Use of morphology index histograms to quantify populations of the fungal pathogen Paracoccidioides brasiliensis

This study developed an objective method to quantify morphological variation in Paracoccidioides brasiliensis, a dimorphic fungal pathogen that switches between spherical yeast cells at 37°C and elongated hyphal forms at lower temperatures. The researchers created a morphology index (Mi) formula based on cell length, diameter, and septal diameter measurements, calibrated so pure yeast cells scored ~1 and true hyphae scored ~4. This mathematical approach, adapted from work on Candida albicans, allowed discrimination of morphological populations using histograms. During temperature-induced transitions between forms, mean Mi values changed linearly with time, indicating a continuous process. Notably, P. brasiliensis showed an inverse relationship between Mi and cell wall chitin and 1,3-α-glucan content, opposite to what occurs in C. albicans. The yeast phase contained three times more chitin than the mycelial phase. Additionally, 1,3-β-glucan accumulated specifically in well-developed hyphae, suggesting its requirement for complete hyphal formation. The method successfully quantified intermediate morphologies in both wild-type and mutant strains that conventional description could not adequately characterize.

Key findings

• A morphology index (Mi) formula was developed for P. brasiliensis using cell measurements, with Mi values of ~1 for yeast and ~4 for hyphae, enabling objective quantification of intermediate forms
• During temperature-induced dimorphic transitions in both directions, mean Mi values varied linearly with time, indicating a continuous morphological process rather than discrete phase changes
• P. brasiliensis showed inverse relationships between Mi and cell wall chitin and 1,3-α-glucan content, with the yeast phase containing three times more chitin than the mycelial phase
• The yeast phase is rich in 1,3-α-glucan while the mycelial phase substitutes 1,3-β-glucan, which accumulates specifically in well-developed hyphae, suggesting distinct polysaccharide requirements for each morphological form
• Mi histograms successfully distinguished pure morphological populations from mixed populations and revealed atypical morphologies in dimorphic mutants that single Mi values alone could not adequately characterize