PrtT-Regulated Proteins Secreted by Aspergillus fumigatus Activate MAPK Signaling in Exposed A549 Lung Cells Leading to Necrotic Cell Death

Aspergillus fumigatus is the most commonly encountered mold pathogen of humans, predominantly infecting the respiratory system. Colonization and penetration of the lung alveolar epithelium is a key but poorly understood step in the infection process. This study focused on identifying the transcriptional and cell-signaling responses activated in A549 alveolar carcinoma cells incubated in the presence of A. fumigatus wild-type and ΔPrtT protease-deficient germinating conidia and culture filtrates (CF). Microarray analysis of exposed A549 cells identified distinct classes of genes whose expression is altered in the presence of germinating conidia and CF and suggested the involvement of both NFkB and MAPK signaling pathways in mediating the cellular response. Phosphoprotein analysis of A549 cells confirmed that JNK and ERK1/2 are phosphorylated in response to CF from wild-type A. fumigatus and not phosphorylated in response to CF from the ΔPrtT protease-deficient strain. Inhibition of JNK or ERK1/2 kinase activity substantially decreased CF-induced cell damage, including cell peeling, actin-cytoskeleton damage, and reduction in metabolic activity and necrotic death. These results suggest that inhibition of MAPK-mediated host responses to treatment with A. fumigatus CF decreases cellular damage, a finding with possible clinical implications

The Essential Thioredoxin Reductase of the Human Pathogenic Mold Aspergillus fumigatus Is a Promising Antifungal Target

The identification of cellular targets for antifungal compounds is a cornerstone for the development of novel antimycotics, for which a significant need exists due to increasing numbers of susceptible patients, emerging pathogens, and evolving resistance. For the human pathogenic mold Aspergillus fumigatus, the causative agent of the opportunistic disease aspergillosis, only a limited number of established targets and corresponding drugs are available. Among several targets that were postulated from a variety of experimental approaches, the conserved thioredoxin reductase (TrxR) activity encoded by the trxR gene was assessed in this study. Its essentiality could be confirmed following a conditional TetOFF promoter replacement strategy. Relevance of the trxR gene product for oxidative stress resistance was revealed and, most importantly, its requirement for full virulence of A. fumigatus in two different models of infection resembling invasive aspergillosis. Our findings complement the idea of targeting the reductase component of the fungal thioredoxin system for antifungal therapy

Arginine Auxotrophy Affects Siderophore Biosynthesis and Attenuates Virulence of Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic human pathogen mainly infecting immunocompromised patients. The aim of this study was to characterize the role of arginine biosynthesis in virulence of A. fumigatus via genetic inactivation of two key arginine biosynthetic enzymes, the bifunctional acetylglutamate synthase/ornithine acetyltransferase (argJ/AFUA_5G08120) and the ornithine carbamoyltransferase (argB/AFUA_4G07190). Arginine biosynthesis is intimately linked to the biosynthesis of ornithine, a precursor for siderophore production that has previously been shown to be essential for virulence in A. fumigatus. ArgJ is of particular interest as it is the only arginine biosynthetic enzyme lacking mammalian homologs. Inactivation of either ArgJ or ArgB resulted in arginine auxotrophy. Lack of ArgJ, which is essential for mitochondrial ornithine biosynthesis, significantly decreased siderophore production during limited arginine supply with glutamine as nitrogen source, but not with arginine as sole nitrogen source. In contrast, siderophore production reached wild-type levels under both growth conditions in ArgB null strains. These data indicate that siderophore biosynthesis is mainly fueled by mitochondrial ornithine production during limited arginine availability, but by cytosolic ornithine production during high arginine availability via cytosolic arginine hydrolysis. Lack of ArgJ or ArgB attenuated virulence of A. fumigatus in the insect model Galleria mellonella and in murine models for invasive aspergillosis, indicating limited arginine availability in the investigated host niches

Ultrashort Peptide Bioconjugates Are Exclusively Antifungal Agents and Synergize with Cyclodextrin and Amphotericin B

Many natural broad-spectrum cationic antimicrobial peptides (AMPs) possess a general mode of action that is dependent on lipophilicity and charge. Modulating the lipophilicity of AMPs by the addition of a fatty acid has been an effective strategy to increase the lytic activity and can further broaden the spectrum of AMPs. However, lipophilic modifications that narrow the spectrum of activity and exclusively direct peptides to fungi are less common. Here, we show that short peptide sequences can be targeted to fungi with structured lipophilic biomolecules, such as vitamin E and cholesterol. The conjugates were active against Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans but not against bacteria and were observed to cause membrane perturbation by transmission electron microscopy and in membrane permeability studies. However, for C. albicans, selected compounds were effective without the perturbation of the cell membrane, and synergism was seen with a vitamin E conjugate and amphotericin B. Moreover, in combination with β-cyclodextrin, antibacterial activity emerged in selected compounds. Biocompatibility for selected active compounds was tested in vitro and in vivo using toxicity assays on erythrocytes, macrophages, and mice. In vitro cytotoxicity experiments led to selective toxicity ratios (50% lethal concentration/MIC) of up to 64 for highly active antifungal compounds, and no in vivo murine toxicity was seen. Taken together, these results highlight the importance of the conjugated lipophilic structure and suggest that the modulation of other biologically relevant peptides with hydrophobic moieties, such as cholesterol and vitamin E, generate compounds with unique bioactivity

Novel Water-Soluble Amphotericin B‑PEG Conjugates with Low Toxicity and Potent <i>in Vivo</i> Efficacy

Systemic fungal infections are an increasingly prevalent health problem, especially among immunocompromised patients. Antifungal drug development lags far behind in comparison to other types of antimicrobial drugs. Current commercially available antifungals are limited by their insufficient potency, side effects, drug–drug interactions, developing drug-resistance, and narrow formulation options. Here, we report the preparation and evaluation of two novel PEG amide conjugates of amphotericin B (AMB (<b>1</b>)): AB1 (<b>4</b>) and AM2 (<b>5</b>). These compounds are nonlabile, they are prepared in only two and three synthetic steps, respectively, and they show antifungal activity against a wide range of clinical fungal isolates. Their toxicity is significantly lower, and their water solubility is up to 5000-fold higher than that of AMB (<b>1</b>). <i>In vivo</i> efficacy studies in a mouse model of systemic candidiasis showed that AM2 (<b>5</b>) successfully cured all the mice at concentrations above 3.5 mg/kg body weight. In conclusion, these properties make AB1 (<b>4</b>) and AM2 (<b>5</b>) promising candidates for clinical use

Mechanisms of bacterial (Serratia marcescens) attachment to, migration along, and killing of fungal hyphae

We have found a remarkable capacity for the ubiquitous Gram-negative rod bacterium Serratia marcescens to migrate along and kill the mycelia of zygomycete molds. This migration was restricted to zygomycete molds and several basidiomycete species. No migration was seen on any molds of the phylum Ascomycota. S. marcescens migration did not require fungal viability or surrounding growth medium, as bacteria migrated along aerial hyphae as well. S. marcescens did not exhibit growth tropism toward zygomycete mycelium. Bacterial migration along hyphae proceeded only when the hyphae grew into the bacterial colony. S. marcescens cells initially migrated along the hyphae, forming attached microcolonies that grew and coalesced to generate a biofilm that covered and killed the mycelium. Flagellum-defective strains of S. marcescens were able to migrate along zygomycete hyphae, although they were significantly slower than the wild-type strain and were delayed in fungal killing. Bacterial attachment to the mycelium does not necessitate type 1 fimbrial adhesion, since mutants defective in this adhesin migrated equally well as or faster than the wild-type strain. Killing does not depend on the secretion of S. marcescens chitinases, as mutants in which all three chitinase genes were deleted retained wild-type killing abilities. A better understanding of the mechanisms by which S. marcescens binds to, spreads on, and kills fungal hyphae might serve as an excellent model system for such interactions in general; fungal killing could be employed in agricultural fungal biocontrol.Fil: Hover, Tal. Tel Aviv University; IsraelFil: Maya, Tal. Tel Aviv University; IsraelFil: Ron, Sapir. Tel Aviv University; IsraelFil: Sandovsky, Hani. Tel Aviv University; IsraelFil: Shadkchan, Yana. Tel Aviv University; IsraelFil: Kijner, Nitzan. Tel Aviv University; IsraelFil: Mitiagin, Yulia. Tel Aviv University; IsraelFil: Fichtman, Boris. Bar Ilan University; IsraelFil: Harel, Amnon. Bar Ilan University; IsraelFil: Shanks, Robert M. Q.. University of Pittsburgh Medical Center; Estados UnidosFil: Bruna, Roberto Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Garcia Vescovi, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Osherov, Nir. Tel Aviv University; Israe

Contribution of ATPase copper transporters in animal but not plant virulence of the crossover pathogen <i>Aspergillus flavus</i>

<p>The ubiquitous fungus <i>Aspergillus flavus</i> is notorious for contaminating many important crops and food-stuffs with the carcinogenic mycotoxin, aflatoxin. This fungus is also the second most frequent <i>Aspergillus</i> pathogen after <i>A. fumigatus</i> infecting immunosuppressed patients. In many human fungal pathogens including <i>A. fumigatus</i>, the ability to defend from toxic levels of copper (Cu) is essential in pathogenesis. In <i>A. fumigatus</i>, the Cu-fist DNA binding protein, AceA, and the Cu ATPase transporter, CrpA, play critical roles in Cu defense. Here, we show that <i>A. flavus</i> tolerates higher concentrations of Cu than <i>A. fumigatus</i> and other <i>Aspergillus</i> spp. associated with the presence of two homologs of <i>A. fumigatus</i> CrpA termed CrpA and CrpB. Both <i>crpA</i> and <i>crpB</i> are transcriptionally induced by increasing Cu concentrations via AceA activity. Deletion of <i>crpA</i> or <i>crpB</i> alone did not alter high Cu tolerance, suggesting they are redundant. Deletion of both genes resulted in extreme Cu sensitivity that was greater than that following deletion of the regulatory transcription factor <i>aceA</i>. The Δ<i>crpA</i>Δ<i>crpB</i> and Δ<i>aceA</i> strains were also sensitive to ROI stress. Compared to wild type, these mutants were impaired in the ability to colonize maize seed treated with Cu fungicide but showed no difference in virulence on non-treated seed. A mouse model of invasive aspergillosis showed Δ<i>crpA</i>Δ<i>crpB</i> and to a lesser degree Δ<i>aceA</i> to be significantly reduced in virulence, following the greater sensitivity of Δ<i>crpA</i>Δ<i>crpB</i> to Cu than Δ<i>aceA.</i></p