title: Cryptococcus neoformans survive and replicate in human microglia.
abstract: BACKGROUND
  Cryptococcus neoformans (CN) is an opportunistic pathogen that can cause fatal meningoencephalitis in patients with immune deficiency. Among the central nervous system (CNS) cells that are infected by CN are perivascular microglia and macrophages. Little is known about the interaction of CN and CNS phagocytes at the cellular level.
  
  
  METHODS
  To better understand the fate of CN in microglia, we followed Ab-opsonized CN in human fetal microglial culture by phase-contrast microscopy, combined lighted microscopy and transmission electron microscopy of plastic-embedded monolayers, and immunocytochemistry for localization of capsular Ag.
  
  
  RESULTS
  Phase-contrast microscopy revealed that microglia initially internalized and contained Ab-opsonized yeast cells within phagolysosomes. However, CN escaped from microglia and resumed extracellular growth 16 to 24 hours after being phagocytosed. Transmission electron microscopy/1-mu epoxy sections revealed that intracellular CN were localized in two types of phagosomes in microglia: spacious phagosomes (SP) and close-fitting phagosomes (CP). Three lines of evidence indicate that SP are the primary sites for intracellular CN survival and replication: (a) SP contained multiple, budding yeast cells, whereas CP contained only single yeast cells within a tightly bound phagosomal membrane; (b) the number of SP and the number of CN within SP increased considerably at 24 hours compared with at 2 hours; and (c) microglial cultures challenged with heat- or amphotericin B-treated CN had significantly fewer SP than those challenged with live CN. Both SP and CP phagosomes fused with lysosomes, suggesting that CN survival in SP was not due to failure of phagolysosomal fusion. In SP, there was attenuation and diffusion of capsular polysaccharide within the phagosome, whereas in CP the fungal capsules remained compact and homogeneous. Immunocytochemistry with an mAb directed to capsular glucoronoxylomannan supported continued synthesis of polysaccharide within SP.
  
  
  CONCLUSIONS
  The results suggest that, in human microglia, CN survive and replicate within SP. Modification of CN capsular polysaccharide within SP may be a contributing factor to this aberrant microglial-CN interaction.
authors.affiliation: Department of Pathology (Neuropathology), Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Lee
authors.forename: Y
authors.initials: Y
authors.lastname: Kress
authors.forename: M L
authors.initials: ML
authors.lastname: Zhao
authors.forename: D W
authors.initials: DW
authors.lastname: Dickson
authors.forename: A
authors.initials: A
authors.lastname: Casadevall
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003455:Cryptococcus neoformans / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D051379:Mice; D017628:Microglia / Q000382:microbiology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008858:Microscopy, Phase-Contrast; D010588:Phagosomes / Q000382:microbiology / Q000648:ultrastructure; D011134:Polysaccharides / Q000032:analysis
chemical_list: D011134:Polysaccharides