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

Abstract

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.

Author summary

Cryptococcus neoformans is a relatively frequent cause of life-threatening infection in severely immunocompromised patients, especially those with AIDS. Persistence of infection involves residence within macrophages, where C. neoformans can survive and replicate while residing in the phagolysosome. New treatments may be developed from a better understanding of how this pathogen resists clearance from and adapts for persistence in host phagocytic cells. In this study, we demonstrate a novel role for urease, a major virulence factor of C. neoformans, in its interaction with macrophages. This enzyme is able to break down urea into ammonia, which is a base, thus raising the surrounding pH. In the context of a mammalian infection, we show that cryptococcal urease increases the phagolysosomal pH which delays yeast replication, therefore causing less damage to macrophages and prolongs intracellular residence. Moreover, urease promotes C. neoformans exit from macrophages without killing the host cells. Overall, our data implies that urease also contributes to virulence by allowing the pathogen to persist and disseminate in macrophages.