PMID- 36744953
OWN - NLM
STAT- Publisher
LR  - 20240216
IS  - 2165-0497 (Electronic)
IS  - 2165-0497 (Linking)
VI  - 11
IP  - 2
DP  - 2023 Feb 6
TI  - Functional Characterization of a Bacillus-Derived Novel Broad-Spectrum Antifungal 
      Lipopeptide Variant against Candida tropicalis and Candida auris and Unravelling 
      Its Mode of Action.
PG  - e0158322
LID - 10.1128/spectrum.01583-22 [doi]
LID - e01583-22
AB  - Limited treatment options, recalcitrance, and resistance to existing therapeutics 
      encourage the discovery of novel antifungal leads for alternative therapeutics. 
      Antifungal lipopeptides have emerged as potential candidates for developing new 
      and alternative antifungal therapies. In our previous studies, we isolated and 
      identified the lipopeptide variant AF(4) and purified it to homogeneity via 
      chromatography from the cell-free supernatant of Bacillus subtilis. AF(4) was 
      found to have broad-spectrum antifungal activity against more than 110 fungal 
      isolates. In this study, we found that clinical isolates of Candida tropicalis 
      and Candida auris exposed to AF(4) exhibited low MICs of 4 to 8 mg/L. Time-kill 
      assays indicated the in vitro pharmacodynamic potential of AF(4). 
      Biocompatibility assays demonstrated ~75% cell viability at 8 mg/L of AF(4), 
      indicating the lipopeptide's minimally cytotoxic nature. In lipopeptide-treated 
      C. tropicalis and C. auris cells, scanning electron microscopy revealed damage to 
      the cell surface, while confocal microscopy with acridine orange(AO)/propidium 
      iodide (PI) and FUN-1 indicated permeabilization of the cell membrane, and DNA 
      damage upon DAPI (4',6-diamidino-2-phenylindole) staining. These observations 
      were corroborated using flow cytometry (FC) in which propidium iodide, 
      2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and rhodamine 123 (Rh123) 
      staining of cells treated with AF(4) revealed loss of membrane integrity, 
      increased reactive oxygen species (ROS) production, and mitochondrial membrane 
      dysfunction, respectively. Membrane perturbation was also observed in the 
      1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence study and the interaction with 
      ergosterol was observed by an ergosterol binding assay. Decreased membrane dipole 
      potential also indicated the probable binding of lipopeptide to the cell 
      membrane. Collectively, these findings describe the mode of action of AF(4) 
      against fungal isolates by membrane disruption and ROS generation, demonstrating 
      its antifungal potency. IMPORTANCE 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. Antifungal 
      lead molecules with low adverse effects are the need of the hour. In this study, 
      we briefly describe the antifungal potential of the AF(4) lipopeptide and its 
      mode of action using microscopy, flow cytometry, and fluorescence-based assays. 
      Our investigation reveals the basic mode of action of the investigated 
      lipopeptide. This lipopeptide with broad-spectrum antifungal potency is 
      apparently membrane-active, and there is a smaller chance that organisms exposed 
      to such a compound will develop drug resistance. It could potentially act as a 
      lead molecule for the development of an alternative antifungal agent to combat 
      candidiasis.
FAU - Ramesh, Swetha
AU  - Ramesh S
AD  - Department of Biological Sciences, BITS Pilani K.K. Birla Goa Campus, Goa, India.
FAU - Madduri, Madhuri
AU  - Madduri M
AD  - Department of Biological Sciences, BITS Pilani K.K. Birla Goa Campus, Goa, India.
FAU - Rudramurthy, Shivaprakash M
AU  - Rudramurthy SM
AUID- ORCID: 0000-0002-9097-9253
AD  - Department of Medical Microbiology, Post Graduate Institute of Medical Education 
      & Research (PGIMER), Chandigarh, India.
FAU - Roy, Utpal
AU  - Roy U
AUID- ORCID: 0000-0001-6315-4523
AD  - Department of Biological Sciences, BITS Pilani K.K. Birla Goa Campus, Goa, India.
LA  - eng
PT  - Journal Article
DEP - 20230206
PL  - United States
TA  - Microbiol Spectr
JT  - Microbiology spectrum
JID - 101634614
SB  - IM
PMC - PMC10100908
OTO - NOTNLM
OT  - CLSM
OT  - antifungal
OT  - antifungal agents
OT  - antifungal susceptibility testing
OT  - antifungal therapy
OT  - flow cytometry
OT  - lipopeptide
OT  - time-kill assay
COIS- The authors declare no conflict of interest.
EDAT- 2023/02/07 06:00
MHDA- 2023/02/07 06:00
PMCR- 2023/02/06
CRDT- 2023/02/06 09:13
PHST- 2023/02/06 09:13 [entrez]
PHST- 2023/02/07 06:00 [pubmed]
PHST- 2023/02/07 06:00 [medline]
PHST- 2023/02/06 00:00 [pmc-release]
AID - 01583-22 [pii]
AID - spectrum.01583-22 [pii]
AID - 10.1128/spectrum.01583-22 [doi]
PST - aheadofprint
SO  - Microbiol Spectr. 2023 Feb 6;11(2):e0158322. doi: 10.1128/spectrum.01583-22.