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, J. Jourde)
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 about 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, E. Mordelet, S. Daou, 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). Our data showed that effective crossing takes place as soon as 6 hours after inoculation, in the cortical capillaries and never in the choroids plexus. During the dissemination process, crossing of various type of endothelium occurs. Dead yeasts are unable to cross the endothelium. Furthermore, we demonstrated that modification of yeast size and capsule structure take place that differ according to tissues. We are currently designing cellular models in order to better analyze the interaction between C. neoformans and endothelial cells as well as monocytes (figure 1).
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.
The Candida albicans β1,2 oligo mannosides (G. janbon, C. Mille)
The C. albicans cell wall represents the contact surface between the yeast and the host cells. It is mostly composed of glucan molecules (polymers of β-1,3 and β-1,-6 linked glucose residues), mannan molecules (polymers of α-1,2 et α-1,3 mannose residues) and mannoproteins. In addition to these α linked mannose residues, some β-1,2 oligomannosides are associated to the cell wall mannoproteins and to the phospholipomannan (PLM). Numerous studies have shown a linkage between the presence of these residues and the virulence of C. albicans. Whereas the biosynthetic pathways of the β-glucans and the mannoproteins are least partially known, no gene involved in the biosynthesis of the β-1,2 oligomannosides was identified. In collaboration the D. Poulain laboratory in Lille, we have identified some genes necessary for the biosynthesis of the β-1,2 oligomannosides in C. albicans, C. glabrata and S. cerevisiae and we are studying the influence of these residues on the candidiosis physiopathology.
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). A genetic screen for Candida glabrata Biofilm mutants was performed using a 96-well plate model of biofilm formation on an insertional mutants library. We identified more than 100 mutant strains affected in their abilities to form biofilm (figure 2). 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.
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.
Study on immune reconstitution inflammatory syndrome during systemic mycoses in AIDS (O. Lortholary, F. Dromer)
Immune reconstitution inflammatory syndrome (IRIS) has been described during cryptococcosis in the setting of HIV infection. The syndrome is characterized by acute symptoms compatible with severe inflammation in the context of negative culture and effective response to treatment by highly active antiretroviral therapy (HAART) in terms of virological or immunological criteria. Our database on the epidemiological survey of cryptococcosis in France and the observational study on the management of secondary prophylaxis for cryptococcosis in HIV-infected patients under highly active antiretroviral therapy (HAART) (collaboration INSERM U593, Bordeaux) were used. We were able to calculate the incidence, to describe the symptoms and analyze the risk factors of developing IRIS-associated cryptococcosis in collaboration with A. Fontanet (Unité d'Epidémiologie des Maladies Emergentes). Similar cases following diagnosis of histoplasmosis in HIV-infected patients treated with HAART were also described. In both context, IRIS occurred in patients who were prescribed HAART shortly after the diagnosis of disseminated mycosis. These data indicate that special care should be taken in the management of these patients.
Study on in vitro and in vivo efficacy of antifungal combinations (E. Dannaoui, P. Schwarz, F. Dromer, O. Lortholary)
Animal models have been used to analyze the in vivo efficacy of antifungal combinations against Aspergillus fumigatus and Cryptococcus neoformans. Amphotericin B, flucytosine, caspofungin, and itraconazole were used in murine models of disseminated cryptococcosis and aspergillosis. Endpoint were either survival or fungal burden in target organs. We were able to confirm data obtained in vitro using antifungal combination against A. fumigatus and to demonstrate the potential interest of some of the combination. In the model of cryptococcosis, we were interested to study combination therapy in the case of infection with isolates resistant to some of the antifungal agents used and were able to demonstrate in vivo/in vitro correlation in this case.
Molecular identification of filamentous fungi (M. Desnos, E. Dannaoui, D. Garcia-Hermoso, S. Bretagne, Jean-Charles Gantier, F. Dromer)
Identification of filamentous 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. Fungal species responsible for black grains mycetoma are especially difficult to identify. We thus sequenced rDNA (ITS1- 5.8S - ITS2 regions) of several isolated from various species responsible for these chronic infections in order to test whether molecular identification could be useful. Analysis is underway but already showed that sequencing can be used to characterize some of these difficult to identify species.
Figure 1 : C. neoformans internalized by murine monocytes in vitro. Strain H99 was labelled with fluorescein prior to incubation with monocytes. Left panel : DIC microscopy, right panel epifluorescence (magnitude x1000).
Figure 2: Biofilm formed by C. glabrata (mutant biofilm++) on a plastic slide (scanning electronic microscopy)
Keywords: Cryptococcus neoformans, Candida albicans, Candida glabrata, Aspergillus, capsule, polysaccharide, mycology, pathophysiology, antifungals, biofilms, blood brain barrier