Discovery of a Role for Hsp82 in Histoplasma Virulence through a Quantitative Screen for Macrophage Lethality

Abstract

The application of forward genetics can reveal new factors required for the virulence of intracellular pathogens. To facilitate such virulence screens, we developed macrophage cell lines with which the number of intact host cells following infection with intracellular pathogens can be rapidly and easily ascertained through the expression of a constitutive lacZ transgene. Using known virulence mutants of Francisella novicida and Histoplasma capsulatum, we confirmed the applicability of these host cells for the quantitative assessment of bacterial and fungal virulence, respectively. To identify new genes required for Histoplasma virulence, we employed these transgenic macrophage cells to screen a collection of individual transfer DNA (T-DNA) insertion mutants. Among the mutants showing decreased virulence in macrophages, we identified an insertion in the locus encoding the Histoplasma Hsp82 homolog. The lesion caused by the T-DNA insertion localizes to the promoter region, resulting in significantly decreased HSP82 expression. Reduced HSP82 expression markedly attenuates the virulence of Histoplasma yeast in vivo. While the HSP82 hypomorph grows normally in vitro at 37°C and under acid and salinity stresses, its ability to recover from high-temperature stress is impaired. These results provide genetic proof of the role of stress chaperones in the virulence of a thermally dimorphic fungal pathogen.

Glycosylation and Immunoreactivity of the Histoplasma capsulatum Cfp4 Yeast-Phase Exoantigen

“…To determine the role of Cfp4 in macrophage killing, we utilized the previously described P338D1-lacZ macrophage killing assay in which macrophage-expressed LacZ is used as an indicator of surviving macrophages (17). Macrophages were maintained in Ham's F-12 with 10% heat-inactivated fetal bovine serum (FBS).…”

“…Within phagocytes, Histoplasma yeasts grow and replicate, ultimately leading to lysis of the phagocyte and spread of the infection. A few factors facilitating the intramacrophage lifestyle of Histoplasma are beginning to be defined (11) and include Cbp1, a secreted factor of unknown function (12), production of siderophores and iron reductase systems that enable iron acquisition within the phagosome (13)(14)(15), de novo synthesis of essential vitamin cofactors (16), and thermotolerance (17).…”

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

“…Flanking sequences were identified by PCR and sequencing. The authors identified a new virulence gene in Histoplasma, a homolog of Hsp82 (Edwards et al, 2011). Marion et al (2006) performed a more comprehensive analysis of insertional mutagenesis in Histoplasma capsulatum using Agrobacterium-mediated transformation.…”

“…Researchers have developed protocols that have improved the efficiency of transformation using T-DNA in C. neoformans (Idnurm et al, 2004), Histoplasma and Blastomyces (Brandhorst et al, 2002;Edwards et al, 2011;Gauthier et al, 2010;Laskowski and Smulian, 2010;Marion et al, 2006;Smulian et al, 2007;. In addition, T-DNA mutagenesis protocols have been developed for Coccidioides (Abuodeh et al, 2000), Trichoderma spp.…”

“…T-DNA was used to identify genes in Histoplasma involved in pathogenesis in a novel highthroughput macrophage-killing screen (Edwards et al, 2011). Transgenic (a novel gene was introduced) macrophage lines were constructed that constitutively expressed bacterial lacZ.…”