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  Director : DROMER Françoise (dromer@pasteur.fr)


  abstract

 

A better understanding of the interactions between the hosts and pathogenic fungi should improve the prognosis of systemic fungal infections. We thus focus our projects on Cryptococcus neoformans and Candida albicans pathogenicity by studying the host (clinical and epidemiological studies, animal models of infection) and the fungus (virulence factors, variability) sides.



  report

cale

Cryptococcosis is a life-threatening infection that occurs in up to 30% of patients with AIDS in tropical area in Africa and South East Asia. The most common clinical feature is a meningoencephalitis associated with a severe prognosis (³ 20% acute mortality) which explains the need for new prophylactic and therapeutic approaches. One of the characteristics of C. neoformans is the presence of a capsule made of polysaccharides, mainly glucuronoxylomannan (GXM). We are studying the pathophysiology of the infection and the virulence factors, among which the capsule has been shown to be the most important.

C. neoformans capsule (G. Janbon, F. Moyrand)

Among the evidence demonstrating a major role for the capsule in the virulence of the yeast, are the avirulence of acapsular strains and the various deleterious effects on the immune response assigned to GXM. Immunofluorescence studies with monoclonal antibodies specific for various epitopes of the GXM allowed us to demonstrate that the capsule structure is highly variable from cell to cell within a given yeast cell population. Our working hypothesis is that the variability of the capsule structure plays a role in the virulence of the yeast. The mechanisms and molecular determinants involved in the antigenic variability have been studied and uncovered for numerous procaryote and eucaryote microorganisms, but no study has been performed for pathogenic fungi. We have cloned different genes coding for proteins involved in the biosynthetic pathway of the cryptococcal capsule and are studying the mechanisms responsible for the capsule structure variability. A program of systematic disruption of the genes potentially involved in the capsule synthesis has been implemented and should provide within 2 years up to 150 mutants strains for study.

Sudy of a protein from C. neoformans involved in the humoral response during cryptococcosis (F. Dromer, S. Jacquelin, J. Lemay)

We have demonstrated that the humoral response to proteic antigens of C. neoformans correlates with the outcome of the infection in a model of disseminated cryptococcosis in outbred mice. We have identified one of the protein associated with a humoral response in mice surviving a lethal challenge and cloned the corresponding gene PEP1. The next step will be to determine if the presence of specific antibodies in the serum from patients with cryptococcosis is predictive of the outcome and to evaluate the potential usefulness of the protein in immunotherapy. This project is done in collaboration with JL Guesdon and S. Rouyre and Y. Germani (IP Bangui) (PTR58). After production of the recombinant protein, we designed two immunoenzymatic assays (ELISA and western-blot) that will now allow us to analyze the anti-GXM and anti-Pep1p antibodies in patients with cryptococcosis. Monoclonal antibodies specific for various epitopes of Pep1p have also been produced by using splenic cells from mice that have been immunized with rPep1p or that have survived a lethal challenge with C. neoformans. Their effect on the course of the infection as well as that of rPep1p will now be tested in the model of disseminated cryptococcosis.

Human cryptococcosis (F. Dromer, O. Lortholary, L. Improvisi, O. Ronin)

A prospective multicentric prospective study on the pathophysiology of C. neoformans infection (Crypto A/D study) was designed in April 1997 to collect clinical data, isolates and sequential biological fluids from 230 patients diagnosed with cryptococcosis in France. We recently collected the last missing files and are starting the analysis.

We have studied the immune response (pro- and anti-inflammatory cytokines measured in plasma) in patients with cryptococcosis (75 HIV-positive patients and 14 HIV-negative patients) and analyzed the data according to the HIV status, the infecting serotype and the extend of the dissemination. We have shown that the inflammatory response in the plasma was increased in HIV-infected patients with cryptococcal fongemia compared to what is observed in patients with other immune defects or in the CSF. This is a further demonstration of the compartimentalization of the immune response during human cryptococcosis as already assessed in the murine model (O. Lortholary).

An observational study on the management of secondary prophylaxis for cryptococcosis in 280 HIV-infected patients under highly active antiretroviral therapy (HAART) has been implemented in 2001 and should be analyzed in 2003 (O. Lortholary, collaboration INSERM U330, Bordeaux).

Candida albicans

C. albicans is a commensal of the digestive tract responsible for a wide range of infections in immunocompetent and immunodeficient patients. Patients with prolonged neutropenia following chemotherapy or bone marrow transplantation, organ transplant recipients are at risk for candidemia. C. albicans is also responsible of nosocomial infections in surgery units, intensive care units.

Biosynthesis of b-1,2 oligomannosides in C. albicans (G. Janbon, C. Mille)

Contact between C. albicans and the environment through the fungal cell wall involves interactions with various molecules, i.e. glucans (b-1,3 et b-1,6 glucose), mannans (a-1,2 et a-1,3 mannose) and mannoproteins. In addition, b-1,2 oligomannosides (associated with mannoproteins or a phospholipomannan, PLM) are found and several studies have shown that these b-1,2 oligomannosides are involved in the virulence of C. albicans. The biosynthetic pathway leading to glucans or mannoproteins have been at least partially described, whereas that leading to the b-1,2 oligomannosides are unknown. In collaboration with D. Poulain (Lille), we have identified some genes responsible for the synthesis of the b-1,2 oligomannosides in C. albicans, Candida glabrata and Saccharomyces cerevisiae in order to facilitate the study of their influence in the pathophysiology of candidiasis.

Prevention of gut colonization by C. albicans with synthetic b-1,2 oligomannosides (F. Dromer, L. Improvisi)

In collaboration with D. Poulain (Lille) and J.-M. Mallet (ENS, Paris), we demonstrated that synthetic b-1,2 oligomannosides but not a-1,2oligomannosides prevented gut colonization in a dose dependent manner in in the infant mouse model of instestinal candidiasis. This first demonstration that a synthetic analogue of a yeast adhesin can prevent yeast colonization in the gut opens the possibility of new prophylactic strategies.

Biofilm formation in C. albicans and Candida glabrata (G. Janbon, F. Dromer, I. Iraqui)

Biofilms are tridimentional structures composed of microorganisms that developed of various surfaces. Candida biofilms could colonize intravascular cathethers or prosthetic devices and be responsible for recurrence despite antifungal therapy. Our objective in collaboration with the groups of C. d'Enfert (UP Biologie et Pathogénicité fongiques) and JM Ghigo (G5, Génétique des biolfilms) is to determine molecular events that lead to the development of biofilms (PTR50). We have isolated several mutants of C. glabrata that are unable to form biofilms and are currently isolating the corresponding genes. We have also started to delete some of the genes of C. albicans which expression have been shown by transcriptome analysis to be induced during biofilm formation.

National Reference Center Mycology and Antifungal Agents (F. Dromer, O. Lortholary)

The National Reference Center has several missions that include (1) expertise in the identification of pathogenic fungi (J.C. Ganthier, D. Garcia-Hermoso, O. Ronin) and their molecular typing (S. Bretagne, D. Garcia-Hermoso), as well as antifungal susceptibility testing (D. Garcia-Hermoso, O. Ronin) and advice for the management of patients with severe mycoses; (2) epidemiological survey of all rare, severe or exotic mycoses as well as the emergence of resistance to antifungal drugs.

A regional observatory for yeasts (Observatoire des Levures) has been implemented in Paris and suburbs. Its objective is to monitor the infections and characterize overtime the yeasts responsible for fungemia (typing and susceptibility testing profiles).

Study of the efficacy of antifungal combinations (double and triple) on Aspergillus fumigatus has started in order to find means of improving the still severe prognosis of invasive aspergillosis (E. Dannaoui). The effect of new molecules (echinocandins and new azoles) is being studied in vitro in liquid medium using checkerboard experiments and in vivo in the disseminated model of aspergillosis in mice. The efficacy of some combinations are also determined in vitro on C. neoformans (E. Dannaoui, P. Schwarz)

Keywords: Cryptococcus neoformans, capsule, Candida albicans, polysaccharide, mycology, pathophysiology, antifungals, biofilms



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  publications

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  personnel

  Office staff Researchers Scientific trainees Other personnel
  Edith Martin, Secretary,emartin@pasteur.fr DROMER, Françoise, IP, chef de laboratoire,dromer@pasteur.fr

JANBON, Guilhem, IP, chargé de recherches,janbon@pasteur.fr

LORTHOLARY, Olivier, AP-HP, olivier.lortholary@avc.ap-hop-paris.fr

GANTIER, Jean-Charles, Pr. des Universités, jc.gantier@cep.u-psud.fr

BRETAGNE, Stéphane, PU-PH, bretagne@univ-paris12.fr
MILLE, Céline, thesis student

LEMAY, Julie,jlemay@pasteur.fr

CHARLIER, Caroline,charlier@pasteur.fr

DANNAOUI, Eric,dannaoui@pasteur.fr

IRAQUI, Ismaïl,iiraqui@pasteur.fr

SCHWARZ, Patrick,pschwarz@pasteur.fr

IMPROVISI, Luce, engineer

JACQUELIN, Sébastien, technician

MOYRAND, Frédérique, technician,

RONIN, Olivier, technician,oronin@pasteur.fr

GARCIA-HERMOSO, Dea, engineer,dea@pasteur.fr

ANFRY, Lucile

TOURNAIRE, Marc


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