Capsule growth in Cryptococcus neoformans is coordinated with cell cycle progression

Abstract

The fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G2/M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G1/S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans.

Importance: Cryptococcus neoformans is a pathogenic fungus that has significant incidence worldwide. Its main virulence factor is a polysaccharide capsule that can increase in size during infection. In this work, we demonstrate that this process occurs in a specific phase of the cell cycle, in particular, in G1. In agreement, mutants that have an abnormal longer G1 phase show larger capsule sizes. We believe that our findings are relevant because they provide a link between capsule growth, cell cycle progression, and virulence in C. neoformans that reveals new aspects about the pathogenicity of this fungus. Moreover, our findings indicate that cell cycle elements could be used as antifungal targets in C. neoformans by affecting both the growth of the cells and the expression of the main virulence factor of this pathogenic yeast.