Extracellular Vesicles of the Plant Pathogen Botrytis cinerea.

Fungal secretomes are known to contain a multitude of components involved in nutrition, cell growth or biotic interactions. Recently, extra-cellular vesicles have been identified in a few fungal species. Here, we used a multidisciplinary approach to identify and characterize extracellular vesicles produced by the plant necrotroph Botrytis cinerea. Transmission electron microscopy of infectious hyphae and hyphae grown in vitro revealed extracellular vesicles of various sizes and densities. Electron tomography showed the co-existence of ovoid and tubular vesicles and pointed to their release via the fusion of multi-vesicular bodies with the cell plasma membrane. The isolation of these vesicles and exploration of their protein content using mass spectrometry led to the identification of soluble and membrane proteins involved in transport, metabolism, cell wall synthesis and remodeling, proteostasis, oxidoreduction and traffic. Confocal microscopy highlighted the capacity of fluorescently labeled vesicles to target cells of B. cinerea, cells of the fungus Fusarium graminearum, and onion epidermal cells but not yeast cells. In addition, a specific positive effect of these vesicles on the growth of B. cinerea was quantified. Altogether, this study broadens our view on the secretion capacity of B. cinerea and its cell-to-cell communication.

Clathrin Is Important for Virulence Factors Delivery in the Necrotrophic Fungus Botrytis cinerea.

Fungi are the most prevalent plant pathogens, causing annually important damages. To infect and colonize their hosts, they secrete effectors including hydrolytic enzymes able to kill and macerate plant tissues. These secreted proteins are transported from the Endoplasmic Reticulum and the Golgi apparatus to the extracellular space through intracellular vesicles. In pathogenic fungi, intracellular vesicles were described but their biogenesis and their role in virulence remain unclear. In this study, we report the essential role of clathrin heavy chain (CHC) in the pathogenicity of Botrytis cinerea, the agent of gray mold disease. To investigate the importance of this protein involved in coat vesicles formation in eukaryotic cells, a T-DNA insertional mutant reduced in the expression of the CHC-encoding gene, and a mutant expressing a dominant-negative form of CHC were studied. Both mutants were strongly affected in pathogenicity. Characterization of the mutants revealed altered infection cushions and an important defect in protein secretion. This study demonstrates the essential role of clathrin in the infectious process of a plant pathogenic fungus and more particularly its role in virulence factors delivery.

The infection cushion of Botrytis cinerea: a fungal 'weapon' of plant-biomass destruction.

The necrotrophic plant-pathogen fungus Botrytis cinerea produces multicellular appressoria dedicated to plant penetration, named infection cushions (IC). A microarray analysis was performed to identify genes upregulated in mature IC. The expression data were validated by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and biochemical assays. 1231 upregulated genes and 79 up-accumulated proteins were identified. The data support the secretion of effectors by IC: phytotoxins, ROS, proteases, cutinases, plant cell wall-degrading enzymes and plant cell death-inducing proteins. Parallel upregulation of sugar transport and sugar catabolism-encoding genes would indicate a role of IC in nutrition. The data also reveal a substantial remodelling of the IC cell wall and suggest a role for melanin and chitosan in IC function. Lastly, mutagenesis of two upregulated genes in IC identified secreted fasciclin-like proteins as actors in the pathogenesis of B. cinerea. These results support the role of IC in plant penetration and also introduce other unexpected functions for this fungal organ, in colonization, necrotrophy and nutrition of the pathogen.

A Similar Secretome Disturbance as a Hallmark of Non-pathogenic Botrytis cinerea ATMT-Mutants?

The gray mold fungus Botrytis cinerea is a necrotrophic pathogen able to infect hundreds of host plants, including high-value crops such as grapevine, strawberry and tomato. In order to decipher its infectious strategy, a library of 2,144 mutants was generated by random insertional mutagenesis using Agrobacterium tumefaciens-mediated transformation (ATMT). Twelve mutants exhibiting total loss of virulence toward different host plants were chosen for detailed analyses. Their molecular characterization revealed a single T-DNA insertion in different loci. Using a proteomics approach, the secretome of four of these strains was compared to that of the parental strain and a common profile of reduced lytic enzymes was recorded. Significant variations in this profile, notably deficiencies in the secretion of proteases and hemicellulases, were observed and validated by biochemical tests. They were also a hallmark of the remaining eight non-pathogenic strains, suggesting the importance of these secreted proteins in the infection process. In the twelve non-pathogenic mutants, the differentiation of infection cushions was also impaired, suggesting a link between the penetration structures and the secretion of proteins involved in the virulence of the pathogen.

Bacteriophages Targeting Adherent Invasive Escherichia coli Strains as a Promising New Treatment for Crohn's Disease.

BACKGROUND AND AIMS: Adherent invasive Escherichia coli [AIEC] are abnormally predominant on the ileal mucosa of Crohn's disease [CD] patients. They bind to the CEACAM6 receptor expressed on the surface of epithelial cells. We aimed to assess the potential of bacteriophages, viruses infecting bacteria, to decrease the levels of AIEC bacteria associated with the intestinal mucosa. METHODS: We combined ex vivo and in vivo experiments with murine and human intestinal samples to quantify the ability of virulent bacteriophages to target the prototype AIEC strain LF82. RESULTS: We found that three virulent bacteriophages were able to replicate in ileal, caecal and colonic sections and faeces homogenates from murine gut samples colonised with the prototype AIEC strain LF82. A single day of per os treatment with the three bacteriophages cocktail given to LF82-colonised CEABAC10 transgenic mice, expressing the human CEACAM6 receptor for AIEC, decreased significantly the number of AIEC in faeces and in the adherent flora of intestinal sections. In addition, a single dose of the cocktail reduced dextran sodium sulphate-induced colitis symptoms on conventional mice colonised with the strain LF82 over a 2-week period. The cocktail targeted also LF82 bacteria in homogenates of ileal biopsies taken from CD patients. CONCLUSIONS: These findings demonstrate that bacteriophages are a new treatment option for targeting AIEC in CD patients and represent a strong basis for a clinical trial evaluation.

GipA Factor Supports Colonization of Peyer's Patches by Crohn's Disease-associated Escherichia Coli.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC) associated with Crohn's disease target M cells lining Peyer's patches (PPs) through the expression of long polar fimbriae (LPF) and survive macrophage killing. Invasion of PPs constitutes a way to colonize the mucosa for bacteria able to escape or resist killing of underlying immune cells. We aimed to identify new virulence factors involved in PPs colonization by AIEC. METHODS: The presence of gipA (Growth in PPs) gene was determined by polymerase chain reaction. In vivo experiments were performed using CEABAC10 transgenic mice. Intramacrophagic behavior of AIEC was assessed in murine bone marrow-derived macrophages and human monocyte-derived macrophages. Cytokines production was quantified by ELISA. RESULTS: A higher prevalence of gipA-positive E. coli was observed in patients with Crohn's disease (27.3%) compared with controls (17.2%). Unlike non-AIEC strains, all gipA-positive AIEC strains also harbored lpfA. GipA deletion impaired AIEC translocation across M cells and their replication inside macrophages. GipA expression was induced by gastrointestinal (bile salts) and phagolysosomal (reactive oxygen species and acid pH) conditions. GipA deletion decreased lpfA mRNA level in AIEC bacteria. Survival of AIEC-ΔgipA bacteria was reduced in medium containing H2O2 or acidic pH. GipA deletion impaired AIEC colonization of PPs and dissemination to mesenteric lymph nodes in mice. CONCLUSIONS: GipA is required for optimal colonization of mouse PPs and survival within macrophages by AIEC, suggesting that this factor plays a role in AIEC promotion of Crohn's disease. Detection of gipA and lpfA could be a predictor for the presence of AIEC.

Monocyte-derived macrophages from Crohn's disease patients are impaired in the ability to control intracellular adherent-invasive Escherichia coli and exhibit disordered cytokine secretion profile.

BACKGROUND: Ileal lesions of Crohn's disease [CD] patients are colonised by adherent-invasive Escherichia coli [AIEC] able to survive in macrophage cell lines. We analysed the ability of monocyte-derived macrophages [MDM] from CD patients to control AIEC intracellular replication and the pro-inflammatory cytokine response of the infected-MDM. METHODS: Peripheral blood MDM were obtained from 24 CD genotyped for NOD2 and ATG16L1 mutations, 5 ulcerative colitis [UC] patients and 12 healthy controls [HC]. The numbers of intracellular bacteria were determined using gentamicin assay. Cytokine secretion was quantified by ELISA assay. RESULTS: We observed that higher levels of bacteria were internalised within MDM from CD patients than MDM from HC or UC patients. MDM from CD patients were unable to restrict AIEC intracellular replication. Infection of MDM from CD patients with AIEC resulted in significantly increased secretion of IL-6 and tumour necrosis factor alpha [TNF α] than did infection with non-pathogenic E. coli. AIEC-infected MDM from CD patients exhibited a disordered cytokines secretion compared with MDM from UC patients and HC. AIEC-infected MDM from patients with quiescent CD released significantly higher amounts of IL-6 and TNF-alpha than those with active disease or those from HC. The level of secreted TNF-alpha was correlated to the number of intracellular AIEC in MDM from CD patients. Treatment of MDM with infliximab did not change the MDM behaviour. CONCLUSIONS: MDM from CD patients are unable to restrict intracellular AIEC replication, leading to disordered inflammatory response influenced by disease activity.

Saccharomyces cerevisiae CNCM I-3856 prevents colitis induced by AIEC bacteria in the transgenic mouse model mimicking Crohn's disease.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of patients with Crohn's disease (CD), are able to adhere to and invade intestinal epithelial cells. Overexpression of the glycoprotein CEACAM6 on host cells favors AIEC attachment and inflammation. We investigated the ability of Saccharomyces cerevisiae CNCM I-3856 to inhibit AIEC adhesion and to reduce colitis. METHODS: Adhesion experiments were performed on T84 cells and on enterocytes from patients with CD with AIEC LF82 in the presence of S. cerevisiae. Colonization and symptoms of colitis were assessed in LF82-infected transgenic CEABAC10 mice treated with live S. cerevisiae or S. cerevisiae derivatives. Proinflammatory cytokines were quantified by enzyme linked immunosorbent assay. Intestinal permeability was assessed by measuring the 4 kDa dextran-FITC flux in the serum. RESULTS: S. cerevisiae strongly inhibited LF82 adhesion to T84 cells and to the brush border of CD enterocytes. Yeasts decreased LF82 colonization and colitis in CEABAC10 mice and restored barrier function through prevention of the LF82-induced expression of pore-forming tight junction claudin-2 at the plasma membrane of intestinal epithelial cells. These effects were accompanied by a decrease in proinflammatory cytokines IL-6, IL-1ß, and KC release by the gut mucosa. Yeast derivatives exerted similar effects on LF82 colonization and colitis demonstrating that yeast viability was not essential to exert beneficial effects. CONCLUSIONS: S. cerevisiae yeasts reduce colitis induced by AIEC bacteria in CEACAM6-expressing mice. Such a probiotic strategy could be envisaged in a subgroup of patients with CD abnormally expressing CEACAM6 at the ileal mucosa and therefore susceptible to being colonized by AIEC bacteria.

Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation.

AIM: To provide further insight into the characterization of mucosa-associated Escherichia coli (E. coli) isolated from the colonic mucosa of cancer patients. METHODS: Phylogroups and the presence of cyclomodulin-encoding genes of mucosa-associated E. coli from colon cancer and diverticulosis specimens were determined by PCR. Adhesion and invasion experiments were performed with I-407 intestinal epithelial cells using gentamicin protection assay. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) expression in T84 intestinal epithelial cells was measured by enzyme-linked immunosorbent assay and by Western Blot. Gut colonization, inflammation and pro-carcinogenic potential were assessed in a chronic infection model using CEABAC10 transgenic mice. Cell proliferation was analyzed by real-time mRNA quantification of PCNA and immunohistochemistry staining of Ki67. RESULTS: Analysis of mucosa-associated E. coli from colon cancer and diverticulosis specimens showed that whatever the origin of the E. coli strains, 86% of cyclomodulin-positive E. coli belonged to B2 phylogroup and most harbored polyketide synthase (pks) island, which encodes colibactin, and/or cytotoxic necrotizing factor (cnf) genes. In vitro assays using I-407 intestinal epithelial cells revealed that mucosa-associated B2 E. coli strains were poorly adherent and invasive. However, mucosa-associated B2 E. coli similarly to Crohn's disease-associated E. coli are able to induce CEACAM6 expression in T84 intestinal epithelial cells. In addition, in vivo experiments using a chronic infection model of CEACAM6 expressing mice showed that B2 E. coli strain 11G5 isolated from colon cancer is able to highly persist in the gut, and to induce colon inflammation, epithelial damages and cell proliferation. CONCLUSION: In conclusion, these data bring new insights into the ability of E. coli isolated from patients with colon cancer to establish persistent colonization, exacerbate inflammation and trigger carcinogenesis.

Crohn disease--associated adherent-invasive E. coli bacteria target mouse and human Peyer's patches via long polar fimbriae.

Crohn disease (CD) is a multifactorial disease in which an abnormal immune response in the gastrointestinal (GI) tract leads to chronic inflammation. The small intestine, particularly the ileum, of patients with CD is colonized by adherent-invasive E. coli (AIEC)--a pathogenic group of E. coli able to adhere to and invade intestinal epithelial cells. As the earliest inflammatory lesions are microscopic erosions of the epithelium lining the Peyer's patches (PPs), we investigated the ability of AIEC bacteria to interact with PPs and the virulence factors involved. We found that AIEC bacteria could interact with mouse and human PPs via long polar fimbriae (LPF). An LPF-negative AIEC mutant was highly impaired in its ability to interact with mouse and human PPs and to translocate across monolayers of M cells, specialized epithelial cells at the surface of PPs. The prevalence of AIEC strains harboring the lpf operon was markedly higher in CD patients compared with controls. In addition, increased numbers of AIEC, but not LPF-deficient AIEC, bacteria were found interacting with PPs from Nod2(-/-) mice compared with WT mice. In conclusion, we have identified LPF as a key factor for AIEC to target PPs. This could be the missing link between AIEC colonization and the presence of early lesions in the PPs of CD patients.

Complete genome sequence of Crohn's disease-associated adherent-invasive E. coli strain LF82.

BACKGROUND: Ileal lesions of Crohn's disease (CD) patients are abnormally colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells and macrophages. PRINCIPAL FINDINGS: We report here the complete genome sequence of E. coli LF82, the reference strain of adherent-invasive E. coli associated with ileal Crohn's disease. The LF82 genome of 4,881,487 bp total size contains a circular chromosome with a size of 4,773,108 bp and a plasmid of 108,379 bp. The analysis of predicted coding sequences (CDSs) within the LF82 flexible genome indicated that this genome is close to the avian pathogenic strain APEC_01, meningitis-associated strain S88 and urinary-isolated strain UTI89 with regards to flexible genome and single nucleotide polymorphisms in various virulence factors. Interestingly, we observed that strains LF82 and UTI89 adhered at a similar level to Intestine-407 cells and that like LF82, APEC_01 and UTI89 were highly invasive. However, A1EC strain LF82 had an intermediate killer phenotype compared to APEC-01 and UTI89 and the LF82 genome does not harbour most of specific virulence genes from ExPEC. LF82 genome has evolved from those of ExPEC B2 strains by the acquisition of Salmonella and Yersinia isolated or clustered genes or CDSs located on pLF82 plasmid and at various loci on the chromosome. CONCLUSION: LF82 genome analysis indicated that a number of genes, gene clusters and pathoadaptative mutations which have been acquired may play a role in virulence of AIEC strain LF82.