ABSTRACT The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection. IMPORTANCE Candida albicans is a natural member of the human mucosal microbiota that can also cause superficial infections and life-threatening systemic infections, both of which are characterized by inflammation. Host defense relies mainly on the ingestion and destruction of C. albicans by innate immune cells, such as macrophages and neutrophils. Although some C. albicans cells are killed by macrophages, most undergo a morphological change and escape by inducing macrophage pyroptosis. Here, we investigated the C. albicans genes and host factors that promote macrophage pyroptosis in response to intracellular fungi. This work provides a foundation for understanding how host immune cells interact with C. albicans and may lead to effective strategies to modulate inflammation induced by fungal infections.

Improved descriptions of the lowest energy excited states of tyrosine and phenylalanine side chains have been developed in order to extend the capabilities of calculating the circular dichroism (CD) spectra of proteins. Four transitions (Lb, La, Bb, and Ba) for each of the side-chain chromophores were considered, and the transition monopole charges were obtained from a CNDO/S calculation on models representing the individual groups. Monopole charges at midpoints of the bonds, corresponding to the maximum transition charge densities in the Lb band, and monopole charges representing the vibronic coupling with the B transitions for the La transition were also included. The aromatic transitions were combined with the peptide transitions (npi, pi0pi n'pi, and pi+pi) and disulfide transitions (n1sigma and n4sigma) in the framework of the origin-independent matrix method to compute the CD spectra of different crystal forms and Y --> L and F --> L mutants of bovine pancreatic trypsin inhibitor (BPTI). The structures of the mutants were obtained by replacing the appropriate tyrosine or phenylalanine residue by leucine in the wild-type crystal structure. The CD calculations were performed on the energy-minimized structures. The CD spectrum calculated for the form II crystal structure of BPTI showed the best agreement with experiment. In the far UV, the calculated and experimental CD spectra agree to various extents for the wild-type and mutant BPTI. Among the mutants, the calculated CD spectra of Y4L, Y10L, Y23L, and F45L showed reasonable agreement with experiment, while those of Y21L and F22L, the two residues interacting with most aromatic groups, showed poor agreement. In the near UV, the negative bands predicted for the wild-type and mutant BPTI have much less intensity than observed experimentally.

Turbidimetric or light scattering assays can be used to determine the extent of aggregation in protein formulations. Using low molecular weight urokinase (LMW-UK) as a model protein, the effect of polymeric additives on heat-induced aggregation was evaluated. Previous work has shown that under 60°C heat treatment, LMW-UK initially denatures and the unfolded protein associates to form soluble aggregates. Eventually, these aggregates associate to form a precipitate. The effects of polymers on the initial aggregation phase was examined. Hydroxyethyl (heta) starch, polyethylene glycol 4000, and gelatin were found to be effective, concentration-dependent inhibitors of aggregation, whereas polyvinylpyrrolidone (PVP) and polyethylene glycol 300 were ineffective. Overall, the effect of polymeric additives on the stability of thermally-stressed LMW-UK can be accounted for by preferential exclusion of the solute from the surface of the protein.

A new technique for enhancing the solubility of peptides and proteins in organic solvents is described. Complexation of polypeptides with stoichiometric amounts of an anionic detergent, such as sodium dodecyl sulfate (SDS), produces diminished aqueous solubility, but enhanced solubility in organic solvents. Consequently, the partitioning of a polypeptide into a nonpolar solvent can be increased by two to four orders of magnitude. In the case of an insulin-SDS complex, the solubility in 1-octanol is more than tenfold greater than in water. In 1-octanol, the native structure of insulin remains intact, as determined by CD spectroscopy, and the thermal denaturation temperature (T(m)) is increased by approximately 50-degrees-C relative to unfolding in water. Finally, peptides and proteins can be extracted back into an aqueous phase provided the chloride concentration is sufficient to displace bound detergent molecules.