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Antifungal effect of triclosan on Aspergillus fumigatus: quorum quenching role as a single agent and synergy with liposomal amphotericin-B.

ABSTRACT: The purpose of this research was to determine Aspergillus fumigatus conidial viability and its biofilm formation upon treatment with triclosan and amphotericin-B loaded liposomes. A. fumigatus was treated with the antimicrobials, triclosan and liposomal amphotericin-B (L-AMB), in single and combined supplementation. To quantify the cells' viability upon treatments, resazurin-based viability assay was performed. Confocal laser scanning microscopy was done by applying FUN-1 stain to screen the role of the agents on extracellular polymeric substances. Total A. fumigatus biomass upon treatments was estimated by using crystal violet-based assay. To study the agents' effect on the conidial viability, flow cytometry analysis was performed. Expression levels of A. fumigatus genes encoding cell wall proteins, α-(1,3)-glucans and galactosaminogalactan were analysed by real-time polymerase chain reaction assay. A synergistic interaction occurred between triclosan and L-AMB when they were added sequentially (triclosan + L-AMB) at their sub-minimum inhibitory concentrations, the triclosan and L-AMB MICs were dropped to 0.6 and 0.2 mg/L, respectively, from 2 to 1 mg/L. Besides, L-AMB and triclosan contributed to the down-regulation of α-(1,3)-glucan and galactosaminogalactan in A. fumigatus conidia and resulted in less conidia aggregation and mycelia adhesion to the biotic/abiotic surfaces; A. fumigatus conidia-became hydrophilic upon treatment, as a result of rodlet layer being masked by a hydrophilic layer or modified by the ionic strength of the rodlet layer. In A. fumigatus, the potential mechanisms of action for L-AMB might be through killing the cells and for triclosan through interrupting the cells' development as a consequence of quorum quenching.

SUBMITTER: Tamimi R

PROVIDER: S-EPMC9206924 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin (CSP) is the second line of therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, CSP induces a tolerance phenotype with partial reestablishment of fungal growth called CSP paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high CSP concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in CSP tolerance and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the ΔcrzACEA17 mutant, the ΔcrzAAf293 mutant fails to activate cell wall remodeling genes upon CSP exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This study describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

Project description:Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

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Antifungal effect of triclosan on Aspergillus fumigatus: quorum quenching role as a single agent and synergy with liposomal amphotericin-B.

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