Bet-hedging antimicrobial strategies in macrophage phagosome acidification drive the dynamics of Cryptococcus neoformans intracellular escape mechanisms

Abstract

The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.

Survival in macrophages induces enhanced virulence in Cryptococcus

“…Surviving and escaping alveolar macrophages might also provide Cn the opportunity to take up residence in a more permissive cell. Such a scheme for reaching permissive macrophages has been shown in mycobacterial infection ( 31 ), and a similar interaction has been suggested for Cn as well ( 32 ). As for manipulating the outcome of subsequent phagocytosis, specific host receptors that initiate recognition and phagocytosis of a microbe have distinct implications for the fate of the ingested organism and the inflammatory state produced.…”

“…Our data reinforce the theory that Cryptococcus emerges from macrophages primed for growth in vivo but also permissive to re-phagocytosis, providing a molecular mechanism for the latter. While the production of the capsule is known to inhibit phagocytosis, this inhibition may be balanced by the exposure of select MAMPs, providing another mechanism by which Cn may exchange a stressful intracellular environment for a more permissive one ( 32 ). These findings add detail and support the long-suggested hypothesis that macrophages are both an opposition to cryptococcal pathogenesis and a necessary collaborator ( 30 , 36 – 38 ).…”

A food color based colorimetric assay forCryptococcus neoformanslaccase activity

“…C. neoformans species complex serotype A strain H99 was obtained from John Perfect in Durham, NC. 9 The lac1 Δ mutant is from the 2007 Lodge library (Fungal Genetics Stock Center) 10 . The H99 GFP strain was obtained from the lab of Dr. Robin May at the University of Birmingham, United Kingdom 11 .…”

Kicking sleepers out of bed: Macrophages promote reactivation of dormant Cryptococcus neoformans by extracellular vesicle release and non-lytic exocytosis

“…For co-cultures using transwell system (0.4 μm; Thincert, Greiner), the inoculum suspension was adjusted to 0.5 mL RPMI and placed in the upper compartment, while the BMDMs were seeded in 1.5 mL RPMI with 2% FBS on the bottom of a 12-well plate. Transwell inserts were included in all conditions to eliminate potential bias related to transwell system usage, and co-culture was conducted at a lower temperature (30°C) optimal for the fungus, based on Dragotakes et al [ 156 ].…”