Laboratoire de Microbiologie Fondamentale et Pathogénicité

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Candida et Pathogénicité

 

Responsables :

Thierry NOEL, Isabelle ACCOCEBERRY.

 

Membres de l'équipe :

 

General information :

Fungi of medical interest constitute the first cause of opportunistic parasitic diseases in industrialized countries. Fungal infections challenge health professionals because of the few number of antifungal molecules available, the growing diversity of emerging pathogens, and antifungal resistance issues. Our team has an expertise in the domain of medical mycology, and, to a lesser extent, in parasitology. Taking advantage of the multidisciplinary composition of the team, the research is based on three domains: basic research, clinical investigations, and transfer of technology with industrial partners.

Basic research

Our team works on Candida yeasts of medical interest with a focus on Candida lusitaniae. The sequence of its haploid genome is available, and we developed several genetic tools to make it accessible to both formal and reverse genetics. Two projects are actually on-going: Candida/phagocytes interactions (A) and antifungal resistance (B).

 

A : Cellular and molecular characterization of the interactions between Candida and phagocytic cells.

Neutrophils and macrophages constitute the main barrier to Candida infections. One of our projects is to characterize the interactions between the yeast Candida and the phagocytes at the cellular and molecular levels, with a particular attention to the fungal lipid metabolism which could produce important signaling molecules during the interaction, and which could serve as the source of nutriment allowing the phagocytosed yeasts to survive phagolysis, to multiply inside phagocytes, and to escape the immune cell.

1 – Development of a cellular model for the multiparametric characterization of the interactions between Candida and phagocytic cells

We developed a new in vitro model for the multi-parameter characterization of the time course interaction of Candida yeast cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Various parameters are measured simultaneously in a single infection experiment: the rate of association of phagocytes to fungal cells, phagocyte survival, and the part of ingested fungal cells among the total fungal biomass. The model was validated with Candida albicans, C. glabrata, and C. lusitaniae, and allowed us to show that all three species were able to survive and to escape macrophage phagocytosis by using very different strategies, either by the intraphagocytic yeast-to-hyphae transition (C. albicans), the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae) (Dementhon et al., Plos One 2012).

 

Fig.1. Macrophage infected with Candida cells.
Left: fluorescence panel showing a macrophage stained with APC-anti-CD16 antibody (red) and calcein (green) containing several yeast cells (blue). Right: bright field.

 

2 – Role of the fungal lipid metabolism during the interaction Candida/macrophage

Yeast survival in macrophages was correlated to a fungal metabolic switch to the peroxisomal metabolism, which houses the fatty acid β-oxidation pathway. Using null mutants in specific genes of C. lusitaniae, catabolism assay of 14Cα-palmitoyl-CoA, organelle purification and immunoelectron microscopy, we demonstrated, for the first time in an ascomycetous yeast, the co-existence of three pathways of fatty acids catabolism, one located in the mitochondria and two others located in the peroxisomes (Gabriel, PLoS One 2014). Other essential steps of ß-oxidation were characterized by reverse genetics, and candidate genes that could be involved in an alternate Fox2p-independent peroxisomal pathway are currently under investigation. The question about the precise role of fatty acid catabolism in the virulence of C. lusitaniae will be answered when all the fatty oxidation pathways will be identified and turned-off.

 

Fig.2. Thin layer chromatogrophy of the products of 14Cα-palmitoyl-CoA catabolism by protein extracts of wild type and fox2? mutant. P : peroxisomal fraction ; M : mitochondrial fraction. T0 : 14Cα-palmitoyl-CoA (PalCoA) before the addition of protein extracts. T2-3CoA : Trans2-3 enoyl CoA (= product of the oxidation of palmitoylCoA, and substrate of Fox2p).

 

3 – Characterization of signaling molecules during the interaction Candida / macrophage

Signaling molecules are often described as important modulators during the interaction between Candida cells and phagocytes. We created a dpp3? mutant in C. lusitaniae impaired for a pyrophosphate phosphatase activity of the sterol pathway. The mutation modified the ratio of the phenetyl alcool (PEA)/tyrosol signaling molecules secreted by the fungal cells, which in turn affected the NO and ROS production by the macrophages, as well as the balance of TNF-a and IL-10 production. In parallel, we demonstrated that Dpp3 is an essential protein for the virulence of C. lusitaniae in a mouse model of candidemia (Sabra et al, Infect. Immun. 2014). We aim now to characterize the molecules of the C. lusitaniae secretome that are involved in this cross-talk.

 

Fig.3. DBA2/J Mouse survival rates after infection with the wild type (WT), the KO dpp3?, and the DPP3 reconstructed  (REC) strains of C. lusitaniae.

 

B - Molecular characterization of the NCS1 membrane transporters of fluoropyrimidine and vitamins B and of their antagonist interactions with azole antifungals in C. lusitaniae


We are interested for several years in the uptake transport of azole and fluoropyrimidines antifungals at the molecular level, and in their apparent antagonist interactions through a possible competition for entry. We characterized recently two additional transporters of the nucleobase cation symporter 1 family in C. lusitaniae, Dal4p, which transports allantoin, and Fur4p, which transports uracil and 5-fluorouracil. The deletion of FUR4 confers not only resistance to 5-fluorouracil, but also resistance to all azole antifungals. We demonstrated that cross-resistance resulted from antagonistic interaction between fluorinated uridylic acid and azole antifungals (Gabriel et al, AAC 2014). Actually, our efforts are concentrated towards the characterization of two new transporters of the NCS1 family which are involved, at least partially, in the uptake transport of azole antifungals.

 

Fig.4. Salvage and de novo pathways of pyrimidine synthesis in C. lusitaniae. 5FUMP is the key molecule that antagonizes the action of azole antifungals in Candida yeasts.

 

Clinical investigations

Clinical investigations are performed by the hospital part of the team, under the reponsibility of I. Accoceberry. It corresponds to short studies undertaken in the domain of both medical mycology and parasitology, for either validating new diagnostic tests or techniques, participating to epidemiological multicentric studies, or reporting new emerging pathogens with a taxonomic validation at the molecular level.

 

Transfer of technology


Collaborations with industrial partners is an important source of funding for the group. The area of expertise of the team led us to manage several industrial contracts during the 2009-2014 period in different domains such as the elaboration of a new range of vaccines managed against the veterinary apicomplexa, the study of the antifungal activity of a new chemical class of molecules, the study of an antifungal activity of a natural antibiotic molecule, or the Improvement of the production of lipid derivatives by mutant strains of an ascomycetous yeast.

 

Vidéo :

Vidéo d'interaction Candida - phagocytes (accès restreint).

Publications :

Navarro-Arias MJ, Defosse TA, Dementhon K, Csonka K, Mellado-Mojica E, Dias Valério A, González-Hernández RJ, Courdavault V, Clastre M, Hernández NV, Pérez-García LA, Singh DK, Vizler C, Gácser A, Almeida RS, Noël T, López MG, Papon N, Mora-Montes HM. Disruption of Protein Mannosylation Affects <i>Candida guilliermondii</i> Cell Wall, Immune Sensing, and Virulence.Front Microbiol. 2016;7:1951

Gabriel F, Accoceberry I, Bessoule JJ, Salin B, Lucas-Guérin M, Manon S, Dementhon K, Noël T. A Fox2-dependent fatty acid ß-oxidation pathway coexists both in peroxisomes and mitochondria of the ascomycete yeast Candida lusitaniae.PLoS One. 2014;9(12):e114531

Gabriel F, Sabra A, El-Kirat-Chatel S, Pujol S, Fitton-Ouhabi V, Brèthes D, Dementhon K, Accoceberry I, Noël T. Deletion of the uracil permease gene confers cross-resistance to 5-fluorouracil and azoles in Candida lusitaniae and highlights antagonistic interaction between fluorinated nucleotides and fluconazole.Antimicrob Agents Chemother. 2014 Aug;58(8):4476-85

Sabra A, Bessoule JJ, Atanasova-Penichon V, Noël T, Dementhon K. Host-pathogen interaction and signaling molecule secretion are modified in the dpp3 knockout mutant of Candida lusitaniae.Infect Immun. 2014 Jan;82(1):413-22

Lacroix C, Gicquel A, Sendid B, Meyer J, Accoceberry I, François N, Morio F, Desoubeaux G, Chandenier J, Kauffmann-Lacroix C, Hennequin C, Guitard J, Nassif X, Bougnoux ME. Evaluation of two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems for the identification of Candida species.Clin Microbiol Infect. 2014 Feb;20(2):153-8

Gabriel F, Noel T, Accoceberry I. Lindnera (Pichia) fabianii blood infection after mesenteric ischemia.Med Mycol. 2012 Apr;50(3):310-4

Millerioux Y, Clastre M, Simkin AJ, Courdavault V, Marais E, Sibirny AA, Noël T, Crèche J, Giglioli-Guivarc'h N, Papon N. Drug-resistant cassettes for the efficient transformation of Candida guilliermondii wild-type strains.FEMS Yeast Res. 2011 Sep;11(6):457-63

Millerioux Y, Clastre M, Simkin AJ, Marais E, Sibirny AA, Noël T, Crèche J, Giglioli-Guivarc'h N, Papon N. Development of a URA5 integrative cassette for gene disruption in the Candida guilliermondii ATCC 6260 strain.J Microbiol Methods. 2011 Feb;84(2):355-8

Ness F, Prouzet-Mauleon V, Vieillemard A, Lefebvre F, Noël T, Crouzet M, Doignon F, Thoraval D. The Candida albicans Rgd1 is a RhoGAP protein involved in the control of filamentous growth.Fungal Genet Biol. 2010 Dec;47(12):1001-11

Vandeputte P, Pineau L, Larcher G, Noel T, Brèthes D, Chabasse D, Bouchara JP. Molecular mechanisms of resistance to 5-fluorocytosine in laboratory mutants of Candida glabrata.Mycopathologia. 2011 Jan;171(1):11-21

Chapeland-Leclerc F, Hennequin C, Papon N, Noël T, Girard A, Socié G, Ribaud P, Lacroix C. Acquisition of flucytosine, azole, and caspofungin resistance in Candida glabrata bloodstream isolates serially obtained from a hematopoietic stem cell transplant recipient.Antimicrob Agents Chemother. 2010 Mar;54(3):1360-2

Gordien JB, Pigneux A, Vigouroux S, Tabrizi R, Accoceberry I, Bernadou JM, Rouault A, Saux MC, Breilh D. Simultaneous determination of five systemic azoles in plasma by high-performance liquid chromatography with ultraviolet detection.J Pharm Biomed Anal. 2009 Dec 5;50(5):932-8

Florent M, Noël T, Ruprich-Robert G, Da Silva B, Fitton-Ouhabi V, Chastin C, Papon N, Chapeland-Leclerc F. Nonsense and missense mutations in FCY2 and FCY1 genes are responsible for flucytosine resistance and flucytosine-fluconazole cross-resistance in clinical isolates of Candida lusitaniae.Antimicrob Agents Chemother. 2009 Jul;53(7):2982-90

Reboutier D, Piednoël M, Boisnard S, Conti A, Chevalier V, Florent M, Gibot-Leclerc S, Da Silva B, Chastin C, Fallague K, Favel A, Noël T, Ruprich-Robert G, Chapeland-Leclerc F, Papon N. Combination of different molecular mechanisms leading to fluconazole resistance in a Candida lusitaniae clinical isolate.Diagn Microbiol Infect Dis. 2009 Feb;63(2):188-93

Review and Book chapters

Accoceberry, I. Aérocontaminants fongiques (moisissures opportunistes et allergisantes). 2013, pp 55-105, dans "Mycologie Médicale", Ripert C., Édition Tec&Doc, Lavoisier, Paris. ISBN 978-2-7430-1488-9.
Noël T. Introduction et Généralités. 2013, pp 1-48, dans "Mycologie Médicale", Ripert C., Édition Tec&Doc, Lavoisier, Paris. ISBN 978-2-7430-1488-9.
Noël T. 2012. The cellular and molecular defense mechanisms of the Candida yeasts against azole antifungal drugs. J Mycol Med 22:173–178.
Michel-Nguyen A, Favel A, Noël T. Candida lusitaniae. Actualités Permanentes en Microbiologie Clinique volume IX, mise à jour n°2, 2010 (section 12 Mycologie, Chapitre 5, pp 1-14), Éditons EKSA, Paris.

Patent

Noël T, Accoceberry I, Bessoule JJ, Manon ST, Gabriel F. 2012. Novel beta-oxidation pathway and hemicascomycetes yeast mutants. U.S. Patent EP2502932.

 

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