RESUMEN
Behavior evolution can promote the emergence of agricultural pests by changing their ecological niche. For example, the insect pest Drosophila suzukii has shifted its oviposition (egg-laying) niche from fermented fruits to ripe, non-fermented fruits, causing significant damage to a wide range of fruit crops worldwide. We investigate the chemosensory changes underlying this evolutionary shift and ask whether fruit sugars, which are depleted during fermentation, are important gustatory cues that direct D. suzukii oviposition to sweet, ripe fruits. We show that D. suzukii has expanded its range of oviposition responses to lower sugar concentrations than the model D. melanogaster, which prefers to lay eggs on fermented fruit. The increased response of D. suzukii to sugar correlates with an increase in the value of sugar relative to a fermented strawberry substrate in oviposition decisions. In addition, we show by genetic manipulation of sugar-gustatory receptor neurons (GRNs) that sugar perception is required for D. suzukii to prefer a ripe substrate over a fermented substrate, but not for D. melanogaster to prefer the fermented substrate. Thus, sugar is a major determinant of D. suzukii's choice of complex substrates. Calcium imaging experiments in the brain's primary gustatory center (suboesophageal zone) show that D. suzukii GRNs are not more sensitive to sugar than their D. melanogaster counterparts, suggesting that increased sugar valuation is encoded in downstream circuits of the central nervous system (CNS). Taken together, our data suggest that evolutionary changes in central brain sugar valuation computations are involved in driving D. suzukii's oviposition preference for sweet, ripe fruit.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Femenino , Drosophila/fisiología , Drosophila melanogaster/fisiología , Oviposición , Frutas , Proteínas de Drosophila/genética , AzúcaresRESUMEN
iASPP is a protein mostly known as an inhibitor of p53 pro-apoptotic activity and a predicted regulatory subunit of the PP1 phosphatase, which is often overexpressed in tumors. We report that iASPP associates with the microtubule plus-end binding protein EB1, a central regulator of microtubule dynamics, via an SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the cortex of mitotic cells, leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of the membrane-to-cortex linker Myosin-Ic (Myo1c), which we identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up upon mitosis because of defective cortical stiffness. We propose that by increasing cortical rigidity, iASPP helps cancer cells maintain a spherical geometry suitable for proper mitotic spindle positioning and chromosome partitioning.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitosis , Proteínas Represoras/metabolismo , Huso Acromático/metabolismo , Secuencias de Aminoácidos , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Miosina Tipo I/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Unión Proteica , Proteínas Represoras/químicaRESUMEN
In vitro- and in vivo-polarised absorptive epithelia (enterocytes) are considered to be non-phagocytic towards bacteria with invasive pathogenic strains relying on virulence factors to 'force' entry. Here, we report a serendipitous discovery that questions these beliefs. Thus, we uncover in well-established models of human small (Caco-2; TC-7) and large (T84) intestinal enterocytes a polarization-dependent mechanism that can transfer millions of bacteria from the basal to apical compartment. Antibiotic-protection assays, confocal imaging and drug inhibitor data are consistent with a transcellular route in which internalized, basolateral-membrane enclosed bacteria are trafficked to and across the apical surface. Basal-to-apical transport of non-pathogenic bacteria (and inert beads) challenged the idea of pathogens relying on virulence factors to force entry. Indeed, studies with Salmonella demonstrated that it's entry-forcing virulence factor (SPI-I) was not required to enter via the basolateral surface but to promote another virulence-associated event (intra-enterocyte accumulation).
Asunto(s)
Traslocación Bacteriana , Enterocitos/microbiología , Transporte Biológico , Células CACO-2 , Línea Celular , Polaridad Celular/fisiología , Enterocitos/efectos de los fármacos , Escherichia coli Enteropatógena/patogenicidad , Escherichia coli Enteropatógena/fisiología , Células HeLa , Humanos , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/fisiología , Factores de Virulencia/farmacologíaRESUMEN
Enteropathogenic E. coli (EPEC) is a human pathogen that targets the small intestine, causing severe and often fatal diarrhoea in infants. A defining feature of EPEC disease is the loss (effacement) of absorptive microvilli (MV) from the surface of small intestinal enterocytes. Much of our understanding of EPEC pathogenesis is derived from studies using cell lines such as Caco-2 - the most extensively used small intestinal model. However, previous work has revealed fundamental differences between Caco-2 cells and in vivo differentiated enterocytes in relation to MV effacement. This, and the high heterogeneity and low transfection efficiency of the Caco-2 cell line prompted the isolation of several sub-clones (NCL-1-12) to identify a more tractable and improved in vivo-like cell model. Along with established Caco-2 clones (TC-7, BBE1), sub-clones were assessed for growth rate, apical surface morphology, epithelial barrier function and transfection efficiency. TC-7 cells provided the best all-round clone and exhibited highest levels of ectopic gene expression following cell polarisation. Novel alterations in EGFP-labelled mitochondria, that were not previously documented in non-polarised cell types, highlighted the potential of the TC-7 model for defining dynamic enterocyte-specific changes during infection. Crucially, the TC-7 cell line also mimicked ex vivo derived enterocytes with regard to MV effacement, enabling a better dissection of the process. Effacement activity caused by the EPEC protein Map in the Caco-2 but not ex vivo model, was linked to a defect in suppressing its Cdc42-dependent functionality. MV effacement activity of the EPEC protein EspF in the TC-7 model was dependent on its N-WASP binding motif, which is also shown to play an essential role in epithelial barrier dysfunction. Together, this study highlights the many advantages of using TC-7 cells as a small intestinal model to study host-pathogen interactions.
Asunto(s)
Enterocitos/microbiología , Escherichia coli Enteropatógena/patogenicidad , Intestino Delgado/citología , Modelos Animales , Análisis de Varianza , Células CACO-2 , Impedancia Eléctrica , Enterocitos/patología , Escherichia coli Enteropatógena/ultraestructura , Interacciones Huésped-Patógeno , Humanos , Immunoblotting , Microscopía Confocal , Microscopía Electrónica de RastreoRESUMEN
The nucleolus is a multifunctional structure within the nucleus of eukaryotic cells and is the primary site of ribosome biogenesis. Almost all viruses target and disrupt the nucleolus--a feature exclusive to this pathogen group. Here, using a combination of bio-imaging, genetic and biochemical analyses, we demonstrate that the enteropathogenic E. coli (EPEC) effector protein EspF specifically targets the nucleolus and disrupts a subset of nucleolar factors. Driven by a defined N-terminal nucleolar targeting domain, EspF causes the complete loss from the nucleolus of nucleolin, the most abundant nucleolar protein. We also show that other bacterial species disrupt the nucleolus, dependent on their ability to deliver effector proteins into the host cell. Moreover, we uncover a novel regulatory mechanism whereby nucleolar targeting by EspF is strictly controlled by EPEC's manipulation of host mitochondria. Collectively, this work reveals that the nucleolus may be a common feature of bacterial pathogenesis and demonstrates that a bacterial pathogen has evolved a highly sophisticated mechanism to enable spatio-temporal control over its virulence proteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Nucléolo Celular/fisiología , Escherichia coli Enteropatógena/patogenicidad , Infecciones por Escherichia coli/patología , Proteínas de Escherichia coli/metabolismo , Mitocondrias/microbiología , Mitocondrias/patología , Secuencia de Aminoácidos , Western Blotting , Proteínas Portadoras/genética , ADN Bacteriano/genética , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/metabolismo , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , NucleolinaRESUMEN
Bacterial pathogens deliver multiple effector proteins into eukaryotic cells to subvert host cellular processes and an emerging theme is the cooperation between different effectors. Here, we reveal that a fine balance exists between effectors that are delivered by enteropathogenic E. coli (EPEC) which, if perturbed can have marked consequences on the outcome of the infection. We show that absence of the EPEC effector Tir confers onto the bacterium a potent ability to destroy polarized intestinal epithelia through extensive host cell detachment. This process was dependent on the EPEC effectors EspG and EspG2 through their activation of the host cysteine protease calpain. EspG and EspG2 are shown to activate calpain during EPEC infection, which increases significantly in the absence of Tir - leading to rapid host cell loss and necrosis. These findings reveal a new function for EspG and EspG2 and show that Tir, independent of its bacterial ligand Intimin, is essential for maintaining the integrity of the epithelium during EPEC infection by keeping the destructive activity of EspG and EspG2 in check.
Asunto(s)
Calpaína/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Infecciones por Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Receptores de Superficie Celular/fisiología , Células CACO-2 , Infecciones por Escherichia coli/microbiología , Interacciones Huésped-Patógeno , Humanos , VirulenciaRESUMEN
Delivery of effector molecules into LMme(v) macrophages by enteropathogenic Escherichia coli, via its type three secretion system (T3SS), inhibits bacterial uptake by a phosphatidylinositol-3 (PI-3) kinase-dependent pathway. The T3SS system, encoded by the locus of enterocyte effacement (LEE) pathogenicity island, delivers LEE- and non-LEE-encoded effector proteins into host cells. Previous studies discounted essential roles for the LEE-encoded Map, EspF, Tir or Intimin proteins in this process but correlated it with loss of phosphorylation of the PI-3 kinase substrate, Akt (Celli et al., 2001, EMBO J 20: 1245-1258). Given the more recent finding that these bacterial proteins are multifunctional and can act together to subvert host cellular processes, we generated a quadruple deletion mutant (Map, Tir, EspF and Intimin deficient) to unearth any cooperativity in inhibiting uptake. The quadruple mutant was as defective as the T3SS-defective strain at preventing bacterial uptake with further studies revealing a surprising dependence on EspF but not Map, Tir or Intimin. Subversive activities previously associated with EspF are disruption of epithelial barrier function and programmed cell death, with the latter linked to EspF targeting mitochondria. Interestingly, the C-terminal domain possesses a polyproline motif associated with protein-protein interactions. We demonstrate that EspF-mediated inhibition of PI-3 kinase-dependent uptake: (i) is independent of mitochondrial targeting, (ii) requires the N-terminal domain with and (iii) the C-terminal domain is sufficient to disrupt barrier function but not inhibition of bacterial uptake. Moreover, loss of PI-3 kinase-dependent phosphorylation of Akt and gross changes in host phosphotyrosine protein profiles could not be linked to inhibition of the PI-3 kinase-dependent uptake process.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/fisiología , Escherichia coli/patogenicidad , Mitocondrias/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Células CACO-2 , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , ADN Bacteriano/análisis , ADN Bacteriano/genética , Escherichia coli/química , Escherichia coli/enzimología , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Islas Genómicas/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Macrófagos/enzimología , Macrófagos/microbiología , Macrófagos/fisiología , Ratones , Mitocondrias/fisiología , Mutación/genética , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación , Fosfotirosina/fisiología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiologíaRESUMEN
In vivo studies with the mouse-specific member of the attaching and effacing (A/E) family of pathogens raised the possibility that these non-invasive enteric pathogens can specifically inhibit inducible nitric oxide synthase (iNOS) expression to prevent the production of antimicrobial nitric oxide (NO). In this study we use polarized Caco-2 cells, a model of human small intestinal epithelia, to (i) demonstrate conclusively that an A/E member, human specific enteropathogenic Escherichia coli (EPEC), can inhibit cytokine-induced iNOS expression, (ii) show that this activity is dependent on the delivery of effector molecules into host cells and (iii) investigate the mechanism of inhibition. Analysis of the level of iNOS-related mRNA, protein and NO production demonstrated that EPEC can inhibit iNOS expression at the transcriptional, by direct and indirect mechanisms, and post-transcriptional levels. This transcriptional block was linked to the failure of the iNOS-related transcriptional factor NF-kappaB, but not STAT1, to undergo phosphorylation-associated activation. A selective pressure to prevent iNOS production was evidenced by the finding that iNOS activity had a potent antimicrobial effect on adherent but not non-adherent bacteria. Moreover, given the central role NF-kappaB plays in transcribing genes associated with early host immune responses, this inhibitory mechanism presumably represents an important role in pathogenesis. Our study also provides insights into the nature of NO production in response to bacterial infection as well as the role of the locus of enterocyte effacement (LEE)-encoded effector molecules in inhibiting iNOS expression.