RESUMO
Bacillus cereus (Bc) is a wide group of Gram-positive and spore-forming bacteria, known to be the etiological agents of various human infections, primarily food poisoning. The Bc group includes enteropathogenic strains able to germinate in the digestive tract and to produce enterotoxins such as Nhe, Hbl, and CytK. One species of the group, Bacillus thuringiensis (Bt), has the unique feature of producing insecticidal crystals during sporulation, making it an important alternative to chemical pesticides to protect crops from insect pest larvae. Nevertheless, several studies have suggested a link between the ingestion of pesticide strains and human cases of food poisoning, calling their safety into question. Consequently, reliable tools for virulence assessment are worth developing to aid decision making in pesticide regulation. Here, we propose complementary approaches based on two biological models, the human intestinal Caco-2 cell line and the insect Drosophila melanogaster, to assess and rank the enteric virulence potency of Bt strains in comparison with other Bc group members. Using a dataset of 48 Bacillus spp. strains, we showed that some Bc group strains, including Bt, were able to induce cytotoxicity in Caco-2 cells with concomitant release of IL-8 cytokine, a landmark of pro-inflammatory response. In the D. melanogaster model, we were able to sort a panel of 39 strains into four different classes of virulence, ranging from no virulence to strong virulence. Importantly, for the most virulent strains, mortality was associated with a loss of intestinal barrier integrity. Interestingly, although strains can share a common toxinotype, they display different degrees of virulence, suggesting the existence of specific mechanisms of virulence expression in vivo in the intestine.
RESUMO
Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. Here we show that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. We show that Cry1A toxins weaken the E-Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, although not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and endocrine functions.
Assuntos
Toxinas de Bacillus thuringiensis , Drosophila melanogaster , Células-Tronco , Animais , Bacillus thuringiensis , Toxinas de Bacillus thuringiensis/efeitos adversos , Adesão Celular , Produtos Agrícolas , Plantas Geneticamente Modificadas , Células-Tronco/efeitos dos fármacosRESUMO
Bacillus thuringiensis (Bt), belonging to the Bacillus cereus (Bc) group, is commonly used as a biopesticide worldwide due to its ability to produce insecticidal crystals during sporulation. The use of Bt, especially subspecies aizawai and kurstaki, to control pests such as Lepidoptera, generally involves spraying mixtures containing spores and crystals on crops intended for human consumption. Recent studies have suggested that the consumption of commercial Bt strains may be responsible for foodborne outbreaks (FBOs). However, its genetic proximity to Bc strains has hindered the development of routine tests to discriminate Bt from other Bc, especially Bacillus cereus sensu stricto (Bc ss), well known for its involvement in FBOs. Here, to develop tools for the detection and the discrimination of Bt in food, we carried out a genome-wide association study (GWAS) on 286 complete genomes of Bc group strains to identify and validate in silico new molecular markers specific to different Bt subtypes. The analyses led to the determination and the in silico validation of 128 molecular markers specific to Bt, its subspecies aizawai, kurstaki and four previously described proximity clusters associated with these subspecies. We developed a command line tool based on a 14-marker workflow, to carry out a computational search for Bt-related markers from a putative Bc genome, thereby facilitating the detection of Bt of interest for food safety, especially in the context of FBOs.
RESUMO
We report here the complete genome sequences of three Bacillus cereus group strains isolated from blood cultures from premature and immunocompromised infants hospitalized in intensive care units in three French hospitals. These complete genome sequences were obtained from a combination of Illumina HiSeq X Ten short reads and Oxford Nanopore MinION long reads.
RESUMO
Bioinsecticides made from the bacterium Bacillus thuringiensis (Bt) are the bestselling bioinsecticide worldwide. Among Bt bioinsecticides, those based on the strain Bt subsp. kurstaki (Btk) are widely used in farming to specifically control pest lepidopteran larvae. Although there is much evidence of the lack of acute lethality of Btk products for non-target animals, only scarce data are available on their potential non-lethal developmental adverse effects. Using a concentration that could be reached in the field upon sprayings, we show that Btk products impair growth and developmental time of the non-target dipteran Drosophila melanogaster. We demonstrate that these effects are mediated by the synergy between Btk bacteria and Btk insecticidal toxins. We further show that Btk bioinsecticides trigger intestinal cell death and alter protein digestion without modifying the food intake and feeding behavior of the larvae. Interestingly, these harmful effects can be mitigated by a protein-rich diet or by adding the probiotic bacterium Lactobacillus plantarum into the food. Finally, we unravel two new cellular mechanisms allowing the larval midgut to maintain its integrity upon Btk aggression: First the flattening of surviving enterocytes and second, the generation of new immature cells arising from the adult midgut precursor cells. Together, these mechanisms participate to quickly fill in the holes left by the dying enterocytes.
RESUMO
Bioinsecticides based on Bacillus thuringiensis (Bt) spores and toxins are increasingly popular alternative solutions to control insect pests, with potential impact of their accumulation in the environment on non-target organisms. Here, we tested the effects of chronic exposure to commercial Bt formulations (Bt var. kurstaki and israelensis) on eight non-target Drosophila species present in Bt-treated areas, including D. melanogaster (four strains). Doses up to those recommended for field application (~ 106 Colony Forming Unit (CFU)/g fly medium) did not impact fly development, while no fly emerged at ≥ 1000-fold this dose. Doses between 10- to 100-fold the recommended one increased developmental time and decreased adult emergence rates in a dose-dependent manner, with species-and strain-specific effect amplitudes. Focusing on D. melanogaster, development alterations were due to instar-dependent larval mortality, and the longevity and offspring number of adult flies exposed to bioinsecticide throughout their development were moderately influenced. Our data also suggest a synergy between the formulation compounds (spores, cleaved toxins, additives) might induce the bioinsecticide effects on larval development. Although recommended doses had no impact on non-target Drosophila species, misuse or local environmental accumulation of Bt bioinsecticides could have side-effects on fly populations with potential implications for their associated communities.
Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Drosophila/efeitos dos fármacos , Controle Biológico de Vetores , Animais , Drosophila melanogaster/efeitos dos fármacos , Feminino , Larva , Masculino , Controle Biológico de Vetores/métodosRESUMO
Bacillus thuringiensis (Bt) produces pore forming toxins that have been used for pest control in agriculture for many years. However, their molecular and cellular mode of action is still unclear. While a first model - referred to as the pore forming model - is the most widely accepted scenario, a second model proposed that toxins could trigger an Mg2+-dependent intracellular signalling pathway leading to cell death. Although Cry1Ca has been shown to form ionic pores in the plasma membrane leading to cell swelling and death, we investigated the existence of other cellular or molecular events involved in Cry1Ca toxicity. The Sf9 insect cell line, derived from Spodoptera frugiperda, is highly and specifically sensitive to Cry1Ca. Through a selection program we developed various levels of laboratory-evolved Cry1Ca-resistant Sf9 cell lines. Using a specific S. frugiperda microarray we performed a comparative transcriptomic analysis between sensitive and resistant cells and revealed genes differentially expressed in resistant cells and related to cation-dependent signalling pathways. Ion chelators protected sensitive cells from Cry1Ca toxicity suggesting the necessity of both Ca2+ and/or Mg2+ for toxin action. Selected cells were highly resistant to Cry1Ca while toxin binding onto their plasma membrane was not affected. This suggested a resistance mechanism different from the classical 'loss of toxin binding'. We observed a correlation between Cry1Ca cytotoxicity and the increase of intracellular cAMP levels. Indeed, Sf9 sensitive cells produced high levels of cAMP upon toxin stimulation, while Sf9 resistant cells were unable to increase their intracellular cAMP. Together, these results provide new information about the mechanism of Cry1Ca toxicity and clues to potential resistance factors yet to discover.
RESUMO
The intestine is endowed with an innate immune system that is required to fight any exogenous bacteria that are swallowed along with the food. The first line of defense that is mounted by the gut epithelium is the release of immune Reactive Oxygen Species (ROS), such as hypochlorous acid (HOCl), into the lumen. HOCl is produced within 1.5 h of bacterial ingestion and is very labile once released. Therefore, to monitor HOCl production upon ingestion of allochthonous bacteria, one needs a detection system that can quickly and efficiently detect HOCl production in the intestine. While most of the ROS-sensitive probes available in the market detect all kinds of ROS without any distinction, the R19-S fluorescent probe has been developed to specifically detect HOCl. Here, we describe a protocol to monitor HOCl production using this probe in the gut lumen of adult Drosophila upon ingestion of the opportunistic bacteria Bacillus thuringiensis.
RESUMO
The digestive tract is the first organ affected by the ingestion of foodborne bacteria. While commensal bacteria become resident, opportunistic or virulent bacteria are eliminated from the gut by the local innate immune system. Here we characterize a new mechanism of defense, independent of the immune system, in Drosophila melanogaster. We observed strong contractions of longitudinal visceral muscle fibers for the first 2 hours following bacterial ingestion. We showed that these visceral muscle contractions are induced by immune reactive oxygen species (ROS) that accumulate in the lumen and depend on the ROS-sensing TRPA1 receptor. We then demonstrate that both ROS and TRPA1 are required in a subset of anterior enteroendocrine cells for the release of the DH31 neuropeptide which activates its receptor in the neighboring visceral muscles. The resulting contractions of the visceral muscles favors quick expulsion of the bacteria, limiting their presence in the gut. Our results unveil a precocious mechanism of defense against ingested opportunistic bacteria, whether they are Gram-positive like Bacillus thuringiensis or Gram-negative like Erwinia carotovora carotovora. Finally, we found that the human homolog of DH31, CGRP, has a conserved function in Drosophila.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Proteínas de Drosophila/fisiologia , Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/fisiologia , Hormônios de Inseto/fisiologia , Animais , Animais Geneticamente Modificados , Bacillus thuringiensis/patogenicidade , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Drosophila melanogaster/fisiologia , Feminino , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/fisiopatologia , Humanos , Imunidade Inata , Canais Iônicos , Lactobacillus plantarum/patogenicidade , Contração Muscular/fisiologia , Infecções Oportunistas/microbiologia , Infecções Oportunistas/fisiopatologia , Infecções Oportunistas/prevenção & controle , Pectobacterium carotovorum/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Canal de Cátion TRPA1/fisiologiaRESUMO
Using pathogens or high levels of opportunistic bacteria to damage the gut, studies in Drosophila have identified many signaling pathways involved in gut regeneration. Dying cells emit signaling molecules that accelerate intestinal stem cell proliferation and progenitor differentiation to replace the dying cells quickly. This process has been named 'regenerative cell death'. Here, mimicking environmental conditions, we show that the ingestion of low levels of opportunistic bacteria was sufficient to launch an accelerated cellular renewal program despite the brief passage of bacteria in the gut and the absence of cell death and this is is due to the moderate induction of the JNK pathway that stimulates stem cell proliferation. Consequently, the addition of new differentiated cells to the gut epithelium, without preceding cell loss, leads to enterocyte overcrowding. Finally, we show that a couple of days later, the correct density of enterocytes is promptly restored by means of a wave of apoptosis involving Hippo signaling and preferential removal of old enterocytes.
Assuntos
Apoptose , Drosophila melanogaster/crescimento & desenvolvimento , Enterócitos/citologia , Intestinos/crescimento & desenvolvimento , Animais , Morte Celular , Diferenciação Celular/fisiologia , Proliferação de Células , Citocinas/metabolismo , Proteínas de Drosophila/metabolismo , Endoderma/citologia , Epitélio/crescimento & desenvolvimento , Feminino , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Regeneração , Transdução de Sinais , Células-Tronco/citologiaRESUMO
The intestine is a central organ required for the digestion of food, the absorption of nutrients and for fighting against aggressors ingested along with the food. Impairment of gut physiology following mucosal damages impacts its digestive capacities that consequently will affect growth, wellbeing or even survival of the individual. Hence, the assessment of intestinal functions encompasses, among others, the monitoring of its integrity, its cellular renewing, its immune defenses, the production of enteroendocrine hormones and its digestive capacities. Here, we describe in detail how to assess the activity of the proteases secreted in the intestinal lumen of adult Drosophila melanogaster flies. This method can also be used for larval intestines. The present protocol is adapted and improved from the Sigma-Aldrich's protocol proposed in the 'Protease Fluorescent Detection Kit' (Product code PF0100).
RESUMO
Most of the detrimental effects of using conventional insecticides to control crop pests are now well identified and are nowadays major arguments for replacing such compounds by the use of biological control agents. In this respect, the bacterium Bacillus thuringiensis var. kurstaki and Trichogramma (Hymenoptera: Trichogrammatidae) parasitic wasp species are both effective against lepidopterous pests and can actually be used concomitantly. In this work, we studied the potential side effects of B. thuringiensis var. kurstaki on Trichogramma chilonis females. We first evidenced an acute toxicity of B. thuringiensis on T. chilonis. Then, after ingestion of B. thuringiensis at sublethal doses, we focused on life history traits of T. chilonis such as longevity, reproductive success and the time spent on host eggs patches. The reproductive success of T. chilonis was not modified by B. thuringiensis while a significant effect was observed on longevity and the time spent on host eggs patches. The physiological and ecological meanings of the results obtained are discussed.
Assuntos
Bacillus thuringiensis/fisiologia , Interações Hospedeiro-Patógeno , Himenópteros/parasitologia , Controle Biológico de Vetores/métodos , Vespas/microbiologia , Animais , Feminino , Himenópteros/microbiologia , Longevidade/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Vespas/crescimento & desenvolvimentoRESUMO
The digestive tract is subjected to many aggressions throughout animal life. Since disruptions of gut physiology impact on animal fitness and survival, maintenance of gut integrity and functionality is essential for the individual. Over the last 40 years, research on rodents has aimed at understanding how cellular homeostasis of the digestive tract is maintained when challenged with disruptions. Following the discovery of stem cells in the digestive tract of Drosophila, a flurry of studies made an important contribution to our understanding of how the proliferation and the differentiation of these cells are controlled and participate in the renewal of the digestive tract. Insights into these mechanisms in Drosophila have revealed many similarities with mammalian intestinal stem cells. For instance, the highly conserved EGFR, JAK/STAT, Wingless/Wnt, Hedgehog, Integrins, BMP/TGFß, Hippo and Insulin pathways all participate in adult intestinal cellular homeostasis. Here, we provide a literature review of recent advances in the field highlighting the adult Drosophila midgut as a convenient model for dissecting mechanisms involved in the maintenance of the cellular homeostasis of the digestive tract in conventionally reared conditions. In addition, we shed light on recently published data putting Drosophila forward as a genetic tool to decipher the mechanisms underlying intestinal diseases and intestinal tumour progression.
Assuntos
Fenômenos Fisiológicos Celulares/fisiologia , Drosophila/fisiologia , Trato Gastrointestinal/citologia , Trato Gastrointestinal/fisiologia , Homeostase/fisiologia , Animais , Enteropatias/patologia , Modelos Biológicos , Células-TroncoRESUMO
During development, secreted morphogens, such as Hedgehog (Hh), control cell fate and proliferation. Precise sensing of morphogen levels and dynamic cellular responses are required for morphogen-directed morphogenesis, yet the molecular mechanisms responsible are poorly understood. Several recent studies have suggested the involvement of a multi-protein Hh reception complex, and have hinted at an understated complexity in Hh sensing at the cell surface. We show here that the expression of the proteoglycan Dally in Hh-receiving cells in Drosophila is necessary for high but not low level pathway activity, independent of its requirement in Hh-producing cells. We demonstrate that Dally is necessary to sequester Hh at the cell surface and to promote Hh internalisation with its receptor. This internalisation depends on both the activity of the hydrolase Notum and the glycosyl-phosphatidyl-inositol (GPI) moiety of Dally, and indicates a departure from the role of the second glypican Dally-like in Hh signalling. Our data suggest that hydrolysis of the Dally-GPI by Notum provides a switch from low to high level signalling by promoting internalisation of the Hh-Patched ligand-receptor complex.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Proteínas Hedgehog/metabolismo , Glicoproteínas de Membrana/metabolismo , Morfogênese/fisiologia , Proteoglicanas/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Western Blotting , Células Cultivadas , Processamento de Imagem Assistida por Computador , Microscopia de FluorescênciaRESUMO
The graded Hedgehog (Hh) signal is transduced by the transmembrane Smoothened (Smo) proteins in both vertebrates and invertebrates. In Drosophila, associations between Smo and the Fused (Fu)/Costal-2 (Cos2)/Cubitus Interruptus (Ci) cytoplasmic complex lead to pathway activation, but it remains unclear how the cytoplasmic complex responds to and transduces different levels of Hh signaling. We show here that, within the Hh gradient field, low- and high-magnitude Smo activations control differentially the phosphorylation of Cos2 on two distinct serines. We also provide evidence that these phosphorylations depend on the Fu kinase activity and lead to a shift of Cos2 distribution from the cytoplasm to the plasma membrane. Moreover, the distinct Cos2 phosphorylation states mediate differential Hh signaling magnitude, suggesting that phosphorylation and relocation of Cos2 to the plasma membrane facilitate high-level Hh signaling through the control of Ci nuclear translocation and transcriptional activity.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Proteínas Hedgehog/metabolismo , Cinesinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Quinase 2 de Receptor Acoplado a Proteína G/genética , Proteínas Hedgehog/genética , Imunoprecipitação , Cinesinas/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Receptor Smoothened , Ativação TranscricionalRESUMO
A major challenge of developmental biology is to understand how cells coordinate developmental behaviors with their neighbors. To achieve this, cells often employ signaling molecules that emanate from a local source and act at a distance on target cells. The Hedgehog morphogen is an essential signaling molecule required for numerous processes during animal development. Emphasizing the importance of this molecule for both growth control and patterning, Hedgehog signaling activity is often deregulated during cancer formation and progression. The secretion and spread of Hedgehog are not passive processes, but require accessory molecules involved in Hedgehog processing, release, spread and reception. In this review, I focus on the factors that are required to control the spread and activity of Hedgehog, highlighting recent data that have shed light on these processes.
Assuntos
Proteínas Hedgehog/metabolismo , Animais , Humanos , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Transdução de SinaisRESUMO
Cell fate determination during developmental patterning is often controlled by concentration gradients of morphogens. In the epithelial field, morphogens like the Hedgehog (Hh) peptides diffuse both apically and basolaterally; however, whether both pools of Hh are sensed at the cellular level is unclear. Here, we show that interfering with the amount of apical Hh causes a dramatic change in the long-range activation of low-threshold Hh target genes, without similar effect on short-range, high-threshold targets. We provide genetic evidence that the glypican Dally upregulates apical Hh levels, and that the release of Dally by the hydrolase Notum promotes apical Hh long-range activity. Our data suggest that several pools of Hh are perceived in epithelial tissues. Thus, we propose that the overall gradient of Hh is a composite of pools secreted by different routes (apical and basolateral), and that a cellular summation of these components is required for appropriate developmental patterning.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Alelos , Animais , Padronização Corporal , Linhagem da Célula , Cruzamentos Genéticos , Drosophila melanogaster , Endocitose , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Modelos Biológicos , Transdução de SinaisRESUMO
Hedgehog (Hh) signalling plays a crucial role in the development and patterning of many tissues in both vertebrates and invertebrates. Aberrations in this pathway lead to severe developmental defects and cancer. Hh signal transduction in receiving cells is a well studied phenomenon; however questions still remain concerning the mechanism of repression of the pathway activator Smoothened (Smo) in the absence of Hh. Here we describe a novel repressor of the Hh pathway, Target of Wingless (Tow). Tow represents the Drosophila homolog of a conserved uncharacterised protein family. We show that Tow acts in Hh receiving cells, where its overexpression represses all levels of Hh signalling, and that this repression occurs upstream or at the level of Smo and downstream of the Hh receptor Patched (Ptc). In addition, we find that like Ptc, overexpression of Tow causes an accumulation of lipophorin in the wing disc. We demonstrate that loss of tow enhances different ptc alleles in a similar manner to another pathway repressor, Suppressor of Fused (SuFu), possibly through mediating Ptc dependant lipophorin internalisation. Combined, these results demonstrate that Tow is an important novel regulator of the Hh pathway in the wing imaginal disc, and may shed light on the mechanism of Ptc repression of Smo.
Assuntos
Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/fisiologia , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Nucleares/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Primers do DNA , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas Hedgehog/genética , Hibridização In Situ , Dados de Sequência Molecular , Proteínas Nucleares/química , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Receptor SmoothenedRESUMO
Hedgehog (Hh) and Wingless (Wg) morphogens specify cell fate in a concentration-dependent manner in the Drosophila wing imaginal disc. Proteoglycans, components of the extracellular matrix, are involved in Hh and Wg stability, spreading, and reception. In this study, we demonstrate that the glycosyl-phosphatidyl-inositol (GPI) anchor of the glypican Dally-like (Dlp) is required for its apical internalization and its subsequent targeting to the basolateral compartment of the epithelium. Dlp endocytosis from the apical surface of Hh-receiving cells catalyzes the internalization of Hh bound to its receptor Patched (Ptc). The cointernalization of Dlp with the Hh/Ptc complex is dynamin dependent and necessary for full-strength Hh signaling. We also demonstrate that Wg is secreted apically in the disc epithelium and that apicobasal trafficking of Dlp allows Wg transcytosis to favor Wg spreading along the basolateral compartment. Thus, Dlp endocytosis is a common regulatory mechanism of both Hh and Wg morphogen action.