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.

The capsule ofCryptococcus neoformansmodulates phagosomal pH through its acid-base properties

“…Furthermore, GXM molecules are large and structurally complex and consequently not all glucuronic acids may be equally exposed to the solvent and in a position to donate or accept hydronium ions equally. Despite these differences, GXM retains considerable weak acid properties that would confer a maximum buffering capacity at the pH range of approximately 4-5, which corresponds to the optimal pH for C. neoformans growth (13).…”

“…Phagolysosomal pH was measured using ratiometric fluorescence imaging involving the use of pH-sensitive probe Oregon green 488. Oregon green 488 was first conjugated to monoclonal antibody 18B7 using Oregon Green 488 Protein Labeling Kit (Molecular Probes, Eugene, OR), as described (13). The Oregon Green 488 dye has a succinimidyl ester moiety that reacts with primary amines of proteins to form stable dye-protein conjugates.…”

“…Recently, we reported a new role for C. neoformans urease in modulating phagosomal pH (13). Urease positive C. neoformans strains hydrolyzed urea to ammonia resulting in pleiotropic changes to the cryptococcal macrophage interaction that included higher phagosomal pH, delayed intracellular growth, and enhanced non-lytic exocytosis (13).…”

“…Recently, we reported a new role for C. neoformans urease in modulating phagosomal pH (13). Urease positive C. neoformans strains hydrolyzed urea to ammonia resulting in pleiotropic changes to the cryptococcal macrophage interaction that included higher phagosomal pH, delayed intracellular growth, and enhanced non-lytic exocytosis (13). C. neoformans is unusual among intracellular pathogens in that it grows faster at lower pH resulting in faster replication inside phagolysosomes than in the extracellular medium (9).…”

Vomocytosis: What we know so far

“…Other virulence factors that have been linked to vomocytosis are secretion of phospholipase B 1 (plb1) and presence of urease. Both defective strains (plb1Δ and ure1Δ) have been shown to be hypovirulent in animal models (Cox et al, 2001;Cox, Mukherjee, Cole, Casadevall, & Perfect, 2000), and both show a decrease in occurrence of vomocytosis (Chayakulkeeree et al, 2011;Fu et al, 2018). It should be noted, though, that these mutants show attenuated virulence in vivo, leading to higher intracellular killing rates, which could confound observations of vomocytosis rate.…”