RESUMO
Staphylococcus aureus is a pathogenic bacterium responsible for a broad spectrum of infections, including cutaneous, respiratory, osteoarticular, and systemic infections. It poses a significant clinical challenge due to its ability to develop antibiotic resistance. This resistance limits therapeutic options, increases the risk of severe complications, and underscores the urgent need for new strategies to address this threat, including the investigation of treatments complementary to antibiotics. The evaluation of novel antimicrobial agents often employs animal models, with the zebrafish embryo model being particularly interesting for studying host-pathogen interactions, establishing itself as a crucial tool in this field. For the first time, this study presents a zebrafish embryo model for the in vivo assessment of bacteriophage efficacy against S. aureus infection. A localized infection was induced by microinjecting either methicillin-resistant S. aureus (MRSA) or methicillin-susceptible S. aureus (MSSA). Subsequent treatments involved administering either bacteriophage, vancomycin (the reference antibiotic for MRSA), or a combination of both via the same route to explore potential synergistic effects. Our findings indicate that the bacteriophage was as effective as vancomycin in enhancing survival rates, whether used alone or in combination. Moreover, bacteriophage treatment appears to be even more effective in reducing the bacterial load in S. aureus-infected embryos post-treatment than the antibiotic. Our study validates the use of the zebrafish embryo model and highlights its potential as a valuable tool in assessing bacteriophage efficacy treatments in vivo.
Assuntos
Antibacterianos , Staphylococcus aureus Resistente à Meticilina , Terapia por Fagos , Infecções Estafilocócicas , Vancomicina , Peixe-Zebra , Animais , Peixe-Zebra/microbiologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/virologia , Terapia por Fagos/métodos , Vancomicina/farmacologia , Vancomicina/uso terapêutico , Infecções Estafilocócicas/terapia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Modelos Animais de Doenças , Embrião não Mamífero/microbiologia , Testes de Sensibilidade MicrobianaRESUMO
Host-associated microbial communities, like other ecological communities, may be impacted by the colonization order of taxa through priority effects. Developing embryos and their associated microbiomes are subject to stochasticity during colonization by bacteria. For amphibian embryos, often developing externally in bacteria-rich environments, this stochasticity may be particularly impactful. For example, the amphibian microbiome can mitigate lethal outcomes from disease for their hosts; however, this may depend on microbiome composition. Here, we examined the assembly of the bacterial community in spring peeper (Pseudacris crucifer) embryos and tadpoles. First, we reared embryos from identified mating pairs in either lab or field environments to examine the relative impact of environment and parentage on embryo and tadpole bacterial communities. Second, we experimentally inoculated embryos to determine if priority effects (i) could be used to increase the relative abundance of Janthinobacterium lividum, an amphibian-associated bacteria capable of preventing fungal infection, and (ii) would lead to observed differences in the relative abundances of two closely related bacteria from the genus Pseudomonas. Using 16S rRNA gene amplicon sequencing, we observed differences in community composition based on rearing location and parentage in embryos and tadpoles. In the inoculation experiment, we found that priority inoculation could increase the relative abundance of J. lividum, but did not find that either Pseudomonas isolate was able to prevent colonization by the other when given priority. These results highlight the importance of environmental source pools and parentage in determining microbiome composition, while also providing novel methods for the administration of a known amphibian probiotic. IMPORTANCE: Harnessing the functions of host-associated bacteria is a promising mechanism for managing disease outcomes across different host species. In the case of amphibians, certain frog-associated bacteria can mitigate lethal outcomes of infection by the fungal pathogen Batrachochytrium dendrobatidis. Successful probiotic applications require knowledge of community assembly and an understanding of the ecological mechanisms that structure these symbiotic bacterial communities. In our study, we show the importance of environment and parentage in determining bacterial community composition and that community composition can be influenced by priority effects. Further, we provide support for the use of bacterial priority effects as a mechanism to increase the relative abundance of target probiotic taxa in a developing host. While our results show that priority effects are not universally effective across all host-associated bacteria, our ability to increase the relative abundance of specific probiotic taxa may enhance conservation strategies that rely on captive rearing of endangered vertebrates.
Assuntos
Anuros , Larva , Microbiota , Probióticos , RNA Ribossômico 16S , Animais , Larva/microbiologia , Larva/crescimento & desenvolvimento , Anuros/microbiologia , Probióticos/administração & dosagem , Probióticos/farmacologia , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/genética , Oxalobacteraceae/fisiologia , Pseudomonas/fisiologia , Embrião não Mamífero/microbiologiaRESUMO
Animals interact with microbes that affect their performance and fitness, including endosymbionts that reside inside their cells. Maternally transmitted Wolbachia bacteria are the most common known endosymbionts, in large part because of their manipulation of host reproduction. For example, many Wolbachia cause cytoplasmic incompatibility (CI) that reduces host embryonic viability when Wolbachia-modified sperm fertilize uninfected eggs. Operons termed cifs control CI, and a single factor (cifA) rescues it, providing Wolbachia-infected females a fitness advantage. Despite CI's prevalence in nature, theory indicates that natural selection does not act to maintain CI, which varies widely in strength. Here, we investigate the genetic and functional basis of CI-strength variation observed among sister Wolbachia that infect Drosophila melanogaster subgroup hosts. We cloned, Sanger sequenced, and expressed cif repertoires from weak CI-causing wYak in Drosophila yakuba, revealing mutations suspected to weaken CI relative to model wMel in D. melanogaster A single valine-to-leucine mutation within the deubiquitylating (DUB) domain of the wYak cifB homolog (cidB) ablates a CI-like phenotype in yeast. The same mutation reduces both DUB efficiency in vitro and transgenic CI strength in the fly, each by about twofold. Our results map hypomorphic transgenic CI to reduced DUB activity and indicate that deubiquitylation is central to CI induction in cid systems. We also characterize effects of other genetic variation distinguishing wMel-like cifs Importantly, CI strength determines Wolbachia prevalence in natural systems and directly influences the efficacy of Wolbachia biocontrol strategies in transinfected mosquito systems. These approaches rely on strong CI to reduce human disease.
Assuntos
Citoplasma/patologia , Drosophila melanogaster/microbiologia , Embrião não Mamífero/microbiologia , Mutação , Simbiose , Ubiquitinação , Wolbachia/fisiologia , Animais , Citoplasma/microbiologia , Enzimas Desubiquitinantes/metabolismo , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Feminino , MasculinoRESUMO
IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ+ B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ+ B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class.
Assuntos
Resistência à Doença/imunologia , Doenças dos Peixes/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Isotipos de Imunoglobulinas/imunologia , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/imunologia , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/fisiologia , Animais , Resistência à Doença/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/imunologia , Embrião não Mamífero/microbiologia , Feminino , Doenças dos Peixes/microbiologia , Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Isotipos de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/metabolismo , Masculino , Herança Materna/genética , Herança Materna/imunologia , Vibrio/classificação , Vibrio/imunologia , Vibrio/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Zigoto/imunologia , Zigoto/metabolismo , Zigoto/microbiologiaRESUMO
To discover new compounds with broad spectrum and high activity, we designed a series of novel benzamides containing 1,2,4-oxadiazole moiety by bioisosterism, and 28 benzamides derivatives with antifungal activity were synthesized. These compounds were evaluated against four fungi: Botrytis cinereal, FusaHum graminearum, Marssonina mali, and Thanatephorus cucumeris. The results indicated that most of the compounds displayed good fungicidal activities, especially against Botrytis cinereal. For example, 10a (84.4%), 10d (83.6%), 10e (83.3%), 10f (83.1%), 10i (83.3%), and 10l (83.6%) were better than pyraclostrobin (81.4%) at 100 mg/L. In addition, the acute toxicity of 10f to zebrafish embryo was 20.58 mg/L, which was classified as a low-toxicity compound.
Assuntos
Antifúngicos/farmacologia , Benzamidas/farmacologia , Oxidiazóis/farmacologia , Peixe-Zebra/microbiologia , Animais , Ascomicetos/efeitos dos fármacos , Basidiomycota/efeitos dos fármacos , Benzamidas/síntese química , Benzamidas/química , Benzamidas/toxicidade , Botrytis/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/microbiologia , Fusarium/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Oxidiazóis/síntese química , Oxidiazóis/química , Oxidiazóis/toxicidade , Peixe-Zebra/embriologiaRESUMO
The development of powerful model systems has been a critical strategy for understanding the mechanisms underlying the progression of an animal through its ontogeny. Here we provide two examples that allow deep and mechanistic insight into the development of specific animal systems. Species of the cnidarian genus Hydra have provided excellent models for studying host-microbe interactions and how metaorganisms function in vivo. Studies of the Hawaiian bobtail squid Euprymna scolopes and its luminous bacterial partner Vibrio fischeri have been used for over 30 years to understand the impact of a broad array of levels, from ecology to genomics, on the development and persistence of symbiosis. These examples provide an integrated perspective of how developmental processes work and evolve within the context of a microbial world, a new view that opens vast horizons for developmental biology research. The Hydra and the squid systems also lend an example of how profound insights can be discovered by taking advantage of the "experiments" that evolution had done in shaping conserved developmental processes.
Assuntos
Decapodiformes/embriologia , Decapodiformes/microbiologia , Hydra/microbiologia , Microbiota , Aliivibrio fischeri , Animais , Decapodiformes/fisiologia , Embrião não Mamífero/microbiologia , Regulação da Expressão Gênica , Hydra/metabolismo , Luz , Simbiose , Via de Sinalização WntRESUMO
14-3-3 proteins are widespread in animals, but their functions and mechanisms remain poorly defined. Here we clearly demonstrate that 14-3-3 ß/α-A is a newly identified PGN-binding protein present abundantly in the eggs/embryos of zebrafish. We also show that recombinant 14-3-3 ß/α-A acts as a pattern recognition receptor capable of identifying the bacterial signature molecule PGN, binding the bacteria, and functions as an antibacterial effector molecule directly killing the bacteria. Importantly, microinjection of r14-3-3 ß/α-A into early embryos significantly enhanced the resistance of the embryos against pathogenic A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-14-3-3 ß/α-A antibody. Collectively, these results indicate that 14-3-3 ß/α-A is a maternal PGN-binding protein that can protect the early embryos of zebrafish against pathogenic attacks, a novel role assigned to 14-3-3 ß/α-A proteins. This work also provides new insights into 14-3-3 proteins that are widely distributed in various animals.
Assuntos
Proteínas 14-3-3/metabolismo , Infecções Bacterianas/veterinária , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Peptidoglicano/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologia , Proteínas 14-3-3/genética , Animais , Anticorpos Bloqueadores/metabolismo , Infecções Bacterianas/microbiologia , Resistência à Doença , Embrião não Mamífero/imunologia , Embrião não Mamífero/microbiologia , Imunidade Materno-Adquirida , Ligação Proteica , Receptores de Reconhecimento de Padrão/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Food-associated outbreaks linked to enteropathogenic Yersinia enterocolitica are of concern to public health. Pigs and their meat are recognized risk factors for transmission of Y. enterocolitica. This study aimed to describe the comparative genomics of Y. enterocolitica along with a number of misclassified Yersinia isolates, now constituting the recently described Yersinia hibernica. The latter was originally cultured from an environmental sample taken at a pig slaughterhouse. Unique features were identified in the genome of Y. hibernica, including a novel integrative conjugative element (ICE), denoted as ICEYh-1 contained within a 255 kbp region of plasticity. In addition, a zebrafish embryo infection model was adapted and applied to assess the virulence potential among Yersinia isolates including Y. hibernica.
Assuntos
Embrião não Mamífero/microbiologia , Genômica/métodos , Yersiniose/diagnóstico , Yersinia enterocolitica/classificação , Yersinia/classificação , Animais , Conjugação Genética , Diagnóstico Diferencial , Modelos Animais de Doenças , Microbiologia de Alimentos , Filogenia , Suínos , Fatores de Virulência/genética , Yersinia/genética , Yersinia/isolamento & purificação , Yersinia/patogenicidade , Yersinia enterocolitica/genética , Yersinia enterocolitica/isolamento & purificação , Yersinia enterocolitica/patogenicidade , Peixe-ZebraRESUMO
Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.
Assuntos
Fibrose Cística/microbiologia , Fibrose Cística/terapia , Embrião não Mamífero/microbiologia , Terapia por Fagos , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/microbiologia , Animais , Antibacterianos/farmacologia , Bacteriófagos/fisiologia , Citocinas/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Mediadores da Inflamação/metabolismo , Microinjeções , Morfolinos/farmacologia , Terapia por Fagos/efeitos adversos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Reprodutibilidade dos TestesRESUMO
Nosema bombycis is an obligate intracellular pathogen that can be transmitted vertically from infected females to eggs, resulting in congenital infections in embryos. Here we investigated the proliferation characteristics of N. bombycis in silkworm embryos using a histopathological approach and deep RNA sequencing. We found that N. bombycis proliferated mainly around yolk granules at the early stage of the embryonic development, 1-2 days post oviposition (dpo). At 4-6 dpo, a portion of N. bombycis in different stages adjacent to the embryo were packaged into the newly formed intestinal lumen, while the remaining parasites continued to proliferate around yolk granules. In the newly hatched larvae (9 dpo), the newly formed spores accumulated in the gut lumen and immediately were released into the environment via the faeces. Transcriptional profiling of N. bombycis further confirmed multiplication of N. bombycis throughout every stage of embryonic development. Additionally, the increased transcriptional level of spore wall proteins and polar tube proteins from 4 dpo indicated an active formation of mature spores. Taken together, our results have provided a characterization of the proliferation of this intracellular microsporidian pathogen in congenitally infected embryos leading to vertical transmission.
Assuntos
Bombyx/microbiologia , Interações Hospedeiro-Patógeno , Nosema/fisiologia , Animais , Bombyx/embriologia , Bombyx/crescimento & desenvolvimento , Embrião não Mamífero/microbiologia , Larva/crescimento & desenvolvimento , Larva/microbiologia , RNA-SeqRESUMO
Coral-associated bacteria are critical for the well-being of their host and may play essential roles during ontogeny, as suggested by the vertical transmission of some bacteria in brooding corals. Bacterial acquisition patterns in broadcast spawners remain uncertain, as 16S rRNA gene metabarcoding of coral early life stages suggests the presence of bacterial communities, which have not been detected by microscopic examinations. Here, we combined 16S rRNA gene metabarcoding with fluorescence in situ hybridization (FISH) microscopy to analyze bacterial assemblages in Acropora tenuis egg-sperm bundles, embryos, and larvae following a spawning event. Metabarcoding results indicated that A. tenuis offspring ≤ 4-day-old were associated with diverse and dynamic bacterial microbiomes, dominated by Rhodobacteraceae, Alteromonadaceae, and Oceanospirillaceae. While FISH analyses confirmed the lack of internalized bacteria in A. tenuis offspring, metabarcoding showed that even the earliest life stages examined (egg-sperm bundles and two-cell stages) were associated with a diverse bacterial community, suggesting the bacteria were confined to the mucus layer. These results can be explained by vertical transmission of certain taxa (mainly Endozoicomonas) in the mucus surrounding the gametes within bundles, or by horizontal bacterial transmission through the release of bacteria by spawning adults into the water column.
Assuntos
Antozoários/microbiologia , Fenômenos Fisiológicos Bacterianos , Estágios do Ciclo de Vida , Microbiota/fisiologia , Animais , Antozoários/crescimento & desenvolvimento , Embrião não Mamífero/microbiologia , Hibridização in Situ Fluorescente , Larva/microbiologia , Masculino , Óvulo/microbiologia , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Reprodução , Espermatozoides/microbiologiaRESUMO
The endosymbiont Wolbachia is known for manipulating host reproduction in selfish ways. However, the molecular mechanisms have not yet been investigated in embryos. Here, we found that Wolbachia had no effect on the number of deposited eggs in Tetranychus urticae Koch (Acari: Tetranychidae) but caused two types of reproductive manipulation: killing uninfected female embryos via cytoplasmic incompatibility (CI) and increasing the hatching ratio of infected female embryos. RNA sequencing analyses showed that 145 genes were differentially expressed between Wolbachia-infected (WI) and Wolbachia-uninfected (WU) embryos. Wolbachia infection down-regulated messenger RNA (mRNA) expression of glutathione S-transferase that could buffer oxidative stress. In addition, 1613 and 294 genes were identified as CI-specific up-/down-regulated genes. Compared to WU and WI embryos, embryos of CI cross strongly expressed genes involved in transcription, translation, tissue morphogenesis, DNA damage and mRNA surveillance. In contrast, most of the genes associated with energy production and metabolism were down-regulated in the CI embryos compared to the WU and WI embryos, which provides some clues as to the cause of death of CI embryos. These results identify several genes that could be candidates for explaining Wolbachia-induced CI. Our data form a basis to help elucidate the molecular consequences of CI in embryos.
Assuntos
Tetranychidae/fisiologia , Transcriptoma , Wolbachia/fisiologia , Animais , Citoplasma , Embrião não Mamífero/microbiologia , Embrião não Mamífero/fisiologia , Feminino , Masculino , Reprodução , Tetranychidae/embriologia , Tetranychidae/crescimento & desenvolvimento , Tetranychidae/microbiologiaRESUMO
Herein, positively surface-charged silver nanoparticles (AgNPs) capped with trimethylchitosan nitrate (TMCN) were synthesized using an environmentally friendly method. Nano-sized TMCN-AgNPs (~80 nm) with high zeta potential (>30 mV) provide sufficient static repulsion to stabilize colloid AgNPs in aqueous solutions without aggregation for >3 months. In in vitro cell cycle assays, TMCN-AgNPs showed low cytotoxicity towards L929 cells. A microdilution inhibition assay demonstrated the antifungal potential of TMCN-AgNPs, with a minimum inhibitory concentration of 0.06 mM against Candida tropicalis ATCC 750, and 0.46 mM against both Candida albicans ATCC 76615 and Candida glabrata ATCC 15545. Moreover, the addition of TMCN-AgNPs at 0.23 mM significantly reduced biofilm formation in 96-well plates with C. albicans and C. tropicalis. Importantly, when zebrafish eggs were infected with Candida cells, 0.23 mM TMCN-AgNPs greatly diminished the amount of biofilm on eggs and rescued the survival of embryos by up to 70%.
Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Quitosana/farmacologia , Embrião não Mamífero/microbiologia , Nanopartículas Metálicas/química , Prata/farmacologia , Peixe-Zebra/microbiologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Embrião não Mamífero/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Camundongos , Testes de Sensibilidade Microbiana , Óvulo/efeitos dos fármacos , Peixe-Zebra/embriologiaRESUMO
Previous studies have shown that ATP synthase α subunit (ATP5A1) plays multiple roles, but our understanding of its biologic functions remains poor and incomprehensive. Here, we clearly demonstrated that zebrafish ATP5A1 was a newly characterized lipoteichoic acid (LTA)- and LPS-binding protein abundantly stored in the eggs and embryos of zebrafish. Zebrafish ATP5A1 acted not only as a pattern recognition receptor, capable of identifying LTA and LPS, but also as an effector molecule, capable of inhibiting the growth of both gram-positive and -negative bacteria. ATP5A1 could disrupt the bacterial membranes by a combined action of membrane depolarization and permeabilization. We also found that the N-terminal 65 residues were critical for the antibacterial activity of zebrafish ATP5A1. In particular, we showed that microinjection of exogenous recombinant (r)ATP5A1 into early embryos could promote their resistance against pathogenic Aeromonas hydrophila challenge, and this pathogen-resistant activity was markedly reduced by the coinjection of anti-ATP5A1 antibody or by the knockdown with morpholino for atp5a1 but not by the coinjection of anti-actin antibody. Moreover, each egg/embryo contains a sufficient amount of ATP5A1 in vivo to kill A. hydrophila. Furthermore, the N-terminal 65 residues 1-65 of ATP5A1 α subunit (rA1-65) with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the N-terminal 69 residues 66-134 (rA66-134) or C-terminal residues 135-551 (rA135-551) of ATP5A1 α subunit without in vitro antibacterial activity did not. Finally, we showed that the antibacterial activity of the N-terminal 65 residues of ATP5A1 α subunit was conserved throughout animal evolution. Collectively, these results indicate that ATP5A1 is a novel maternal immunocompetent factor that can protect the early embryos of zebrafish from bacterial infection. This work also provides a new viewpoint for understanding the biologic roles of ATP5A1, which is ubiquitously present in animals.-Ni, S., Zhou, Y., Chen, Y., Du, X., Zhang, S. Identification of ATP synthase α subunit as a new maternal factor capable of protecting zebrafish embryos from bacterial infection.
Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/prevenção & controle , Embrião não Mamífero/microbiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Citoplasma/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , Homologia de Sequência de AminoácidosRESUMO
Maternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. In the present study, we investigated whether heritable endosymbionts that cause different reproductive phenotypes in Drosophila melanogaster influence the mRNA transcriptome of early embryos. We used mRNA-seq to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. We found no consistent evidence of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, we capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. We found small but statistically significant signals of depurination of Drosophila rRNA in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.
Assuntos
Drosophila melanogaster/embriologia , Drosophila melanogaster/microbiologia , Embrião não Mamífero/microbiologia , Simbiose , Transcriptoma/genética , Animais , Drosophila melanogaster/genética , Feminino , Genes de Insetos/genética , Interações Hospedeiro-Patógeno/genética , Masculino , Fenótipo , RNA Ribossômico , Reprodução/genética , Proteínas Inativadoras de Ribossomos/genética , Proteínas Inativadoras de Ribossomos/fisiologia , Análise de Sequência de RNA , Spiroplasma/enzimologia , WolbachiaRESUMO
Microbial symbioses exhibit astounding adaptations, yet all symbionts face the problem of how to reliably associate with host offspring every generation. A common strategy is vertical transmission, in which symbionts are directly transmitted from the female to her offspring. The diversity of symbionts and vertical transmission mechanisms is as expansive as the diversity of eukaryotic host taxa that house them. However, there are several common themes among these mechanisms based on the degree to which symbionts associate with the host germline during transmission. In this review, we detail three distinct vertical transmission strategies, starting with associations that are transmitted from host somatic cells to offspring somatic cells, either due to lacking a germline or avoiding it. A second strategy involves somatically-localized symbionts that migrate into the germline during host development. The third strategy we discuss is one in which the symbiont maintains continuous association with the germline throughout development. Unexpectedly, the vast majority of documented vertically inherited symbionts rely on the second strategy: soma-to-germline migration. Given that not all eukaryotes contain a sequestered germline and instead produce offspring from somatic stem cell lineages, this soma-to-germline migration is discussed in the context of multicellular evolution. Lastly, as recent genomics data have revealed an abundance of horizontal gene transfer events from symbiotic and non-symbiotic bacteria to host genomes, we discuss their impact on eukaryotic host evolution.
Assuntos
Células Germinativas/fisiologia , Simbiose , Animais , Movimento Celular , Embrião não Mamífero/microbiologia , Células-Tronco/citologiaRESUMO
An important area of modern biology consists of understanding the relationship between genotype and phenotype. However, to understand this relationship it is essential to investigate one of the principal links between them: the proteome. With the development of recent mass-spectrometry approaches, it is now possible to quantify entire proteomes and thus relate them to different phenotypes. Here, we present a comparison of the proteome of two extreme developmental states in the well-established model organism Drosophila melanogaster: adult and embryo. Protein modules such as ribosome, proteasome, tricarboxylic acid cycle, glycolysis, or oxidative phosphorylation were found differentially expressed between the two developmental stages. Analysis of post-translation modifications of the proteins identified in this study indicates that they generally follow the same trend as their corresponding protein. Comparison between changes in the proteome and the transcriptome highlighted patterns of post-transcriptional regulation for the subunits of protein complexes such as the ribosome and the proteasome, whereas protein from modules such as TCA cycle, glycolysis, and oxidative phosphorylation seem to be coregulated at the transcriptional level. Finally, the impact of the endosymbiont Wolbachia pipientis on the proteome of both developmental states was also investigated.
Assuntos
Drosophila melanogaster/genética , Biossíntese de Proteínas/genética , Proteoma/genética , Transcriptoma/genética , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Drosophila melanogaster/microbiologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/microbiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteólise , Proteoma/metabolismo , Proteômica/métodos , Wolbachia/patogenicidadeRESUMO
Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response.
Assuntos
Citocinas/metabolismo , Lectinas/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/microbiologia , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Códon sem Sentido/genética , Citocinas/genética , Dexametasona/farmacologia , Resistência à Doença/imunologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/microbiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Lectinas/genética , Depleção Linfocítica , Morfolinos/farmacologia , Mutação/genética , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/efeitos dos fármacos , Análise de Sobrevida , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Cystic fibrosis (CF) is a hereditary disease due to mutations in the CFTR gene and causes mortality in humans mainly due to respiratory infections caused by Pseudomonas aeruginosa. In a previous work we used phage therapy, which is a treatment with a mix of phages, to actively counteract acute P. aeruginosa infections in mice and Galleria mellonella larvae. In this work we apply phage therapy to the treatment of P. aeruginosa PAO1 infections in a CF zebrafish model. The structure of the CFTR channel is evolutionary conserved between fish and mammals and cftr-loss-of-function zebrafish embryos show a phenotype that recapitulates the human disease, in particular with destruction of the pancreas. We show that phage therapy is able to decrease lethality, bacterial burden, and the pro-inflammatory response caused by PAO1 infection. In addition, phage administration relieves the constitutive inflammatory state of CF embryos. To our knowledge, this is the first time that phage therapy is used to cure P. aeruginosa infections in a CF animal model. We also find that the curative effect against PAO1 infections is improved by combining phages and antibiotic treatments, opening a useful therapeutic approach that could reduce antibiotic doses and time of administration.
Assuntos
Fibrose Cística/complicações , Modelos Animais de Doenças , Embrião não Mamífero/imunologia , Infecções por Pseudomonas/terapia , Fagos de Pseudomonas/crescimento & desenvolvimento , Pseudomonas aeruginosa/virologia , Infecções Respiratórias/terapia , Animais , Antibacterianos/uso terapêutico , Embrião não Mamífero/microbiologia , Embrião não Mamífero/virologia , Camundongos , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/virologia , Fagos de Pseudomonas/isolamento & purificação , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Infecções Respiratórias/microbiologia , Peixe-ZebraRESUMO
In ovo injection of probiotics has been of interest for achieving early health benefits. However, there is limited research demonstrating where bacteria could migrate within the embryo after injection. The objective of this study was to evaluate bacterial colonization or migration after in ovo injection of broiler embryo with bioluminescent Escherichia coli. Injection using 106 CFU/mL nonpathogenic E. coli was applied to amniotic and air cell regions on day 18 of incubation. On days 18, 19, 20, and 21 the amnion, skin, lung, gastrointestinal tract (GIT), bursa, and spleen were collected. On day 21, the GIT was separated into crop, duodenum, jejunum, ileum, and ceca sections. All tissues were visualized using anin vivo imaging system to confirm the presence of bioluminescent E. coli. Samples were homogenized, 10-fold serially diluted, and spread onto appropriate agar to determine bacterial loads in all tissues. Results indicated that eggs injected into the amnion had significantly high numbers of E. coli cells in all tissues compared to air cell injected and control treatments 2 h post-injection (P < 0.0001). E. coli was also found on the lungs, spleen, and bursa of eggs injected either in the amnion or air cell (P < 0.05). Results indicated that in ovo injection into the amnion was more efficient than air cell injection, yielding a higher bacterial concentration in the evaluated tissues, specifically the ileum and ceca. Future research using bioluminescent probiotic bacteria may establish sites of preference for different probiotics leading to site-specific application that can maximize their overall impact when in ovo injected.