Deciphering the Intracellular Niche of Cryptococcus neoformans in Alveolar Macrophages

Cryptococcus neoformans is a fungal pathogen responsible for ~200,000 deaths yearly. This ubiquitously present yeast affects immunocompromised individuals, which left untreated is invariably fatal. Once C. neoformans particles are inhaled and make their way into the lungs they encounter alveolar macrophages. Here, interactions influence whether the infection is controlled or disseminates to cause disease. Since C. neoformans can survive and replicate inside host macrophages, they represent a safe haven, hidden from the immune response of the body. The mechanisms C. neoformans use to mediate intracellular parasitism are not fully understood. Even less is known about how immune signals impact this niche. Therefore, we aim to gain a better understanding of the macrophage–cryptococcal interactions and decipher the properties of the cryptococcal-containing phagosome (CCP), which may be key to determining infection outcome. We identified three populations of CCPs with different behaviors: one that gains acidification after phagocytosis but subsequently loses it; some that acidify and remain acidic; and some that never acidify. We also directly observed phagosomal membrane damage, suggesting a possible mechanism for pH manipulation. This contrasts with phagosomes containing S. cerevisiae, which rapidly acidify, stay acidic, and have low levels of membrane damage. Moreover, we have identified a population of CCPs that display both early endosomal lipid and lysosomal protein characteristics, a combination not normally observed. This suggests C. neoformans can alter its phagosome and provides a potential mechanism for intracellular survival that may be driving pathogenesis. We have demonstrated a delay in acquisition of lysosomal markers to CCPs. When macrophages are stimulated with interferon-?, acquisition of lysosomal markers occurs more rapidly. Host Rab20 has been shown to be interferon-? inducible and appears to facilitate this promotion of phago-lysosomal fusion and may be key in influencing the infection, thereby linking host immune status and phagosomal properties during cryptococcal infection.