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, N. Lee, 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. 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 we have already constructed more than 60 mutant strains. These mutants are used to study the involvement of the capsule structure on the cryptococcosis physiopathology but also for more basic projects on different aspect of the biosynthesis of the polysaccharide in the eukaryotes (regulation of the nucleotide-sugars in the vesicles, relationship between the splicing of some specific genes and the capsule structure ). Thus, C. neoformans could be considered as a very good model for the study of the biosynthesis of the polysaccharides biosynthetic pathway in the higher eukaryotes.
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. In collaboration with F. Chrétien (INSERM EMI 0011, Hôpital H. Mondor, Créteil) we are analyzing the mechanisms allowing C. Neoformans crossing of the BBB. We are currently designing cellular models in order to better analyze the interaction between C. neoformans and endothelial cells as well as monocytes.
Analysis of the host response to CNS infection by C. neoformans ( S. Mémet)
Very few data are available as regards to the role of host cells, namely endothelial and glial cells, in crossing of the blood brain barrier (BBB), dissemination of C. neoformans, as well as in induction/repression of an inflammatory reponse. We have therefore initiated a thorough analysis of the inflammatory signalling triggered by C. neoformans, with particular emphasis on the role of the transcription factor NF-κB, a major regulator of .inflammation, innate and acquired immunity. Experiments are being conducted with two fungal pathogens that induce either a scanty (C. neoformans) or a very strong (C. albicans) inflammatory response, C. neoformans being responsible for meningoencephalitis. Comparing the impact of these two pathogens should shed light on the molecular mechanisms responsible for similarities and specificities of these two fungi in mounting an innate immune host response, and possibly elucidate the reasons for their differences in BBB crossing efficacy and subsequent constitution of meningoencephalitis. Preliminary results with a human BBB endothelial cell model indicate that although both fungi activate NF-κB in these cells, the time-course of activation is different.
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, M. Riera)
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 different units within the Institut Pasteur (UP Biologie et Pathogénicité fongiques, U Chimie Organique, PF Cristallogenèse et diffraction des rayons X and PF Production de protéines recombinantes et anticorpsis) to determine molecular events that lead to the development of biofilms (PTR173). A genetic screen for Candida glabrata biofilm mutants was performed using a 96-well plate model of biofilm formation and an insertional mutants library. We identified more than 100 mutant strains affected in their ability to form biofilm. The study of some insertion sites allowed the identification of an adhesin necessary for biofilm formation and to understand its regulation. We have thus demonstrated that this adhesin encoding gene was regulated by the sub-telomeric silencing machinery, by a signal activated during biofilm growth and the Yak1p kinase. We have also demonstrated that Yak1p is necessary for biofilm formation in C. albicans, thus confirming the validity of the C. glabrata model.
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. Garcia-Hermoso, J.C. Ganthier, D. Raoux, D. Hoinard, O. Ronin) and their molecular typing (S. Bretagne, D. Garcia-Hermoso), as well as antifungal susceptibility testing 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 (Active Surveillance Program on Yeast Related bloodstream Infections) has been implemented in Paris and suburbs. Its objective is to monitor the infections and characterize overtime the yeasts responsible for fungemia (identification, typing and susceptibility testing profiles).
A secured web site called RESOMYC only accessible to clinician and microbiologists/mycologists in charge of patients with invasive mycoses in France has recently been implemented in order to allow on-line notification and retrieval of current data on the epidemiology of invasive mycoses surveyed at the Reference Center.
Epidemiological studies on fungal infections (O. Lortholary, F. Dromer, S. Bretagne)
Study of the immune reconstitution inflammatory syndrome during systemic mycoses (IRIS): We studied the risk factors for IRIS in HIV-infected patients with incident cryptococcosis or histoplamosis. In collaboration with Nina Singh (Pittsburgh University), we also described a similar entity occurring in solid transplant recipients for whom immunosuppressive therapy is decreased in the context of opportunistic fungal infections.
Study of fungal endocarditis: In collaboration with Agnès Lefort (Internal Medicine, Hôpital Beaujon), we have designed a prospective multicenter study (MYCENDO) to analyze the epidemiology of fungal endocarditis and to assess the usefulness of serological and molecular tools in the diagnosis and the prognosis evaluation.
Combination of amphotericin B with flucytosine against flucytosine-susceptible or -resistant isolates of C. neoformans (P. Schwarz, F. Dromer, O. Lortholary, E. Dannaoui)
Amphotericin B in combination with flucytosine is currently the first-line therapy for cryptococcal meningitis. This combination is not recommended and could be even deleterious when the infecting isolate of C. neoformans is resistant to flucytosine. Nevertheless, few data are available to support this hypothesis. For these reasons, the combination has been evaluated against both flucytosine-susceptible and -resistant isolates. Two strains have been tested in vivo in a murine model of disseminated cryptococcosis and the results have shown that a synergistic interaction was obtained against both the flucytosine-susceptible and -resistant isolate. The combination, subsequently tested in vitro by different methodologies against a large panel of flucytosine-resistant isolates with various mechanisms of resistance, has been shown to be either synergistic or indifferent but never antagonistic. These results suggest that the combination of amphotericin B with flucytosine can be used in patients even if the infecting isolate is resistant to flucytosine.
Molecular identification of pathogenic fungi (D. Garcia-Hermoso, M. Desnos, S. Bretagne, Jean-Charles Gantier, F. Dromer)
Identification of fungi is classically based on morphology (microscopic examination and growth characteristics). For several groups of fungi, these characteristics are not satisfactory to allow identification because of poor or delayed fructifications and furthermore very few experts are available. We systematically sequence rDNA (ITS1- 5.8S - ITS2 & 26S regions) of every isolate in order to test whether molecular identification could be useful. We are building a large database combining epidemiological data, numeric images, physiological characteristics and sequences for all pathogenic isolates sent to the NRCMA.
Molecular identification of zygomycetes (P. Schwarz, E. Dannaoui): Zygomycetes are responsible for life-threatening infections (zygomycosis) in immunocompromized patients, in diabetic patients but also in immucompetent individuals after severe traumatic injuries. About 20 species belonging to about 10 genera are known to cause infections in humans. A large number of zygomycetes strains has been studied by molecular biology and the results showed that a rapid and reliable identification to the species level is possible. Moreover, this study has shown that some zygomycetes species are misplaced taxonomically. The availability of a molecular identification tool suggest that diagnosis of these infections directly from infected tissues, which are often negative in culture, would be possible.
Keywords: Cryptococcus neoformans, Candida albicans, Candida glabrata, NF-kappaB, capsule, Zygomycete, mycology, pathophysiology, antifungals, biofilms, blood brain barrier