Introduction
Fungal diseases are an increasing public health concern and present a significant global economic challenge across various agribusiness sectors, including agriculture, livestock farming, and aquaculture. The limited arsenal of available antifungal agents is proving inadequate in the face of emerging drug resistance and the broad diversity of fungal pathogens (Fisher et al., Reference Fisher, Hawkins, Sanglard and Gurr2018). Consequently, the development of novel antifungal agents is essential to overcome the limitations of existing treatments and counteract evolving resistance mechanisms (Perfect, Reference Perfect2017).
Antimicrobial peptides (AMPs) have emerged as a promising alternative due to their broad-spectrum activity, minimal host toxicity, and low propensity for inducing microbial resistance (Huan et al., Reference Huan, Kong, Mou and Yi2020). These small, naturally occurring molecules play a crucial role in the innate immune system, typically exhibiting cationic and amphipathic properties that enable them to target and disrupt microbial membranes. Beyond their classical antimicrobial function, some AMPs also modulate immune responses and contribute to host defence mechanisms across diverse organisms (Matos and Rosa, Reference Matos and Rosa2022).
AMPs derived from aquatic organisms, in particular, hold significant biotechnological potential for therapeutic development. Their diverse mechanisms of action and ability to remain stable in saline and highly alkaline environments make them attractive candidates for drug design (To et al., Reference To, Zhang and Tam2023). Among these, tachyplesins and magainins are two well-characterized classes of cationic amphipathic peptides that exhibit a broad spectrum of biological activities (Kobayashi et al., Reference Kobayashi, Hirakura and Matsuzaki2001; Maloy and Kari, Reference Maloy and Kari1995). Despite their distinct structural conformations (i.e., tachyplesins adopting a disulfide-stabilized β-hairpin structure and magainins forming linear α-helices), both peptides are known for their pore-forming activity, leading to membrane permeabilization and subsequent microbial death (Bulet et al., Reference Bulet, Stöcklin and Menin2004).
Herein, we advanced our understanding of the antifungal potential of aquatic AMPs by evaluating the synergistic activity and mode of action of synthetic peptides inspired by tachyplesins and magainins. Our findings demonstrate that these modified AMPs exhibit broad-spectrum antifungal activity, particularly against filamentous fungi and yeasts relevant to agriculture and aquaculture. Furthermore, we show that these peptides display combined effects and possess the ability to disrupt fungal cell membranes, reinforcing their potential as innovative antifungal agents.
Materials and methods
Peptide synthesis and purification
Carboxy-terminally amidated peptides based on the mature form of the tachyplesin-I from the Japanese horseshoe crab Tachypleus tridentatus (T-I: KWC1FRVC2YRGIC3YRRC4R-NH2; cysteine pairing pattern: C1-C4, C2-C3; MW = 2263.42 Da), and on a lysine-rich analogue of magainin-2 from the African clawed frog Xenopus laevis (MSI-94: GIGKFLKKAKKFGKAFVKIMKK-NH2; MW = 2495.97 Da) were chemically synthesized by Altergen (Schiltigheim, France). The peptides were purified using semi-preparative C18 reversed-phase high-performance liquid chromatography (RP-HPLC), and their molecular masses were analysed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Voyager).
Fungistatic and fungicidal tests
The antifungal activity of the synthetic peptides was tested against 17 strains of filamentous fungi and yeasts of agricultural/aquaculture, biotechnological, and veterinary/human health interest (Table 1). Full details of the origin, source, growth media, and culture conditions of the fungal strains were described earlier (Matos et al., Reference Matos, Garcia-Teodoro, Martins, Schmitt, Guzmán, de Freitas, Stoco, Ferreira, Stadnik, Robl, Perazzolo and Rosa2023). The fungistatic tests were conducted in duplicate using the liquid growth inhibition assay (Matos et al., Reference Matos, Garcia-Teodoro, Martins, Schmitt, Guzmán, de Freitas, Stoco, Ferreira, Stadnik, Robl, Perazzolo and Rosa2023) by incubating 10 μL of peptides (20 to 0.3 μM, final concentration) with 90 μL of filamentous fungi spores (104 spores/mL) or 90 μL of yeast cell suspensions at the exponential growth phase (A600nm = 0.001). In negative controls, peptides were replaced with sterile ultrapure water. Cultures were incubated with shaking for 48 h at 28 °C. Spore germination was observed under an inverted microscope, while yeast growth was monitored by optical density at 600 nm. Minimum inhibitory concentration (MIC) values are expressed as the lowest AMP concentration tested that resulted in complete fungal growth inhibition. The minimum fungicidal concentration (MFC) was determined by inoculating potato dextrose agar plates with samples from each well of the MIC test that showed no growth.
Antifungal spectrum of activity of carboxy-terminally amidated peptides derived from tachyplesin-I (T-I) and magainin-2 (MSI-94), expressed as minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC)
MIC values are reported in micromolar and refer to the minimum concentration required to achieve 100% growth inhibition. MFC values (in μM) refer to the minimum concentration required to inactivate 100% of the fungi. NA stands for not active at the tested concentrations. Nakaseomyces glabratus was formerly known as Candida glabrata, while Pichia kudriavzevii is the teleomorph of the Candida krusei.
Antifungal synergy assays
Synergies between T-I and MSI-94 in inhibiting the growth of the yeasts Candida albicans 12A (MDM8) and Rhodotorula mucilaginosa were evaluated using the checkerboard microtiter assay, as previously described (Matos et al., Reference Matos, Garcia-Teodoro, Martins, Schmitt, Guzmán, de Freitas, Stoco, Ferreira, Stadnik, Robl, Perazzolo and Rosa2023). Results of the synergy were expressed as fractional inhibitory concentration (FIC) index values according to the formula: FIC = [T-I]/MICT-I + [MSI-94]/MICMSI-94, where MICT-I and MICMSI-94 are the MICs of T-I and MSI-94 tested alone, and [T-I] and [MSI-94] are the MICs of the peptides tested in combination. FIC index values were defined as strong synergy for FIC ≤ 0.5, synergy for 0.5 < FIC < 1, additivity for 1 ≤ FIC < 2, no synergy for FIC = 2, and antagonism effect for FIC > 2.
Sytox green uptake assay
Spore solutions of Fusarium oxysporum MUCL 909 (106 spores/mL) or exponential-phase cultures of R. mucilaginosa (A600nm = 0.2) containing 1 μM Sytox Green (Invitrogen) were dispensed into 0.1 mL MicroAmp Fast 96-well reaction plates (Applied Biosystems) with different concentrations of each peptide in triplicate. In positive controls, peptides were substituted with 0.5% Triton X-100. In negative controls, peptides were replaced with sterile ultrapure water. Sytox Green uptake was measured every 30 s over 3 h (λ excitation = 480 nm; λ emission = 550 nm) at 28 °C using a StepOnePlus Real-time PCR System (Applied Biosystems).
Results and discussion
In this study, we expanded our understanding of the antifungal potential of antimicrobial peptides (AMPs) by evaluating the activity spectrum of carboxy-terminally amidated peptides derived from aquatic animals. Against filamentous fungi, both T-I and MSI-94 exhibited fungistatic and fungicidal activity against two agriculturally and aquaculturally relevant Fusarium species, as well as the soil-derived Rhizopus sp. Notably, MSI-94 demonstrated greater potency than T-I (Table 1). Interestingly, most of these filamentous fungi were resistant to α-helical AMPs inspired by shrimp anti-lipopolysaccharide factors (ALFs) (Matos et al., Reference Matos, Garcia-Teodoro, Martins, Schmitt, Guzmán, de Freitas, Stoco, Ferreira, Stadnik, Robl, Perazzolo and Rosa2023). This resistance may be attributed to extracellular proteases secreted by the fungi, which can degrade and inactivate various classes of AMPs (Satala et al., Reference Satala, Bras, Kozik, Rapala-Kozik and Karkowska-Kuleta2023).
Regarding yeasts, both T-I and MSI-94 exhibited a broader spectrum of activity (Table 1). T-I displayed yeasticidal activity against all tested strains, whereas MSI-94 was ineffective only against Candida parapsilosis ATCC 22019 and Nakaseomyces glabratus CCT 0728. Notably, both modified peptides showed potent activity against the clinical isolate Candida tropicalis and the marine yeast R. mucilaginosa (MIC ≤ 2.5 μM). Additionally, T-I and MSI-94 were effective against the clinical isolate 12A (MDM8) and the reference strains ATCC 10231 (Table 1) and ATCC 14053 (Löfgren et al., Reference Löfgren, Smânia, Smânia, Bachère and Barracco2009) of C. albicans, suggesting they target strain-independent cellular components within this species.
The antifungal activity spectrum further underscores the biotechnological potential of these carboxy-terminally amidated peptides, which are also recognized for their antibacterial, antiviral, and antiparasitic properties (Carriel-Gomes et al., Reference Carriel-Gomes, Kratz, Barracco, Bachére, Barardi and Simões2007; Löfgren et al., Reference Löfgren, Miletti, Steindel, Bachère and Barracco2008, Reference Löfgren, Smânia, Smânia, Bachère and Barracco2009; Maloy and Kari, Reference Maloy and Kari1995). Modifying naturally occurring AMPs is a promising strategy for developing safer and more effective biomolecules with enhanced stability, reduced toxicity, and improved antimicrobial properties (Chen and Jiang, Reference Chen and Jiang2023). Designing novel therapeutic agents inspired by aquatic AMPs could have broad applications, including combating drug-resistant infections in agribusiness sectors such as aquaculture (Gerdol et al., Reference Gerdol, Schmitt, Venier, Rocha, Rosa and Destoumieux-Garzón2020; Matos and Rosa, Reference Matos and Rosa2022).
Having established the activity spectrum of both modified AMPs, we investigated whether their combination could enhance antiyeast properties. The combination of T-I and MSI-94 demonstrated an additive effect against C. albicans 12A (MDM8) (FIC = 1.1) and R. mucilaginosa (FIC = 1.0). Additionally, previous studies have evaluated the combined effects of tachyplesins and magainins against bacteria. Specifically, the cyclic analogue T-SS exhibited strong synergistic activity with the magainin-2 derivative MG2 against Gram-negative Escherichia coli ATCC 25922 and Gram-positive Staphylococcus epidermidis ATCC 12228 (Kobayashi et al., Reference Kobayashi, Hirakura and Matsuzaki2001). Given the rise of drug-resistant pathogens and the limited availability of new antifungal agents, enhancing AMP efficacy through combination therapy offers a promising strategy due to their diverse mechanisms of action (Buda De Cesare et al., Reference Buda De Cesare, Cristy, Garsin and Lorenz2020).
Finally, we explored the antifungal mode of action of T-I and MSI-94 by assessing their ability to permeabilize fungal cell membranes. The spores of F. oxysporum MUCL 909 showed membrane permeability following treatment with T-I at 20 μM and MSI-94 at concentrations above 0.6 μM (Figure 1). In R. mucilaginosa, membrane permeabilization was observed with T-I at 5 μM and MSI-94 at 2.5 μM (Figure 1). Fungal cell membranes were effectively disrupted with 0.5% Triton X-100, whereas no increase in fluorescence was observed in negative controls (Figure 1). These findings indicate that T-I and MSI-94 act as membrane-disrupting peptides capable of permeabilizing fungal membranes.
Membrane disruption of fungal spores (Fusarium oxysporum MUCL 909) and yeast cells (Rhodotorula mucilaginosa) was quantified with the Sytox Green uptake assay. For positive controls, 0.5% Triton X-100 was used instead of peptides, while for negative controls, sterile ultrapure water was substituted.
In conclusion, we demonstrated that carboxy-terminally amidated peptides inspired by tachyplesins and magainins exhibit broad-spectrum antifungal activity, effectively targeting filamentous fungi and yeasts relevant to agriculture and aquaculture. Our findings highlight the potential of modified peptides derived from aquatic AMPs as promising antifungal agents with applications across various fields.
Acknowledgements
We are grateful to Dr Margherita Anna Barracco (UFSC, Brazil) and Dr Evelyne Bachère (IHPE, France) for providing the synthetic peptides and to the Laboratório Multiusuário de Estudos em Biologia at the Universidade Federal de Santa Catarina (LAMEB/UFSC) for granting access to its infrastructure.
Author contributions
B. G.-T.: Methodology, Formal analysis. C.P.M.: Methodology, Formal analysis. K.S.V.: Methodology, Funding acquisition. F.W.M.S.: Methodology, Funding acquisition. D.R.: Conceptualization, Supervision, Formal analysis, Writing. L.M.P.: Methodology, Funding acquisition, Writing. R.D.R.: Conceptualization, Supervision, Formal analysis, Writing.
Funding
This work was funded by the Maricá Development Company (CODEMAR) and the National Council for Scientific and Technological Development – CNPq (grants: 406530/2016-5 and 312047/2021-5). B.G.-T. was supported by an MSc scholarship provided by CAPES.
Competing interest
The authors declare no conflict of interests.
