The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Raw data have been deposited in the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE54810. Funding: This work was supported in part by grants to EMB from the MRC (G0501164) and BBSRC (BB/G009619/1), to EMB and NDR from the Wellcome Trust (WT093596MA), to MB from Imperial College London (Division of Investigative Sciences PhD Studentship), to HH from the ERA-NET PathoGenoMics project TRANSPAT, Austrian Science Foundation (FWF I282-B09), to SGF from the National Institutes of Health, USA (R01AI073829). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

A Salmonella toxin promotes persister formation through acetylation of tRNA

The recalcitrance of many bacterial infections to antibiotic treatment is thought to be due to the presence of persisters that are non-growing, antibiotic-insensitive cells. Eventually, persisters resume growth, accounting for relapses of infection. Salmonella is an important pathogen that causes disease through its ability to survive inside macrophages. After macrophage phagocytosis, a significant proportion of the Salmonella population forms non-growing persisters through the action of toxin-antitoxin modules. Here we reveal that one such toxin, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation. Furthermore, we report the crystal structure of TacT and note unique structural features, including two positively charged surface patches that are essential for toxicity. Finally, we identify a detoxifying mechanism in Salmonella wherein peptidyl-tRNA hydrolase counteracts TacT-dependent growth arrest, explaining how bacterial persisters can resume growth

The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis.

Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 β-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy

Impact of <i>pacC</i> deletion upon <i>A. fumigatus</i> growth.

<p><b>(A and B)</b> Colonial growth phenotypes on supplemented DMEM, 48 hr of growth, 10³ conidia. <b>(C)</b> Immunofluorescence-mediated visualisation of <i>A. fumigatus</i> growth in co-culture with A549 epithelial cells, 16 hours, supplemented DMEM pH 7.4. Fungal cells are labelled with calcoflour white and host cells are labelled with FITC-conjugated concanavalin A, 10⁵ spores/ml, 200× magnification. <b>(D)</b> Quantitation of cell size (μM²) at 16 hr of co-culture with A549 cells, 10⁴ spores/ml, growth conditions as for panel C. <b>(E)</b> Branching frequency of <i>A. fumigatus</i> hyphae, 10⁴ spores/ml, growth conditions as for panel C.</p

<i>ΔpacC</i> mutants are less avidly internalised by A549 and spore internalisation depends on Dectin-1.

<p><b>(A)</b> Percent internalisation by nystatin protection assay. Epithelial monolayers were incubated with 10⁶ spores/ml for 4 hr, n  =  9 for wild types and <i>ΔpacC</i>. <b>(B)</b> Percent internalisation, when blocking the Dectin-1 receptor (0.3 µg/ml α-Dectin-1 antibody) or actin-mediated internalisation (0.2 µM CD), or in the presence of an isotype control antibody (0.3 µg/ml) technical and biological duplicates. <b>(C)</b> Percent detachment, relative to PBS challenge, of A549 upon infection with <i>A. fumigatus</i> (10⁵ spores/ml) for 16 hr and inhibition of actin-mediated internalisation via Cytochalasin D (CD, 0.2 µM), when blocking the Dectin-1 receptor (0.3 µg/ml α-Dectin-1 antibody) or in the presence of an isotype control antibody (0.3 µg/ml), technical and biological triplicates. <b>(D)</b> Use of a soluble Fc Dectin-1 protein for immunofluorescence-mediated imaging of β-glucan in <i>A. fumigatus</i> spores, 4 hr, supplemented DMEM. <b>(E)</b><i>A. fumigatus</i> spore size (μM²), 4 hr, supplemented DMEM. A, B, C: unpaired <i>t</i> test (unless otherwise indicated) *** <i>p</i><0.001, ** 0.001<<i>p</i><0.01, and * 0.01<<i>p</i><0.05.</p

PacC is required for pathogenicity and epithelial invasion in leukopenic mice.

<p><b>(A)</b> Kaplan-Meier curve for murine survival (n ≥ 9) after infection with 6 × 10⁵ and 5 × 10⁴ spores for ATCC46645 and CEA10 respectively. <b>(B)</b> Histopathology of leukopenic murine lungs after 4, 8, 12, hr of infection with ATCC46645 and <i>ΔpacC</i>^ATCC (10⁸ spores), Grocott's Methenamine silver (GMS) and light green staining, 200× magnification. <b>(C)</b><i>ΔpacC</i>^ATCC hyphae (black arrows) are unable to penetrate the respiratory epithelium (RE), BA indicates bronchial airway space, 20 hr post-infection, 400× magnification.</p

Distinct and sequential PacC-dependent activities elicit epithelial decay.

<p><b>(A)</b> Percent detachment, relative to PBS challenge, of A549 cells exposed to antipain-treated and non-treated fungal culture supernatants (48 hr cultures, 10⁵ spores/ml). <b>(B)</b> Integrity of A549 monolayers following exposure to fungal culture supernatants (16 hr cultures, 10⁵ spores/ml). 200× magnification. <b>(C)</b> Percent aberrant morphology, amongst fungus-proximal ECs, per unit of hyphal length, relative to wild-type challenged monolayers. Monolayers were incubated for 18 hr or 36 hr (<i>ΔpacC</i> mutants). A and B: technical and biological triplicates and C: performed twice in triplicate, 1-way ANOVA test, *** <i>p</i><0.001 and ** 0.001 <<i>p</i><0.01.</p