Research Article: Cryptococcus neoformans urease affects the outcome of intracellular pathogenesis by modulating phagolysosomal pH

Date Published: June 15, 2018

Publisher: Public Library of Science

Author(s): Man Shun Fu, Carolina Coelho, Carlos M. De Leon-Rodriguez, Diego C. P. Rossi, Emma Camacho, Eric H. Jung, Madhura Kulkarni, Arturo Casadevall, Robin Charles May.


Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized. Consequently, we analyzed the effect of urease on fungal-macrophage interaction using wild-type, urease-deficient and urease-complemented strains of C. neoformans. The frequency of non-lytic exocytosis events was reduced in the absence of urease. Urease-positive C. neoformans manifested reduced and delayed intracellular replication with fewer macrophages displaying phagolysosomal membrane permeabilization. The production of urease was associated with increased phagolysosomal pH, which in turn reduced growth of urease-positive C. neoformans inside macrophages. Interestingly, the ure1 mutant strain grew slower in fungal growth medium which was buffered to neutral pH (pH 7.4). Mice inoculated with macrophages carrying urease-deficient C. neoformans had lower fungal burden in the brain than mice infected with macrophages carrying wild-type strain. In contrast, the absence of urease did not affect survival of yeast when interacting with amoebae. Because of the inability of the urease deletion mutant to grow on urea as a sole nitrogen source, we hypothesize urease plays a nutritional role involved in nitrogen acquisition in the environment. Taken together, our data demonstrate that urease affects fitness within the mammalian phagosome, promoting non-lytic exocytosis while delaying intracellular replication and thus reducing phagolysosomal membrane damage, events that could facilitate cryptococcal dissemination when transported inside macrophages. This system provides an example where an enzyme involved in nutrient acquisition modulates virulence during mammalian infection.

Partial Text

C. neoformans, a major life-threatening fungal pathogen predominantly infects severely immunocompromised patients and causes over 180,000 deaths per year worldwide [1]. C. neoformans is ubiquitous, although is most frequently found in soils contaminated with bird excreta or from trees [2–11]. Current treatments for Cryptococcosis often fail, are inadequate and/or unavailable for these infections, especially in developing countries. Therefore, it is important to study the fundamental pathogenic processes of C. neoformans to discover new treatments against this pathogen.

Urease is an important virulence factors of C. neoformans [40]. However, most studies have focused on urease role in brain invasion and its effects on the host immune response. Cryptococcal urease facilitates transmission of C. neoformans across the blood-brain barrier [44–46] and polarizes the immune system to a Th2 response, which translates into greater fungal burden in lung in mouse model [52]. Yet the effect of urease in macrophage interaction has not been explored despite the fact that the outcome of the interaction of C. neoformans with macrophages is a key determinant of the outcome of infection [13,78,79]. In this study, we analyzed the role of urease in C. neoformans-macrophage interactions. Our results provide new insights on how this enzyme can affect the pathogenesis of Cryptococcus spp since we show that urease influences the intracellular growth of C. neoformans, affects non-lytic exocytosis from macrophages, is critical for growth at mammalian physiological pH and confers upon the yeast the potential for using urea as a nitrogen source in nutrition.