Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function.

Saccharomyces cerevisiae vacuoles are functionally analogous to mammalian lysosomes. Both also serve as physical platforms for Tor Complex 1 (TORC1) signal transduction, the master regulator of cellular growth and proliferation. Hygromycin B is a eukaryotic translation inhibitor. We recently reported on hygromycin B hypersensitive (hhy) mutants that fail to grow at subtranslation inhibitory concentrations of the drug and exhibit vacuolar defects (Banuelos et al. in Curr Genet 56:121-137, 2010). Here, we show that hhy phenotype is not due to increased sensitivity to translation inhibition and establish a super HHY (s-HHY) subgroup of genes comprised of ARF1, CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52, and VPS54 that function exclusively or inclusively at trans-Golgi and late endosome interface. Live cell imaging of s-hhy mutants revealed that hygromycin B treatment disrupts vacuolar morphology and the localization of late endosome marker Pep12, but not that of late endosome-independent vacuolar SNARE Vam3. This, along with normal post-late endosome trafficking of the vital dye FM4-64, establishes that severe hypersensitivity to hygromycin B correlates specifically with compromised trans-Golgi and late endosome interface. We also show that Tor1p vacuolar localization and TORC1 anabolic functions, including growth promotion and phosphorylation of its direct substrate Sch9, are compromised in s-hhy mutants. Thus, an intact trans-Golgi and late endosome interface is a requisite for efficient Tor1 vacuolar localization and TORC1 function.

Aspergillus fumigatus AcuM regulates both iron acquisition and gluconeogenesis.

Relatively few transcription factors that govern the virulence of Aspergillus fumigatus are known. We constructed 11 A. fumigatus transcription factor mutants and screened them for altered virulence in Galleria mellonella larvae. We discovered that the zinc cluster transcription factor, AcuM, is essential for maximal virulence in this model, as well as in murine models of haematogenously disseminated and invasive pulmonary aspergillosis. Transcriptional profiling experiments suggested that AcuM suppresses sreA and induces hapX to stimulate expression of genes involved in both reductive iron assimilation and siderophore-mediated iron uptake. Consistent with these results, a ΔacuM mutant had reduced iron incorporation, decreased extracellular siderophore production and impaired capacity to grow under iron-limited conditions. Interestingly, an Aspergillus nidulansΔacuM mutant had normal extracellular siderophore production and growth under iron-limited conditions, indicating that AcuM does not govern iron acquisition in this organism. A. fumigatus AcuM also regulated genes involved in gluconeogenesis, and the ΔacuM mutant had impaired growth on gluconeogenic carbon sources. Deletion of sreA in the ΔacuM mutant restored iron uptake, extracellular siderophore production and virulence, but not the defect in gluconeogenesis. Thus, AcuM represses SreA and thereby induces iron acquisition, a process that is essential for the maximal virulence of A. fumigatus.

Role of Aspergillus fumigatus DvrA in host cell interactions and virulence.

The transcription factors that regulate Aspergillus fumigatus interactions with host cells and virulence are incompletely defined. We investigated the role of the putative C2H2 transcription factor DvrA in governing these processes. Although DvrA was identified by its limited homology to Candida albicans Bcr1, a ΔdvrA mutant strain of A. fumigatus had wild-type adherence to host constituents in vitro. However, it had increased capacity to damage both endothelial cells and a pulmonary epithelial cell line compared to the ability of the wild-type strain and a ΔdvrA::dvrA-complemented strain. This increase in damage required direct contact between the mutant and host cells. The ΔdvrA mutant also stimulated greater CCL20, interleukin-8, and tumor necrosis factor mRNA expression in a pulmonary epithelial cell line compared to levels induced by the control strains. Also, it was resistant to nikkomycin Z, suggesting an altered cell wall composition. As predicted by these in vitro results, the ΔdvrA mutant had increased virulence and stimulated a greater pulmonary inflammatory response than the wild-type strain and ΔdvrA::dvrA-complemented strains in the nonneutropenic mouse model of invasive pulmonary aspergillosis. These results indicate that DvrA influences A. fumigatus virulence as well as its capacity to damage host cells and stimulate a proinflammatory response.

Aspergillus fumigatus MedA governs adherence, host cell interactions and virulence.

In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatusDeltamedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The DeltamedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro-inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatusDeltamedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively, these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis.

The Aspergillus fumigatus transcription factor Ace2 governs pigment production, conidiation and virulence.

Aspergillus fumigatus causes serious and frequently fatal infections in immunocompromised patients. To investigate the regulation of virulence of this fungus, we constructed and analysed an A. fumigatus mutant that lacked the transcription factor Ace2, which influences virulence in other fungi. The Deltaace2 mutant had dysmorphic conidiophores, reduced conidia production and abnormal conidial cell wall architecture. This mutant produced an orange pigment when grown on solid media, although its conidia had normal pigmentation. Conidia of the Deltaace2 mutant were larger and had accelerated germination. The resulting germlings were resistant to hydrogen peroxide, but not other stressors. Non-neutropenic mice that were immunosuppressed with cortisone acetate and infected with the Deltaace2 mutant had accelerated mortality, greater pulmonary fungal burden, and increased pulmonary inflammatory responses compared with mice infected with the wild-type or Deltaace2::ace2-complemented strains. The Deltaace2 mutant had reduced ppoC, ecm33 and ags3 mRNA expression. It is known that A. fumigatus mutants with absent or reduced expression of these genes have increased virulence in mice, as well as other phenotypic similarities to the Deltaace2 mutant. Therefore, reduced expression of these genes likely contributes to the increased virulence of the Deltaace2 mutant.

Gliotoxin production in Aspergillus fumigatus contributes to host-specific differences in virulence.

BACKGROUND: Gliotoxin is a epipolythiodioxopiperazine toxin that is made by the filamentous fungus Aspergillus fumigatus. Gliotoxin has a wide range of effects on metazoan cells in culture, including induction of apoptosis through inhibition of Nf-kappaB, and inhibition of superoxide production by phagocytes. These activities have led to the proposal that gliotoxin contributes to pathogenesis during invasive aspergillosis. We tested this hypothesis by creating isogenic strains of gliotoxin-producing and nonproducing strains. METHODS: We deleted gliP, the gene that encodes the nonribosomal peptide synthetase GliP. GliP catalyzes the first biosynthetic step in the synthesis of gliotoxin. We then tested for gliotoxin production and virulence in different animal models. RESULTS: Deletion of gliP resulted in strains that were wild type for growth, but they did not synthesize gliotoxin. Transformation of gliP deletion mutants with a full copy of gliP restored gliotoxin production. The gliP deletion strain had attenuated virulence in nonneutropenic mice immunosuppressed with corticosteroids, but had normal virulence in neutropenic mice. It also had reduced virulence in a Drosophila melanogaster model. CONCLUSIONS: Gliotoxin only contributes to the virulence of A. fumigatus in nonneutropenic mice and in fruit flies with functional phagocytes. These results suggest that the principal targets of gliotoxin are neutrophils or other phagocytes.

Efficacy of ambruticin analogs in a murine model of invasive pulmonary aspergillosis.

Ambruticins are a family of polyketides. The antifungal activity of an ambruticin, KOSN-2079, was tested in the mouse model of invasive aspergillosis. KOSN-2079 significantly reduced pulmonary fungal burdens and improved survival over that with the vehicle control. These results support the continued development of ambruticins as antifungal agents.