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Chapter 45 : Histoplasma capsulatum
Category: Microbial Genetics and Molecular Biology; Fungi and Fungal Pathogenesis
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Molecular phylogenetic analyses reveal that Histoplasma capsulatum comprises at least seven distinct phylogenetic groups associated with different geographical locations. Molecular differences between Histoplasma strains have been identified using restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA, ribosomal DNA, and the YPS3 locus. The yeast phase of Histoplasma is the morphological form found in infected tissues and is the phase devoted to pathogenesis. When subjected to a temperature of 37⁰C, germinating Histoplasma conidia or hyphal cells undergo a morphological conversion to produce yeast phase cells. Genetic evidence demonstrating the role of these potential iron acquisition mechanisms during intramacrophage growth of Histoplasma yeast has only been demonstrated for siderophores (SID1). Consistent with its role in pathogenesis, α-glucan is synthesized solely by yeast phase cells; AGS1 is only expressed by Histoplasma in the yeast phase. The molecular details underlying the mechanisms promoting Histoplasma virulence are now beginning to be revealed with the application of molecular genetics. Investigation of geographically overlapping yet nonrecombining species at the genome level should also provide an important perspective on Histoplasma evolution. Genome comparisons with other species may also enhance our understanding of the issue of dimorphism. The virulence factors identified for Histoplasma have all relied upon the development of reverse genetics methodology to functionally demonstrate the role of suspected genes in virulence. The ability of forward genetics to identify novel or unsuspected genes involved in the biology of Histoplasma is now possible through insertional mutagenesis techniques.
Gene inactivation methodology for H. capsulatum. Three methods have been developed for inactivating gene functions in Histoplasma cells. Deletion of a target gene is accomplished through allelic replacement with a disruption allele. To enrich for recovery of cells in which the desired homologous recombination event has occurred, positive and negative selections are employed. RNAi can be used to deplete gene functions posttranscriptionally by transformation of Histoplasma yeast with a linear plasmid containing inverted copies of the target locus. Subsequent transcription leads to formation of double-stranded RNAs that trigger the RNAi effect. Genes can also be disrupted by Agrobacterium-mediated transfer of a T-DNA element that inserts randomly into Histoplasma chromosomes.
Transition of mycelial phase to yeast phase of H. capsulatum. Two signals are required for the complete transition of mycelial phase to the yeast phase: elevated temperature and cysteine. Cysteine plays a twofold role by providing reduced sulfhydryls/low redox potential necessary to complete stage II as well as supplying organic sulfur to complement the cysteine auxotrophy of yeast cells. Additional characteristics of stages in the conversion process are presented. The DRK1 kinase and RYP1 DNA-binding protein promote differentiation to the yeast phase and control expression of the yeast phase regulon including transcription of Histoplasma virulence factors.
Intracellular pathogenesis of H. capsulatum. H. capsulatum yeast cells (Hc) bind to macrophage CD18 family complement receptors (CR3) via surface-localized HSP60. Cell wall β-glucans of nonpathogenic yeast are recognized by host Dectin-1 surface receptors. In contrast, the cell wall α-glucan layer masks Histoplasma from detection. Uptake of Histoplasma yeast by phagocytes can trigger production of reactive oxygen compounds (ROS) depending on host species and opsonization, but Histoplasma is not killed. In some macrophage cells, Histoplasma yeast cells impair phagolysosomal fusion (PL fusion). Phagosomes containing nonpathogenic yeast become acidified and readily fuse with lysosomes, leading to destruction of the nonpathogenic yeast cells. For Histoplasma, in situations where phagolysosomal fusion occurs, Histoplasma yeast cells escape lysosomal degradation by actively maintaining a luminal pH of >6. In the phagosome or phagolysosome, Histoplasma yeast cells secrete iron-chelating siderophores and a CBP, which further promote Histoplasma virulence.
Classification of Histoplasma strains
Histoplasma mating types