RESUMEN
Caenorhabditis elegans is one of the major model systems in biology based on advantageous properties such as short life span, transparency, genetic tractability and ease of culture using an Escherichia coli diet. In its natural habitat, compost and rotting plant material, this nematode lives on bacteria. However, C. elegans is a predator of bacteria, but can also be infected by nematopathogenic coryneform bacteria such Microbacterium and Leucobacter species, which display intriguing and diverse modes of pathogenicity. Depending on the nematode pathogen, aggregates of worms, termed worm-stars, can be formed, or severe rectal swelling, so-called Dar formation, can be induced. Using the human and animal pathogens Corynebacterium diphtheriae and Corynebacterium ulcerans as well as the non-pathogenic species Corynebacterium glutamicum, we show that these coryneform bacteria can also induce star formation slowly in worms, as well as a severe tail-swelling phenotype. While C. glutamicum had a significant, but minor influence on survival of C. elegans, nematodes were killed after infection with C. diphtheriae and C. ulcerans. The two pathogenic species were avoided by the nematodes and induced aversive learning in C. elegans.
Asunto(s)
Caenorhabditis elegans/microbiología , Caenorhabditis elegans/fisiología , Quimiotaxis , Corynebacterium/fisiología , Animales , Conducta Animal , Femenino , MasculinoRESUMEN
Corynebacterium diphtheriae is typically recognized as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of Cor. diphtheriae are poorly understood. In this study, we investigated the role of DIP0733 as virulence factor to elucidate how it contributes to the process of pathogen-host cell interaction. Based on in vitro experiments, it was suggested recently that the DIP0733 protein might be involved in adhesion, invasion of epithelial cells and induction of apoptosis. A corresponding Cor. diphtheriae mutant strain generated in this study was attenuated in its ability to colonize and kill the host in a Caenorhabditis elegans infection model system. Furthermore, the mutant showed an altered adhesion pattern and a drastically reduced ability to adhere and invade epithelial cells. Subsequent experiments showed an influence of DIP0733 on binding of Cor. diphtheriae to extracellular matrix proteins such as collagen and fibronectin. Furthermore, based on its fibrinogen-binding activity, DIP0733 may play a role in avoiding recognition of Cor. diphtheriae by the immune system. In summary, our findings support the idea that DIP0733 is a multi-functional virulence factor of Cor. diphtheriae.
Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium diphtheriae/metabolismo , Difteria/microbiología , Factores de Virulencia/metabolismo , Animales , Apoptosis , Adhesión Bacteriana , Proteínas Bacterianas/genética , Caenorhabditis elegans , Línea Celular , Corynebacterium diphtheriae/clasificación , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/patogenicidad , Difteria/fisiopatología , Células Epiteliales/citología , Células Epiteliales/microbiología , Humanos , Filogenia , Factores de Virulencia/genéticaRESUMEN
Enteroaggregative Escherichia coli (EAEC) is a significant cause of diarrhoeal illness in both children and adults. Genetic heterogeneity and recovery of EAEC strains from both healthy and diseased individuals complicates our understanding of EAEC pathogenesis. We wished to establish if genetic or phenotypic attributes could be used to distinguish between strains asymptomatically colonising healthy individuals and those which cause disease. Genotypic screening of a collection of twenty four EAEC isolates from children with and without diarrhoea revealed no significant differences in the repertoire of putative virulence factors present in either group of strains. In contrast, EAEC strains from phylogroup A were more strongly associated with asymptomatic groups whereas strains from phylogroup D were more associated with cases of diarrhoea. Phenotypic screening revealed no differences in the ability of strains from either cohort of children to form biofilms, to adhere to and invade cells in tissue culture or to cause disease in the Caenorhabditis elegans model of infection. However, the latter assay did reveal significant reduction in nematode killing rates when specific virulence factors were deleted from human pathogenic strains. Our results suggest that current models of infection are not useful for distinguishing avirulent from pathogenic strains of EAEC but can be useful in studying the effect of specific virulence factors.
Asunto(s)
Diarrea/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/fisiología , Genotipo , Fenotipo , Animales , Adhesión Bacteriana/genética , Biopelículas , Brasil , Caenorhabditis elegans/microbiología , Estudios de Casos y Controles , Línea Celular , Preescolar , Escherichia coli/clasificación , Escherichia coli/patogenicidad , Eliminación de Gen , Humanos , Lactante , Recién Nacido , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
The plasmid-encoded toxin, Pet, a prototypical member of the serine protease autotransporters of the Enterobacteriaceae, possesses an unusually long signal peptide, which can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions correspond to a conserved N-terminal extension previously designated the extended signal peptide region (ESPR), while the N2, H2 and C regions resemble typical Sec-dependent signal sequences and exhibit considerable sequence variability. We have shown previously that the ESPR directs Sec-dependent, post-translational translocation of Pet across the bacterial inner membrane. In this study, we demonstrate that the ESPR is not essential for the secretion or the function of Pet.
Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Enterotoxinas/química , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Señales de Clasificación de Proteína , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Toxinas Bacterianas/genética , Enterotoxinas/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de Secuencia , Eliminación de Secuencia , Serina Endopeptidasas/genéticaRESUMEN
Enteroaggregative Escherichia coli (EAEC) is an emerging pathogen associated to cases of acute or persistent diarrhea in children and adults from developed and developing countries. These microorganisms also have been isolated from human immunodeficiency virus-infected patients. EAEC exhibits aggregative adherence (AA) in HEp-2 cells. This pattern is characterized by the production of bacteria aggregates adhered to monolayer cultured cells with a "stacked brick" phenotype. The AA pattern is related to the presence of a 60MDa plasmid (pAA). In the present study, we evaluated the adherence, invasion and persistent survival of five EAEC strains with Caco-2 and T84 cells, by a bacteria invasion assay and transmission electron microscopy. EAEC isolated from cases of acute infantile diarrhea can be internalized by intestinal epithelial cells cultivated in vitro, suggesting that these strains may employ a mechanism of host cell invasion to colonize the intestinal mucosa. Results showed that EAEC strains could survive intracellularly up to 72h. Our data support evidence that EAEC is able to invade, persist and replicate within intestinal cells for extended time. This strategy may be advantageous to EAEC in colonization and survival, favoring the exploitation of an intracellular niche where these strains are protected against host clearance mechanisms, immune system and antibiotic treatment. Intracellular persistence of EAEC may be associated with development of persistent diarrhea associated to these microorganisms. To our knowledge, this is the first report of EAEC intracellular survival in cultured intestinal epithelial cells.
Asunto(s)
Células Epiteliales/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/fisiología , Intestinos/microbiología , Adhesión Bacteriana , Línea Celular Tumoral , Diarrea Infantil/microbiología , Células Epiteliales/ultraestructura , Escherichia coli/patogenicidad , Humanos , Lactante , Intestinos/ultraestructura , Viabilidad MicrobianaRESUMEN
Abstract Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells.