Cell wall composition in Cryptococcus neoformans is media dependent and alters host response, inducing protective immunity

Abstract

IntroductionCryptococcus neoformans is a basidiomycete fungus that can cause meningoencephalitis, especially in immunocompromised patients. Cryptococcus grows in many different media, although little attention has been paid to the role of growth conditions on the cryptococcal cell wall or on virulence.ObjectiveThe purpose of this study was to determine how different media influenced the amount of chitin and chitosan in the cell wall, which in turn impacted the cell wall architecture and host response.MethodsYeast extract, peptone, and dextrose (YPD) and yeast nitrogen base (YNB) are two commonly used media for growing Cryptococcus before use in in vitro or in vivo experiments. As a result, C. neoformans was grown in either YPD or YNB, which were either left unbuffered or buffered to pH 7 with MOPS. These cells were then labeled with cell wall-specific fluorescent probes to determine the amounts of various cell wall components. In addition, these cells were employed in animal virulence studies using the murine inhalation model of infection.ResultsWe observed that the growth of wild-type C. neoformans KN99 significantly changes the pH of unbuffered media during growth. It raises the pH to 8.0 when grown in unbuffered YPD but lowers the pH to 2.0 when grown in unbuffered YNB (YNB-U). Importantly, the composition of the cell wall was substantially impacted by growth in different media. Cells grown in YNB-U exhibited a 90% reduction in chitosan, the deacetylated form of chitin, compared with cells grown in YPD. The decrease in pH and chitosan in the YNB-U-grown cells was associated with a significant increase in some pathogen-associated molecular patterns on the surface of cells compared with cells grown in YPD or YNB, pH 7. This altered cell wall architecture resulted in a significant reduction in virulence when tested using a murine model of infection. Furthermore, when heat-killed cells were used as the inoculum, KN99 cells grown in YNB-U caused an aberrant hyper-inflammatory response in the lungs, resulting in rapid animal death. In contrast, heat-killed KN99 cells grown in YNB, pH 7, caused little to no inflammatory response in the host lung, but, when used as a vaccine, they conferred a robust protective response against a subsequent challenge infection with the virulent KN99 cells.ConclusionThese findings emphasize the importance of culture media and pH during growth in shaping the content and organization of the C. neoformans cell wall, as well as their impact on fungal virulence and the host response.

Immune evasion by Cryptococcus gattii in vaccinated mice coinfected with C. neoformans

“…It is worth considering that both Cn-cda1 Δ 2 Δ 3 Δ and Cg-cda1 Δ 2 Δ 3 Δ, are capsule-producing strains in vivo . Also of interest, heat-killed KN99 that had been grown in yeast nitrogen base medium buffered to pH 7 also provided protection against a subsequent challenge ( 70 ). Despite the well-established immunosuppressive nature of the cryptococcal capsule, both the live-attenuated strains and the heat-killed WT strain, which are inoculated directly into the lungs of mice, generated immunity to KN99 challenge.…”

Antifungal activity of eumelanin-inspired indoylenepheyleneethynylene against Cryptococcus neoformans

“…Combining all the data from the RNA sequencing and the electron microscopy, we composed a putative model based on the effects of EIPE-1 against C. neoformans as displayed in Figure 6 . As found in all living eukaryotic cells, beneath the fungal cell wall, C. neoformans possesses a plasma membrane that consists of a phospholipid bilayer ( Rodrigues et al, 2008 ; Agustinho et al, 2018 ; Upadhya et al, 2023 ; Zhukov and Popov, 2023 ). This membrane maintains the viability of a cell and prevents the free exchange of molecules from the cytoplasm to the cell’s environment and vice versa ( Upadhya et al, 2023 ).…”

“…As found in all living eukaryotic cells, beneath the fungal cell wall, C. neoformans possesses a plasma membrane that consists of a phospholipid bilayer ( Rodrigues et al, 2008 ; Agustinho et al, 2018 ; Upadhya et al, 2023 ; Zhukov and Popov, 2023 ). This membrane maintains the viability of a cell and prevents the free exchange of molecules from the cytoplasm to the cell’s environment and vice versa ( Upadhya et al, 2023 ). Previously, microorganism membrane permeability to hydrophobic molecules was identified as being pertinent for susceptibility to the molecule ( Reed et al, 2023 ).…”

Vaccination with a ZNF2 oe Strain of Cryptococcus Provides Long-Lasting Protection against Cryptococcosis and Is Effective in Immunocompromised Hosts

“…Mice are highly susceptible to infections by virulent cryptococcal strains, which is advantageous in the laboratory setting but also makes it challenging to study latent or asymptomatic infections. Despite their susceptibility to cryptococcal infections, mice of different backgrounds respond differently to vaccines ( 44 – 49 ). Here, we used both A/Jcr mice and CBA/J mice.…”

“…In contrast, CBA/J mice are C5 sufficient ( 50 ). Both mouse strains are widely used in Cryptococcus pathogenesis and vaccination studies ( 37 , 44 , 45 ).…”