Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition

Abstract

The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.

Sterol-response pathways mediate alkaline survival in diverse fungi

“…The sre1 Δ mutant strain was unable to grow in the presence of Congo Red, whereas the rim101 Δ mutant strain only showed a subtle growth defect due to this chitin polymer inhibitor (Fig 1C). Also, SDS completely inhibited growth of the sre1 Δ strain, whereas the rim101 Δ appeared to be hyper-resistant to the membrane targeting effects of SDS, as evident in the more robust growth of this strain compared to wildtype (29, 30) (Fig 1C).…”

“…We also tested the sensitivity of the sre1 Δ and rim101 Δ mutant strains to cell wall stressors such as Congo Red (inhibits cell wall polymers such as chitin), caffeine (affects cell wall integrity), high salt (osmotically stresses the cell wall), and SDS (stresses the cell membrane) (29). Similar to alkaline pH, high salt resulted in complete growth inhibition for both mutant strains (Fig 1C).…”

“…Media was supplemented with 20% glucose following autoclaving unless otherwise noted. Cell wall stress phenotypes were assessed by growth on various stress media agar plates as previously described (29). Congo Red (0.5%) and NaCl (1.5M) were added to YPD media prior to autoclaving.…”

A fungal arrestin protein contributes to cell cycle progression and pathogenesis

“…5A). We then performed in vivo studies in a murine inhalation model of cryptococcal infection (38, 48, 49). Following intranasal inoculation of C57BL/6 mice (n = 10) with 10 4 colony forming units (CFU) of each strain, we observed no differences between the WT strain and the ali1 Δ mutant in their abilities to cause lethal infection (Fig.…”

“…CO 2 -independent tissue culture (TC, Gibco) medium was used to mimic an in vivo environment, as described previously (84). To assess mutant strain cell surface phenotypes, NaCl (1.5 M) and Congo red (0.5%) were added to YPD medium before autoclaving, while caffeine (1 mg/mL), calcofluor white (1 mg/mL), and SDS (0.03%) were filter sterilized and added to YPD medium after autoclaving (38). Synthetic low-ammonium dextrose (SLAD) medium (0.17% yeast nitrogen base without amino acids and without ammonium sulfate, 50 μM ammonium sulfate, 2% dextrose, and 2% agar) was used as a nitrogen deprivation medium to induce autophagy.…”