Antibody-mediated immobilization of Cryptococcus neoformans promotes biofilm formation

Abstract

Most microbes, including the fungal pathogen Cryptococcus neoformans, can grow as biofilms. Biofilms confer upon microbes a range of characteristics, including an ability to colonize materials such as shunts and catheters and increased resistance to antibiotics. Here, we provide evidence that coating surfaces with a monoclonal antibody to glucuronoxylomannan, the major component of the fungal capsular polysaccharide, immobilizes cryptococcal cells to a surface support and, subsequently, promotes biofilm formation. We used time-lapse microscopy to visualize the growth of cryptococcal biofilms, generating the first movies of fungal biofilm growth. We show that when fungal cells are immobilized using surface-attached specific antibody to the capsule, the initial stages of biofilm formation are significantly faster than those on surfaces with no antibody coating or surfaces coated with unspecific monoclonal antibody. Time-lapse microscopy revealed that biofilm growth was a dynamic process in which cells shuffled position during budding and was accompanied by emergence of planktonic variant cells that left the attached biofilm community. The planktonic variant cells exhibited mobility, presumably by Brownian motion. Our results indicate that microbial immobilization by antibody capture hastens biofilm formation and suggest that antibody coating of medical devices with immunoglobulins must exclude binding to common pathogenic microbes and the possibility that this effect could be exploited in industrial microbiology.

FIG. 1.

C. neoformans motility in presence and absence of specific MAb. Glass bottom petri dishes were uncoated or coated with either MAb 18B7 or MOPC-21, and C. neoformans (Cn) cells in PBS were added. The number of motile cells entering the view of the microscope was counted over 10 h. * denotes that the number of motile cells in the 18B7 + Cn group was zero.

FIG. 2.

Time-lapse microscopic study of C. neoformans biofilm formation. C. neoformans cells were anchored to a glass bottom petri dish, using MAb 18B7, and biofilm formation was induced. Photographs of biofilm formation were taken every 4 min using a 20× (numerical optovar of 1.6) objective. Variant cells (indicated by white arrows) were released from the biofilm community and exhibited motility.

FIG. 3.

Biofilm formation as measured by XTT reduction assay. Cryptococcus neoformans (Cn) cells were grown in 96-well plates that were coated with MAb 18B7 or MAb MOPC-21 or left untreated. As a control, MAb 18B7-coated wells were inoculated with heat-killed C. neoformans (HK Cn). Cell viability was measured at intervals over 12 h. Conditions were measured for six wells, and the average was plotted. Error bars represent standard deviations, and P values were calculated using unpaired t tests. Assays were performed three times, and they generated similar results.

FIG. 4.

Biofilm formation as determined by spot ELISA. (a) Spot ELISA was used to determine polysaccharide shedding during the initial stages of biofilm formation. (b) Area of polysaccharide shedding, as determined by spot ELISAs. (c) C. neoformans cell deposition onto plastic surfaces was determined using spot ELISAs. C. neoformans (Cn) cells were added to 96-well plates coated with MAb 18B7 or to plates left untreated. Heat-killed (HK) cells were also added to wells containing MAb 18B7. Spot area and number were measured over 6 h. Bars represent the averages of 9 to 30 spots (Fig. 5b) and the average number of spots from three wells (Fig. 5c). Error bars indicate standard deviation, and P values were calculated using Student's t tests with the Bonferroni correction for multiple comparisons. * denotes P values of <0.0001, corresponding to the time points in 18B7-treated wells.

FIG. 5.

Fluorescence microscopy of GXM within biofilm matrix. (a) Bright-field microscopy results of mature biofilms. (b) Biofilms were stained with Alexa Fluor 488-labeled 18B7 and viewed using green fluorescent light. Arrows indicate halos of GXM surrounding biofilm microcolonies. A 10× objective (numerical optovar of 1.6) was used.