A role for LHC1 in higher order structure and complement binding of the Cryptococcus neoformans capsule.

Polysaccharide capsules are important virulence factors for many microbial pathogens including the opportunistic fungus Cryptococcus neoformans. In the present study, we demonstrate an unusual role for a secreted lactonohydrolase of C. neoformans, LHC1 in capsular higher order structure. Analysis of extracted capsular polysaccharide from wild-type and lhc1Δ strains by dynamic and static light scattering suggested a role for the LHC1 locus in altering the capsular polysaccharide, both reducing dimensions and altering its branching, density and solvation. These changes in the capsular structure resulted in LHC1-dependent alterations of antibody binding patterns, reductions in human and mouse complement binding and phagocytosis by the macrophage-like cell line J774, as well as increased virulence in mice. These findings identify a unique molecular mechanism for tertiary structural changes in a microbial capsule, facilitating immune evasion and virulence of a fungal pathogen.

Formation of Hirano bodies after inducible expression of a modified form of an actin-cross-linking protein.

Hirano bodies are cytoplasmic inclusions composed mainly of actin and actin-associated proteins. The formation of Hirano bodies during various neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis, has been reported. Although the underlying molecular mechanisms that lead to the formation of these inclusions in the brain are not known, expression of the C-terminal fragment (CT) (amino acids 124 to 295) from the endogenous 34-kDa actin-binding protein of Dictyostelium discoideum leads to the formation of actin inclusions in vivo. In the current study, we report the development of an inducible expression system to study the early phases of Hirano body formation using an inducible promoter system (rnrB). By fusing the CT to a green fluorescent protein (CT-GFP), we monitored protein expression and localization by fluorescence microscopy, flow cytometry, and Western blot analysis. We observed an increase in the number and size of inclusions formed following induction of the CT-GFP vector system. Time-lapse microscopy studies revealed that the CT-GFP foci associated with the cell cortex and fused to form a single large aggregate. Transmission electron microscopy further demonstrates that these inclusions have a highly ordered ultrastructure, a pathological hallmark of Hirano bodies observed in postmortem brain samples from patients with various neurodegenerative disorders. Collectively, this system provides a method to visualize and characterize the events that surround early actin inclusion formation in a eukaryotic model.

The DEAD-box RNA helicase Vad1 regulates multiple virulence-associated genes in Cryptococcus neoformans.

The study of fungal regulatory networks is essential to the understanding of how these pathogens respond to host environmental signals with effective virulence-associated traits. In this study, a virulence-associated DEAD-box RNA helicase-encoding gene (VAD1) was isolated from a mutant defective in the virulence factor laccase. A Deltavad1 mutant exhibited a profound reduction in virulence in a mouse model that was restored after reconstitution with WT VAD1. Loss of VAD1 resulted in upregulation of NOT1, a gene encoding a global repressor of transcription. NOT1 was found to act as an intermediary transcriptional repressor of laccase. Vad1 was located within macromolecular complexes that formed cytoplasmic granular bodies in mature cells and during infection of mouse brain. In addition, VAD1 was shown by in situ hybridization to be expressed in the brain of an AIDS patient coinfected with C. neoformans. To understand the role of VAD1 in virulence, a functional genomics approach was used to identify 3 additional virulence determinants dependent on VAD1: PCK1, TUF1, and MPF3, involved in gluconeogenesis, mitochondrial protein synthesis, and cell wall integrity, respectively. These data show that fungal virulence-associated genes are coordinately regulated and that an analysis of such transcriptomes allows for the identification of important new genes involved in the normal growth and virulence of fungal pathogens.