The yeast-phase virulence requirement for α-glucan synthase differs among Histoplasma capsulatum chemotypes

Histoplasma capsulatum strains can be classified into two chemotypes based on cell wall composition. The cell wall of chemotype II yeast contains a layer of α-(1,3)-glucan that masks immunostimulatory β-(1,3)-glucans from detection by the Dectin-1 receptor on host phagocytes. This α-(1,3)-glucan cell wall component is essential for chemotype II Histoplasma virulence. In contrast, chemotype I yeast cells lack α-(1,3)-glucan in vitro, yet they remain fully virulent in vivo. Analysis of the chemotype I α-glucan synthase (AGS1) locus revealed a 2.7-kb insertion in the promoter region that diminishes AGS1 expression. Nonetheless, AGS1 mRNA can be detected during respiratory infection with chemotype I yeast, suggesting that α-(1,3)-glucan could be produced during in vivo growth despite its absence in vitro. To directly test whether AGS1 contributes to chemotype I strain virulence, we prevented AGS1 function by RNA interference and by insertional mutation. Loss of AGS1 function in chemotype I does not impair the cytotoxicity of ags1(-) mutant yeast to cultured macrophages, nor does it affect the intracellular growth of yeast. In a murine model of histoplasmosis, the ags1(-) chemotype I mutant strains show no defect in lung infection or in extrapulmonary dissemination. Together, these studies demonstrate that AGS1 expression is dispensable for chemotype I yeast virulence, in contrast to the case for chemotype II yeast. Despite the absence of cell wall α-(1,3)-glucan, chemotype I yeast can avoid detection by Dectin-1 in a growth stage-dependent manner. This suggests the production of a unique Histoplasma chemotype I factor that, at least partially, circumvents the α-(1,3)-glucan requirement for yeast virulence.     

An alpha-(1,4)-amylase is essential for alpha-(1,3)-glucan production and virulence in Histoplasma capsulatum

Histoplasma capsulatum is a dimorphic fungus that causes respiratory and systemic disease and is capable of surviving and replicating within macrophages. The virulence of Histoplasma has been linked to cell wall alpha-(1,3)-glucan; however, the role of this polysaccharide during infection, its organization within the cell wall, and its synthesis and regulation remain poorly understood. To identify genes involved in the biosynthesis of alpha-(1,3)-glucan, we employed a forward genetics strategy to isolate physically marked mutants with reduced alpha-(1,3)-glucan. Insertional mutants were generated in a virulent strain of H. capsulatum by optimization of Agrobacterium tumefaciens-mediated transformation. Approximately 90% of these mutants possessed single insertions with no chromosomal rearrangements or deletions in the host genome. To confirm the role and specificity of identified candidate genes, we phenocopied the disrupted locus by either RNA interference or targeted gene deletion. Our findings indicate alpha-(1,3)-glucan production requires the function of the AMY1 gene product, a novel protein with homology to the alpha-amylase family of glycosyl hydrolases, and UGP1, a UTP-glucose-1-phosphate uridylyltransferase which synthesizes UDP-glucose monomers. Loss of AMY1 function attenuated the ability of Histoplasma to kill macrophages and to colonize murine lungs.     

Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma farciminosum

The cell wall of the yeast form of Histoplasma farciminosum contains 13.2% beta-1,3-glucan, 1.0% galactomannan, and 25.8% chitin, whereas the cell wall of mycelial form has 21.8, 4.5, and 40%, respectively, for the same polymers. Also, the cell wall of the yeast form contains alpha-1,3-glucan (13.5%) and an unidentified polymer (21.5%). Chitin, one of the structural polymers of both yeast and mycelial cell walls, is identified as thin isolated fibers (4 nm wide) or in thick bundles (50 nm wide) of fibers. beta-(1-3)-Glucan is also found as thin isolated fibers indistinguishable from isolated fibers of chitin. Fibers 14 nm wide and resembling alpha-(1-3)-glucan fibers of other fungi are found in the yeast form. The results reported here do not give support to the proposal for a different taxonomic classification.     

Definition of the extracellular proteome of pathogenic-phase Histoplasma capsulatum

The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease. Within the mammalian host, pathogenic Histoplasma yeast infect, replicate within, and ultimately kill host phagocytes. Surprisingly, few factors have been identified that contribute to Histoplasma virulence. To address this deficiency, we have defined the constituents of the extracellular proteome using LC-MS/MS analysis of the proteins in pathogenic-phase culture filtrates of Histoplasma. In addition to secreted Cbp1, the extracellular proteome of pathogenic Histoplasma yeast consists of 33 deduced proteins. The proteins include glycanases, extracellular enzymes related to oxidative stress defense, dehydrogenase enzymes, chaperone-like factors, and five novel culture filtrate proteins (Cfp's). For independent verification of proteomics-derived identities, we employed RNA interference (RNAi)-based depletion of candidate factors and showed loss of specific proteins from the cell-free culture filtrate. Quantitative RT-PCR revealed the expression of 10 of the extracellular factors was particularly enriched in pathogenic yeast cells as compared to nonpathogenic Histoplasma mycelia, suggesting that these proteins are linked to Histoplasma pathogenesis. In addition, Histoplasma yeast express these factors within macrophages and during infection of murine lungs. As extracellular proteins are positioned at the interface between host and pathogen, the definition of the pathogenic-phase extracellular proteome provides a foundation for the molecular dissection of how Histoplasma alters the host-pathogen interaction to its advantage.     

Validation of RNAi silencing specificity using synthetic genes: salicylic acid-binding protein 2 is required for innate immunity in plants

RNA interference (RNAi) is widely used to specifically silence the expression of any gene to study its function and to identify and validate therapeutic targets. Despite the popularity of this technology, recent studies have shown that RNAi may also silence non-targeted genes. Here we demonstrate the utility of a quick, efficient and robust approach to directly validate the specificity of RNAi as an alternative to indirect validation of RNAi through gene expression profiling. Our approach involves reversing (complementing) the RNAi-induced phenotype by introducing a synthetic version of the target gene that is designed to escape silencing. This synthetic gene complementation approach can also be used for mutational analysis of the target gene, or to provide a functional version of a defective protein after silencing the defective gene by RNAi. Using this approach we demonstrate that the loss of systemic acquired resistance, a form of innate immunity in plants, is indeed due to the silencing of salicylic acid-binding protein 2 rather than to off-target effects.     

Macrophage specific delivery of TNF-α siRNA complexed with β-1,3-glucan inhibits LPS-induced cytokine production in a murine acute hepatitis model

RNA interference therapy utilizes physiological gene silencing that is originally found as a defense function against foreign RNAs. To silence the target gene, short double stranded RNA has to be delivered to cytosol. However, lack of a suitable delivering carrier is the major obstacle to practical usage. In this study, we present a novel complex consisting of β-1,3-glucan and short interference RNA (siRNA) as a solution for the problem. We used a β-1,3-glucan schizophyllan (SPG) and a siRNA (dA-siTNFα) that is designed to suppress tumor necrosis factor alpha (TNF-α), where the sense strand of siRNA has (dA₄₀) tail to induce complexation with SPG. The dA-siTNFα/SPG complex showed higher affinity to recombinant dectin-1 than SPG itself, where dectin-1 is a β-1,3-glucan receptor expressed on antigen presenting cells and can be a target for specific delivery. The complex suppressed lipopolysaccharide (LPS)-induced TNF-α secretion by peritoneal macrophages in vitro. When the complex was intravenously injected, the oligonucleotide accumulated in liver; especially distributed into Kupffer cells. The complex significantly decreased the serum TNF-α level for the mouse model of LPS-induced acute hepatitis. This new siRNA delivery system may overcome the problem for RNA interference therapy because of its non-toxicity and high target specificity.     

Photodynamic induction of a bacterial cell surface polypeptide.

A mutation in Aspergillus nidulans led to a loss of both melanin and alpha-(1,3)-glucan, a major wall polysaccharide. In addition, the mutation prevented the formation of cleistothecia. Mutant walls contained increased amounts of beta-(1,3)-glucan and galactose polymers, and electron micrographs indicated that they had lost the outermost wall layer. Such walls were more readily digested by lytic enzymes, and this increased susceptibility to hydrolysis was due to the absence of alpha-(1,3)-glucan and not of melanin. The pleiotropic effects of the mutation are discussed, with particular reference to the hypothesis that alpha-(1,3)-glucan acts as the endogenous carbon source for biosynthetic processes in the stationary phase of growth. In this view, glucan synthesis would be the primary target of the mutation, and the absence of glucan would result in the lack of melanin and cleistothecia, formed after nutrients are exhausted. Two other mutations that lowered themycelial alpha-(1,3)-glucan content also inhibited melanin and cleistothecia production.     

Dynamics of gene silencing by RNA interference

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.     

Conditional gene silencing in mammalian cells mediated by a stress-inducible promoter

MicroRNAs (miRNAs) are endogenous non-coding small RNAs, which negatively regulate gene expression in a sequence-specific manner through the RNA interference (RNAi) pathway. Here we describe a new miRNA-based conditional RNAi expression system that relies on cellular stress-response mechanisms in mammalian cells. In our constructs, expression of miRNA mimics is tightly controlled by a heat shock-inducible promoter. This system is highly effective in silencing permanently or conditionally expressed luciferase. The stress inducible vectors also effectively deplete co-expressed pro-apoptotic protein CHOP with heat shock. Furthermore, we demonstrate cloning of a protein-coding sequence between the stress-inducible promoter and the miRNA expression cassette allows simultaneous silencing of a target gene and activation of synthesis of a protein of choice in response to stress stimulation. This new conditional gene silencing approach could be an invaluable tool for various areas of basic and applied research and for therapeutic intervention.