Biofilm formation by Cryptococcus neoformans: Effect of specific antibody and consequences for drug therapy and immune responses

Abstract

Cryptococcus neoformans is an encapsulated yeast-like fungus that is a relatively frequent cause of life-threatening meningoencephalitis in immunosuppresed individuals. This fungus forms biofilms on prosthetic medical devices. In fact, the increasing use of ventriculoperitoneal shunts to manage intracranial hypertension associated with cryptococcal meningoencephalitis highlights the importance of investigating the biofilm-forming properties of this organism.;We developed a system to study cryptococcal biofilm formation in vitro using a microtiter plate model, microscopic examinations and a colorimetric XTT reduction assay to observe the metabolic activity of cryptococci within a biofilm. C. neoformans biofilm formation was inferred from the observation that cells grown in polystyrene plates formed a tenacious attachment to the plastic surface consisting of metabolically active microcolonies enmeshed in a polysaccharide-containing matrix. C. neoformans biofilm formation was dependent on the presence and release of the polysaccharide capsule onto the polystyrene solid support.;Since C. neoformans can form biofilms on medical devices using polysaccharide components, this raised the possibility that antibody to Glucurunoxylomannan (GXM) would interfere with cryptococcal biofilm formation. Addition of GXM-binding monoclonal antibodies (mAb) to C. neoformans cultures did not affect fungal cell adhesion but prevented biofilm formation by interference with release of GXM. Moreover, we associated antibody-mediated inhibition of biofilm formation with protective antibodies, suggesting the possibility that this effect is involved in antibody protective efficacy against C. neoformans.;Since biofilm-forming microbes, including C. neoformans, are resistant to antimicrobial agents and host defense mechanisms, the use of indwelling medical devices is often complicated by high rates of infections. To target cryptococcal biofilms, we used GXM-specific mAbs as delivery vehicles for cytocidal alpha-radiation. We demonstrated that mAb 18137 penetrated the exopolymeric matrix of a biofilm and bound to metabolically active C. neoformans cells, which were susceptible to alpha-radiation. Our results indicate that C. neoformans biofilms are susceptible to treatment with antibody-targeted alpharadiation, suggesting a novel strategy for treatment of microbial biofilms on indwelling medical devices.;This thesis provides evidence that biofilm formation by C. neoformans may play an important role in fungal pathogenesis and proposes a novel specific antibody-mediated strategy for the prevention and treatment of microbial biofilms.