Functional Characterization of a Bacillus -Derived Novel Broad-Spectrum Antifungal Lipopeptide Variant against Candida tropicalis and Candida auris and Unravelling Its Mode of Action

Abstract

C. tropicalis is a major concern for candidiasis in India and C. auris has emerged as a resistant yeast causing difficult-to-treat infections. Currently, amphotericin B (AMB) and 5-flucytosine (5-FC) are the main therapeutics for systemic fungal infections; however, the nephrotoxicity of AMB and resistance to 5-FC is a serious concern.

Two promising natural lipopeptides from Bacillus subtilis effectively induced membrane permeabilization in Candida glabrata

“…In the lipopeptide-treated and AMB-treated cells CIVS were not observed. Collectively, it may be surmised that the prime targets of AF 4 and AF 5 might be the components of the cell membrane ( Ramesh et al, 2023 ). The presence of a long acyl chain in antifungal cyclic lipopeptides such as iturin and fenzycin enables oligomer formation and subsequent insertion into the plasma membrane ( Malina and Shai, 2005 ) and the organisation of aggregates in biological membranes triggering early membrane damage and membrane disintegration was also reported by Horn et al (2013) .…”

A pipeline for rapidly evaluating activity and inferring mechanisms of action of prospective antifungal compounds

“…42,73 The combination of FUN-1 application and microscopic analysis enabled us to evaluate metabolic activity at individual-cell resolution, which is not possible in plate reader analyses. 34,64,66 FUN-1 only localizes in the subcellular region (as CIVS) if the cell is metabolically active and the membrane is intact. 74 Thus, using it as a stain also provides information on the level of membrane integrity and general cellular stress.…”

“…Therefore, our pipeline complemented the quantitative multiwell‐based assays with microscopic assays that evaluated viability, morphology and metabolic activity of treated FORC conidia and hyphae, bearing in mind that certain antifungal compounds are more effective during specific phases of the fungal growth cycle 42,73 . The combination of FUN‐1 application and microscopic analysis enabled us to evaluate metabolic activity at individual‐cell resolution, which is not possible in plate reader analyses 34,64,66 . FUN‐1 only localizes in the subcellular region (as CIVS) if the cell is metabolically active and the membrane is intact 74 .…”

“…FUN‐1 is a membrane‐permeable, nucleic acid‐binding dye that emits green fluorescence in both live and dead cells. In metabolically active fungal cells with intact plasma membranes, FUN‐1 is intracellularly converted into red cylindrical intravacuolar structures (CIVS) 64–66 . In contrast, dead cells exhibit bright, diffuse green fluorescence, while cells with intact membranes but little or no metabolic activity have diffuse green cytoplasmic fluorescence and lack fluorescent CIVS.…”

The elucidation of the multimodal action of the investigational anti-Candida lipopeptide (AF4) lead from Bacillus subtilis

“…As controls, Candida cells were cultivated without any antifungal treatment and treated with conventional antifungal amphotericin B (AMB) at 1 mg/L (2X MIC) for 3 h. The samples were prepared using glutaraldehyde as a fixative, followed by osmium tetroxide staining. Excess fixative and osmium tetroxide were washed off after each step using sodium cacodylate buffer and sterile water as described in ( Semis et al, 2013 ; Ramesh et al, 2023 ). The samples were dried using gradient ethanol dehydration and subjected to critical point drying, sputter coated with gold and visualised at a magnification of 20000X using a Quanta FEG 250 (Thermo Fisher Scientific, United States) instrument.…”