Unit: Molecular Mycology
Director: DROMER Françoise
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.
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 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. These mutants will be used to study the epitopes involved in the crossing of the blood brain barrier.
Sudy of a protein from C. neoformans involved in the humoral response during cryptococcosis (F. Dromer, 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. In collaboration with JL Guesdon and S. Rouyre we designed two immunoenzymatic assays (ELISA and western-blot) that allowed us to analyze in patients with cryptococcosis the baseline production anti-GXM and anti-recombinant Pep1p antibodies. We found that anti-rPep1 antibodies were only detected in patients who survived the infection. Monoclonal antibodies specific for various epitopes of Pep1p were produced. One of them significantly prolonged the survival of infected mice in the model of disseminated cryptococcosis. Other studies are warranted to further elucidate the protective effect of Pep1 antibodies.
Study of C. neoformans crossing of the blood brain barrier (BBB) (C. Charlier, F. Dromer)
Little is known on the mechanisms allowing C. neoformans to cross the BBB. The model of disseminated cryptococcosis in outbred mice and classical fungal burden evaluation and histophatology are used to analyze the kinetics of BBB crossing and dissemination after fungemia. The study is done in collaboration with F. Chrétien (INSERM EMI 0011, Hôpital H. Mondor, Créteil). Preliminary results showed that effective crossing takes place within 24 hours after inoculation. Other techniques are currently designed to better analyze the events.
Human cryptococcosis (F. Dromer, O. Lortholary, O. Ronin)
We recently analyzed in collaboration with A. Fontanet (Epidemiology of Emerging Diseases) and S. Mathoulin-Pélissier (Institut Bergonié, Bordeaux) the trends in the epidemiology of cryptococcosis following the introduction of highly active antiretroviral therapy (HAART) in 1996 in France. Using the database of 1644 cases of cryptococcosis recorded among HIV-positive patients at the National Reference Center for Mycoses in France, we found that the incidence of cryptococcosis among HIV-positive patients decreased by 46% after 1997. Furthermore, independent risk factor for the development of cryptococcosis were an African origin, an age older than 45 years, an heterosexual HIV contamination, no previous AIDS-defining illness, no previous diagnosis of HIV infection. These results suggested that failure to consult and considering oneself not at risk were determinant in the current epidemiology of HIV-related cryptococcosis in France
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 (O. Lortholary, collaboration INSERM U593, Bordeaux). The initial mortality observed during HIV-associated cryptococcosis is still 17%, but the overall mortality has decreased since 1997 and the introduction of HAART. In patients with prolonged immune reconstitution and adequate antifungal therapy for several months, the interruption of maintenance antifungal therapy can be proposed.
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.
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 either unable to form biofilms or forming more biofilms than the wild type strain. Study of the mutants allowed us to identify an adhesin necessary for the biolfilm formation in C. glabrata and to understand the regulation of the corresponding gene's transcription. We have also deleted two genes of C. albicans coding for cell wall β-glucosidases of C. albicans which expression have been shown by transcriptome analysis to be induced during biofilm formation. Their study confirmed their essential role in the formation of biolfilm formation by C. albicans.
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 (D. Hoinard, J.C. Ganthier, D. Garcia-Hermoso, O. Ronin) and their molecular typing (S. Bretagne, D. Garcia-Hermoso), as well as antifungal susceptibility testing (D. Hoinard, 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.
An active surveillance system (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 on in vitro and in vivo efficacy of antifungal combinations (E. Dannaoui, P. Schwarz, F. Dromer, O. Lortholary)
We used several approaches to test the in vitro activity of double and triple combination of antifungal drugs against Aspergillus fumigatus and C. neoformans. The antifungal tested were amphotéricine B, 5 fluorocytosine, caspofungine, and azoles (itraconazole, fluconazole or voriconazole). The checkerboard technique, a microdilution method adapted from the standard established for the study of one drug by the National Committee for Clinical Laboratory Standards was used to study double, and also for the first time to our knowledge to study triple combinations on Aspergillus. Other techniques were used to evaluate double combinations (agar diffusion techniques, and killing curves (this latter technique only on C. neoformans). These studies allowed us to demonstrate the potential interest of combinations that have not been evaluated yet. Studies in experimental models of disseminated aspergillosis and cryptococcosis will allow us to determine the relevance of these findings.
Keywords: Cryptococcus neoformans, capsule, Candida albicans, polysaccharide, mycology, pathophysiology, antifungals, biofilms, blood brain barrier