Objectives:
Cryptococcus neoformans is the etiological agent of cryptococcosis, one of the major invasive fungal diseases across the world. C. neoformans is known for its remarkable ability to parasitize immune system phagocytic cells, exploiting them as a site for replication, protection from host recognition and immune response,  and/or even as a vehicle for dissemination from the lungs, the primary site of inoculation,  to secondary infection organs such as the brain, promoting cryptococcal meningitis, generally with fatal outcomes. In this context, the improvement of methodologies for the study of the interaction between C. neoformans and phagocytes of the mammalian host are essential for the clarification of the mechanisms of the disease. The aim of this work was to construct and characterize C. neoformans reporter strains containing GFP or mCherry fluorescent proteins to better investigate the dynamics of C. neoformans-host cells interaction both in vitro and in vivo.

Methods:
We used biolistic-mediated transformation to develop H99 C. neoformans (serotype A type strain H99) reporter strains containing GFP (Green Fluorescent Protein) or mCherry (red fluorescent protein) fused to histone H3 genes (HIS3) to target fluorophores to the cell nucleus, or Poly-A Binding Protein (PABP) to direct fluorophores to the cytoplasm. Vehicle plasmids which carries only GFP or mCherry genes were used as control. We employed flow cytometry and microscopy analysis for evaluation of fungal fluorescence intensity and intracellular distribution, respectively. We also checked fungal phenotypes including growth at osmotic, oxidative and cell wall stress conditions, as well as expression of C. neoformans virulence factors such as urease and phospholipase activity, capsule formation and melanin production. Strains were tested for virulence in vitro using murine bone marrow derived-macrophages, and in vivo using a Galleria mellonella infection model. Finally, we evaluated fungal phagocytosis by light and fluorescence microscopy and flow cytometry.

Results:
Using fluorescent microscopy, we were able to confirm the expected pattern of intracellular distribution of fluorophores in the engineered strains. Flow cytometry analysis indicated that we achieve transformants with significantly greater fluorescence than parent strain (WT), especially GFP transformants, which showed more than 50 fold-increase. C. neoformans strains exhibited unaltered responses to stress conditions. In addition, the transformation did not affected the expression of C. neoformans major virulence factors. Using the G. mellonella infection model, we observed that HIS3-GFP and PABP-mCherry-infected larvae presented a slightly higher survival rate in comparison to WT-infected animals. However, there was no difference in fungal survival among all tested strains in macrophage infection, a more established and suitable model system for evaluation of C. neoformans virulence. Furthermore, GFP transformants not only exhibited unaltered phagocytosis by primary macrophages in comparison to the parental strain, but also allowed a more accurate and/or faster analysis of phagocytes-C. neoformans interaction than conventional techniques.

Conclusion:
Our results indicates a promising use of engineered C. neoformans HIS3 and PABP fluorescent reporter strains coupled to flow cytometry and fluorescent microscopy for the improvement of investigation of pathogen-host interplay.

Construction of Cryptococcus neoformans reporter strains containing GFP or mCherry fluorescent proteins for pathogen-host interaction studies