Daniel Poulain

Colonization of Mice by Candida albicans Is Promoted by Chemically Induced Colitis and Augments Inflammatory Responses through Galectin-3

BACKGROUNDnLittle is known about the relationship between colonic inflammation and Candida albicans colonization. Galectin-3 (Gal-3) is an intestinal lectin that binds to specific C. albicans glycans and is involved in inflammation.nnnMETHODSnColitis was experimentally induced in wild-type and Gal3(-/-) mice using dextran sulfate sodium (DSS) before oral administration of C. albicans. Yeast recovered from stools was quantified. The presence of yeast and inflammation were evaluated in sections of colon by histologic examination, quantification of myeloperoxidase (MPO) activity, and by gene expression for cytokines and innate immune receptors. Serum from mice was collected for determination of anti-yeast mannan antibodies, including anti-Saccharomyces cerevisiae antibodies (ASCA), which are biomarkers of an inflammatory bowel disease.nnnRESULTSnInflammation strongly promoted C. albicans colonization. Conversely, C. albicans augmented inflammation induced by DSS, as assessed by histologic scores, MPO activity, and tumor necrosis factor (TNF)-alpha and Toll-like receptor (TLR)-2 expression. C. albicans colonization generated ASCA. The absence of Gal-3 reduced DSS inflammation and abolished the response of TLR-2 and TNF-alpha to C. albicans colonization.nnnCONCLUSIONSnDSS-induced colitis provides a model for establishing C. albicans colonization in mice. This model reveals that C. albicans augments inflammation and confirms the role of Gal-3 in both inflammation and the control of host responses to C. albicans.

Bacteriome and Mycobiome Interactions Underscore Microbial Dysbiosis in Familial Crohn's Disease.

ABSTRACT Crohn’s disease (CD) results from a complex interplay between host genetic factors and endogenous microbial communities. In the current study, we used Ion Torrent sequencing to characterize the gut bacterial microbiota (bacteriome) and fungal community (mycobiome) in patients with CD and their nondiseased first-degree relatives (NCDR) in 9 familial clusters living in northern France-Belgium and in healthy individuals from 4 families living in the same area (non-CD unrelated [NCDU]). Principal component, diversity, and abundance analyses were conducted, and CD-associated inter- and intrakingdom microbial correlations were determined. Significant microbial interactions were identified and validated using single- and mixed-species biofilms. CD and NCDR groups clustered together in the mycobiome but not in the bacteriome. Microbiotas of familial (CD and NCDR) samples were distinct from those of nonfamilial (NCDU) samples. The abundance of Serratia marcescens and Escherichia coli was elevated in CD patients, while that of beneficial bacteria was decreased. The abundance of the fungus Candida tropicalis was significantly higher in CD than in NCDR (P = 0.003) samples and positively correlated with levels of anti-Saccharomyces cerevisiae antibodies (ASCA). The abundance of C. tropicalis was positively correlated with S. marcescens and E. coli, suggesting that these organisms interact in the gut. The mass and thickness of triple-species (C. tropicalis plus S. marcescens plus E. coli) biofilm were significantly greater than those of single- and double-species biofilms. C. tropicalis biofilms comprised blastospores, while double- and triple-species biofilms were enriched in hyphae. S. marcescens used fimbriae to coaggregate or attach with C. tropicalis/E. coli, while E. coli was closely apposed with C. tropicalis. Specific interkingdom microbial interactions may be key determinants in CD. IMPORTANCE Here, we characterized the gut bacterial microbiota (bacteriome) and fungal community (mycobiome) in multiplex families with CD and healthy relatives and defined the microbial interactions leading to dysbiosis in CD. We identified fungal (Candida tropicalis) and bacterial (Serratia marcescens and Escherichia coli) species that are associated with CD dysbiosis. Additionally, we found that the level of anti-Saccharomyces cerevisiae antibodies (ASCA; a known CD biomarker) was associated with the abundance of C. tropicalis. We also identified positive interkingdom correlations between C. tropicalis, E. coli, and S. marcescens in CD patients and validated these correlations using in vitro biofilms. These results provide insight into the roles of bacteria and fungi in CD and may lead to the development of novel treatment approaches and diagnostic assays. Here, we characterized the gut bacterial microbiota (bacteriome) and fungal community (mycobiome) in multiplex families with CD and healthy relatives and defined the microbial interactions leading to dysbiosis in CD. We identified fungal (Candida tropicalis) and bacterial (Serratia marcescens and Escherichia coli) species that are associated with CD dysbiosis. Additionally, we found that the level of anti-Saccharomyces cerevisiae antibodies (ASCA; a known CD biomarker) was associated with the abundance of C. tropicalis. We also identified positive interkingdom correlations between C. tropicalis, E. coli, and S. marcescens in CD patients and validated these correlations using in vitro biofilms. These results provide insight into the roles of bacteria and fungi in CD and may lead to the development of novel treatment approaches and diagnostic assays.

Evaluation of MALDI-TOF mass spectrometry for the identification of medically-important yeasts in the clinical laboratories of Dijon and Lille hospitals

Conventional identification (CI) of yeasts is based on morphological, biochemical and/or immunological methods. Matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF or MT-MS) mass spectrometry has been proposed as a new method for the identification of microorganisms. This prospective study compared the performance of MT-MS and CI for the identification of yeasts isolated from clinical samples. Sequencing of the internal transcribed spacer (ITS) regions of ribosomal DNA was used as the reference method in the analysis of a total of 1207 yeast isolates. Concordance between MT-MS and CI was observed for 1105 isolates (91.5%), while 74 isolates (6.1%) were misidentified. Molecular identification revealed that 73 of these 74 isolates were identified correctly by MT-MS and CI correctly identified the last one. Concordance between the two techniques was excellent for the medically-important species (98-100%), including the identification of closely-related species (Candida albicans/C. dubliniensis; C. inconspicua/C. norvegensis; C. parapsilosis/C. metapsilosis/C. orthopsilosis). Only 2.3% of isolates belonging to C. famata, C. lambica and C. magnoliae or to Geotrichum spp. and Trichosporon spp. were not identified by MT-MS. This investigation highlights the potential of MT-MS-based yeast identification as a reliable, time and cost-efficient alternative to CI.

Molecular Identification of Closely Related Candida Species Using Two Ribosomal Intergenic Spacer Fingerprinting Methods

Recent changes in the epidemiology of candidiasis highlighted an increase in non- Candida albicans species emphasizing the need for reliable identification methods. Molecular diagnostics in fungal infections may improve species characterization, particularly in cases of the closely related species in the Candida complexes. We developed two PCR/restriction fragment length polymorphism assays, targeting either a part of the intergenic spacer 2 or the entire intergenic spacer (IGS) of ribosomal DNA using a panel of 270 isolates. A part of the intergenic spacer was used for discrimination between C. albicans and C. dubliniensis and between species of the C. glabrata complex (C. glabrata/C. bracarensis/C. nivariensis). The whole IGS was applied to C. parapsilosis, C. metapsilosis, and C. orthopsilosis, and to separate C. famata (Debaryomyces hansenii) from C. guilliermondii (Pichia guilliermondii) and from the other species within this complex (ie, C. carpophila, C. fermentati and C. xestobii). Sharing similar biochemical patterns, Pichia norvegensis and C. inconspicua exhibited specific IGS profiles. Our study confirmed that isolates of C. guilliermondii were frequently mis-identified as C. famata. As much as 67% of the clinical isolates phenotypically determined as C. famata were recognized mostly as true P. guilliermondii. Conversely, 44% of the isolates initially identified as C. guilliermondii were corrected by the IGS fingerprints as C. parapsilosis, C. fermentati, or C. zeylanoides. These two PCR/restriction fragment length polymorphism methods may be used as reference tools [either alternatively or adjunctively to the existing ribosomal DNA (26S or ITS) sequence comparisons] for unambiguous determination of the Candida species for which phenotypic characterization remains problematic.

Candida albicans Airway Exposure Primes the Lung Innate Immune Response against Pseudomonas aeruginosa Infection through Innate Lymphoid Cell Recruitment and Interleukin-22-Associated Mucosal Response

ABSTRACT Pseudomonas aeruginosa and Candida albicans are two pathogens frequently encountered in the intensive care unit microbial community. We have demonstrated that C. albicans airway exposure protected against P. aeruginosa-induced lung injury. The goal of the present study was to characterize the cellular and molecular mechanisms associated with C. albicans-induced protection. Airway exposure by C. albicans led to the recruitment and activation of natural killer cells, innate lymphoid cells (ILCs), macrophages, and dendritic cells. This recruitment was associated with the secretion of interleukin-22 (IL-22), whose neutralization abolished C. albicans-induced protection. We identified, by flow cytometry, ILCs as the only cellular source of IL-22. Depletion of ILCs by anti-CD90.2 antibodies was associated with a decreased IL-22 secretion and impaired survival after P. aeruginosa challenge. Our results demonstrate that the production of IL-22, mainly by ILCs, is a major and inducible step in protection against P. aeruginosa-induced lung injury. This cytokine may represent a clinical target in Pseudomonas aeruginosa-induced lung injury.

Candida albicans, plasticity and pathogenesis

Abstract The yeast Candida albicans has emerged as a major public health problem during the past two decades. The spectrum of diseases caused by this species ranges from vaginal infections, which affect up to 75% of the women at least once in their lifetime, to deep infections in hospitalized patients which lead to high morbidity and mortality rates. Candida albicans may also play a role in the persistence or worsening of some chronic inflammatory bowel diseases. Active research is now improving our understanding of the molecular mechanisms and genetic factors in the yeast and its host which influence the development of disease. Despite these advances and the availability of a more extensive therapeutic arsenal, current progress in the control of nosocomial infections due to Candida remains limited, mainly due to the difficulties in diagnosing these infections. The biologist has a key role to play in establishing a dialogue with the clinician in order to identify the saprophyte/pathogen transition in patients as early as possible. This review provides a quick synopsis of the modern concepts of Candida pathogenesis with some representative examples illustrating the specifics traits of this yeast in terms of pathogenic adaptation.

Yeasts: neglected pathogens.

Background: Current research on Crohn’s disease (CD) concerns molecular events related to loss of tolerance to microbes that could trigger or maintain inflammation in genetically susceptible individuals. CD is also associated with antimicrobial antibodies, including the antibodies we described against yeast oligomannosides (ASCA). This prompted us to investigate a role for another yeast, Candida albicans, a very common commensal of the human digestive tract and an important opportunistic pathogen. Clinical and Experimental Data: It has been revealed that the major oligomannose epitopes supporting ASCA are expressed by C. albicans in human tissues, suggesting that C. albicans is the immunogen for ASCA. This link has been reinforced by the demonstration that novel serological markers of CD (ALCA and ACCA), consisting of antibodies against chitin and glucan (two components of the C. albicans cell wall), are also generated during C. albicans infection. Mycological investigation of families with multiple cases of CD shows that patients with CD and their healthy relatives are colonized with C. albicans more commonly than control families. In healthy relatives, C. albicans colonization correlates with ASCA levels, whereas the onset of CD is associated with ASCA stability and is independent of the C. albicans intestinal load. Experimental studies show that chemically-induced colitis promotes C. albicans colonization in mice. In turn, C. albicans colonization generates ASCA, increases inflammation, histological scores and pro-inflammatory cytokine expression. Perspectives: Current investigations focus on interactions of TLRs and lectins with yeast epitopes that differently polarize the immune response to C. albicans cell wall glycans, which are the targets of an ‘excessive’ adaptive response associated with CD.

An immunological link between Candida albicans colonization and Crohn’s disease

Abstract The etiology of Crohn’s disease (CD), an autoimmune, inflammatory bowel disease (IBD) which affects approximately one million people in Europe, is still unclear. Nevertheless, it is widely accepted that CD could result from an inappropriate inflammatory response to intestinal microorganisms in a genetically susceptible host. Most studies to date have concerned the involvement of bacteria in disease progression. In addition to bacteria, there appears to be a possible link between the commensal yeast Candida albicans and disease development. In this review, in an attempt to link the gut colonization process and the development of CD, we describe the different pathways that are involved in the progression of CD and in the host response to C. albicans, making the yeast a possible initiator of the inflammatory process observed in this IBD.

Synthetic biotinylated tetra β(1→5) galactofuranoside for in vitro aspergillosis diagnosis

The synthesis of a tetra β(1→5) galactofuranoside was achieved using a thioglycoside donor with a methyl tert-butyl phenyl thio leaving group. This tetrasaccharide was conjugated to biotin and validated as antigen with the monoclonal antibody used for clinical detection of Aspergillus fumigatus galactomannan on streptavidin-coated microplates. Then we have shown its ability to detect antibodies associated with A. fumigatus induced disease by using sera from patients with Allergic broncho-pulmonary aspergillosis (ABPA) and correlated the results of antibody detection with those gained with a commercially available diagnostic test.

Biotin sulfone tagged oligomannosides as immunogens for eliciting antibodies against specific mannan epitopes

Biotinylated tri and tetrasaccharide: α Man (1→3) α Man (1→2) α Man; α Man (1→3) α Man (1→2) α Man (1→2) α Man were prepared using methyl tertbutyl phenyl thioglycosides glycosyl donors (MBP) and biotin sulfone strategy. Three key mannosyl thioglycosidic donors have been prepared: one for 1→2 linkage and two for the 1→3 linkage (protected with a 4,6-O-benzylidene or a 4,6-di-O-benzyl). The benzyliden protected one was not found reactive enough, and the benzylated donor was preferred. These biotinylated oligomanosides were evaluated as antigen in Crohn disease diagnosis and used coupled to streptavidin as hapten for eliciting polyclonal antibodies in mice.