption, suggesting an active engulfment. Significantly, germline invasion occurs only in females, explaining the lack of Wolbachia in the male germline. Once in the syncytial environment of the ovaries, Wolbachia rely on the rachis to multiply and disperse into the germ cells. The utilization of cell-to-cell invasion for germline colonization may indicate an ancestral mode of horizontal transfer that preceded the acquisition of the mutualism.

14.
Int J Parasitol ; 42(11): 1025-36, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23041355

RESUMO

Wolbachia are vertically transmitted endosymbiotic bacteria of arthropods and onchocercid nematodes. It is commonly accepted that they co-evolved with their filarial hosts, and have secondarily been lost in some species. However, most of the data on the Wolbachia/Onchocercidae relationship have been derived from studies on two subfamilies, the Dirofilariinae and the Onchocercinae, which harbour parasites of humans and domestic animals. Within the last few years, analyses of more diverse material have suggested that some groups of Onchocercidae do not have Wolbachia, such as recently studied Splendidofilariinae from birds. This study takes advantage of the analysis of additional Splendidofilariinae, Rumenfilaria andersoni from a Finnish reindeer and Madathamugadia hiepei from a South African gecko, using PCR, immunohistochemical staining and whole-mount fluorescent analysis to detect Wolbachia and describe its strains. A DNA barcoding approach and phylogenetic analyses were used to investigate the symbiosis between Wolbachia and the Onchocercidae. A new supergroup F Wolbachia was demonstrated in M. hiepei, representing the first filarial nematode harbouring Wolbachia described in a non-mammalian host. In the adult, Wolbachia infects the female germline but not the hypodermis, and intestinal cells are also infected. The phylogenetic analyses confirmed a recent emergence of supergroup F. They also suggested several events of horizontal transmission between nematodes and arthropods in this supergroup, and the existence of different metabolic interactions between the filarial nematodes and their symbionts.


Assuntos
Nematoides/microbiologia , Wolbachia/classificação , Wolbachia/genética , Animais , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Variação Genética , Dados de Sequência Molecular , Filogenia , RNA Bacteriano/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo
15.
PLoS Pathog ; 8(9): e1002922, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23028321

RESUMO

Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts.


Assuntos
Albendazol/análogos & derivados , Brugia Malayi/microbiologia , Drosophila melanogaster/microbiologia , Filariose/tratamento farmacológico , Wolbachia/efeitos dos fármacos , Albendazol/farmacologia , Animais , Brugia Malayi/efeitos dos fármacos , Linhagem Celular , Drosophila melanogaster/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microtúbulos/efeitos dos fármacos , Simbiose
16.
Parasit Vectors ; 5: 16, 2012 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-22243803

RESUMO

BACKGROUND: RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans. RESULTS: We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week. CONCLUSIONS: We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.


Assuntos
Brugia Malayi/genética , Filariose/parasitologia , Imunofluorescência , Interferência de RNA , Animais , Brugia Malayi/embriologia , Brugia Malayi/ultraestrutura , Caenorhabditis elegans/genética , Feminino , Genes de Helmintos/genética , Humanos , Fenótipo , RNA de Cadeia Dupla/genética , RNA de Helmintos/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
PLoS Pathog ; 7(11): e1002351, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22072969

RESUMO

Filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility and viability and thus has great promise as a novel approach for treating filarial diseases. However, little is known concerning the basis for this mutualistic relationship. Here we demonstrate using whole mount confocal microscopy that an immediate response to Wolbachia depletion is extensive apoptosis in the adult germline, and in the somatic cells of the embryos, microfilariae and fourth-stage larvae (L4). Surprisingly, apoptosis occurs in the majority of embryonic cells that had not been infected prior to antibiotic treatment. In addition, no apoptosis occurs in the hypodermal chords, which are populated with large numbers of Wolbachia, although disruption of the hypodermal cytoskeleton occurs following their depletion. Thus, the induction of apoptosis upon Wolbachia depletion is non-cell autonomous and suggests the involvement of factors originating from Wolbachia in the hypodermal chords. The pattern of apoptosis correlates closely with the nematode tissues and processes initially perturbed following depletion of Wolbachia, embryogenesis and long-term sterilization, which are sustained for several months until the premature death of the adult worms. Our observations provide a cellular mechanism to account for the sustained reductions in microfilarial loads and interruption of transmission that occurs prior to macrofilaricidal activity following antibiotic therapy of filarial nematodes.


Assuntos
Antibacterianos/farmacologia , Apoptose , Brugia Malayi/microbiologia , Filariose/parasitologia , Wolbachia/efeitos dos fármacos , Wolbachia/fisiologia , Animais , Antibacterianos/uso terapêutico , Brugia Malayi/citologia , Brugia Malayi/efeitos dos fármacos , Brugia Malayi/metabolismo , Caspase 3/biossíntese , Doxiciclina/farmacologia , Feminino , Filariose/tratamento farmacológico , Gerbillinae/parasitologia , Proteínas de Helminto/biossíntese , Humanos , Marcação In Situ das Extremidades Cortadas , Larva/microbiologia , Masculino , Simbiose , Tetraciclina/farmacologia
18.
PLoS One ; 6(6): e20843, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21731626

RESUMO

BACKGROUND: Wolbachia are intriguing symbiotic endobacteria with a peculiar host range that includes arthropods and a single nematode family, the Onchocercidae encompassing agents of filariases. This raises the question of the origin of infection in filariae. Wolbachia infect the female germline and the hypodermis. Some evidences lead to the theory that Wolbachia act as mutualist and coevolved with filariae from one infection event: their removal sterilizes female filariae; all the specimens of a positive species are infected; Wolbachia are vertically inherited; a few species lost the symbiont. However, most data on Wolbachia and filaria relationships derive from studies on few species of Onchocercinae and Dirofilariinae, from mammals. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the Wolbachia distribution testing 35 filarial species, including 28 species and 7 genera and/or subgenera newly screened, using PCR, immunohistochemical staining, whole mount fluorescent analysis, and cocladogenesis analysis. (i) Among the newly screened Onchocercinae from mammals eight species harbour Wolbachia but for some of them, bacteria are absent in the hypodermis, or in variable density. (ii) Wolbachia are not detected in the pathological model Monanema martini and in 8, upon 9, species of Cercopithifilaria. (iii) Supergroup F Wolbachia is identified in two newly screened Mansonella species and in Cercopithifilaria japonica. (iv) Type F Wolbachia infect the intestinal cells and somatic female genital tract. (v) Among Oswaldofilariinae, Waltonellinae and Splendidofilariinae, from saurian, anuran and bird respectively, Wolbachia are not detected. CONCLUSIONS/SIGNIFICANCE: The absence of Wolbachia in 63% of onchocercids, notably in the ancestral Oswaldofilariinae estimated 140 mya old, the diverse tissues or specimens distribution, and a recent lateral transfer in supergroup F Wolbachia, modify the current view on the role and evolution of the endosymbiont and their hosts. Further genomic analyses on some of the newly sampled species are welcomed to decipher the open questions.


Assuntos
Evolução Biológica , Filarioidea/isolamento & purificação , Filarioidea/microbiologia , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Wolbachia/fisiologia , Animais , Sequência de Bases , DNA Ribossômico/genética , Feminino , Filarioidea/citologia , Fluorescência , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Filogenia , Reação em Cadeia da Polimerase , Propídio/metabolismo , Especificidade da Espécie , Wolbachia/citologia , Wolbachia/genética
19.
Nature ; 471(7336): 99-103, 2011 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-21368832

RESUMO

Mechanotransduction refers to the transformation of physical forces into chemical signals. It generally involves stretch-sensitive channels or conformational change of cytoskeleton-associated proteins. Mechanotransduction is crucial for the physiology of several organs and for cell migration. The extent to which mechanical inputs contribute to development, and how they do this, remains poorly defined. Here we show that a mechanotransduction pathway operates between the body-wall muscles of Caenorhabditis elegans and the epidermis. This pathway involves, in addition to a Rac GTPase, three signalling proteins found at the hemidesmosome: p21-activated kinase (PAK-1), the adaptor GIT-1 and its partner PIX-1. The phosphorylation of intermediate filaments is one output of this pathway. Tension exerted by adjacent muscles or externally exerted mechanical pressure maintains GIT-1 at hemidesmosomes and stimulates PAK-1 activity through PIX-1 and Rac. This pathway promotes the maturation of a hemidesmosome into a junction that can resist mechanical stress and contributes to coordinating the morphogenesis of epidermal and muscle tissues. Our findings suggest that the C. elegans hemidesmosome is not only an attachment structure, but also a mechanosensor that responds to tension by triggering signalling processes. We suggest that similar pathways could promote epithelial morphogenesis or wound healing in other organisms in which epithelial cells adhere to tension-generating contractile cells.


Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Epiderme/embriologia , Mecanotransdução Celular/fisiologia , Morfogênese , Contração Muscular/fisiologia , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Células Epidérmicas , Hemidesmossomos/metabolismo , Filamentos Intermediários/metabolismo , Músculos/embriologia , Músculos/fisiologia , Fenótipo , Fosforilação , Transdução de Sinais , Quinases Ativadas por p21/metabolismo
20.
PLoS Negl Trop Dis ; 4(7): e758, 2010 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-20689574

RESUMO

Wolbachia are required for filarial nematode survival and fertility and contribute to the immune responses associated with human filarial diseases. Here we developed whole-mount immunofluorescence techniques to characterize Wolbachia somatic and germline transmission patterns and tissue distribution in Brugia malayi, a nematode responsible for lymphatic filariasis. In the initial embryonic divisions, Wolbachia segregate asymmetrically such that they occupy only a small subset of cells in the developing embryo, facilitating their concentration in the adult hypodermal chords and female germline. Wolbachia are not found in male reproductive tissues and the absence of Wolbachia from embryonic germline precursors in half of the embryos indicates Wolbachia loss from the male germline may occur in early embryogenesis. Wolbachia rely on fusion of hypodermal cells to populate adult chords. Finally, we detect Wolbachia in the secretory canal lumen suggesting living worms may release bacteria and/or their products into their host.


Assuntos
Brugia Malayi/crescimento & desenvolvimento , Brugia Malayi/microbiologia , Wolbachia/isolamento & purificação , Estruturas Animais/microbiologia , Animais , Feminino , Imunofluorescência , Masculino , Coloração e Rotulagem/métodos
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