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Today, Candida albicans is still the most common cause of both local and life-threatening systemic candidiasis. The spread of resistant fungal strains has resulted in an urgent need to search for new promising antimycotics. Here, we investigated the antifungal action of the tobacco defensin NaD1 against susceptible and resistant to azoles and echinocandins strains of C. albicans. We demonstrated that NaD1 was equally effective and fungicidal against all tested strains. The MIC and MFC values were 6.25 and 12.5 µM, respectively. We showed for the first time that NaD1 could act synergistically not only with caspofungin but also with human host defense antimicrobial peptides cathelicidin LL-37 and β-defensin-2 (HBD2) against susceptible and resistant fungal strains. Using flow cytometry, we demonstrated that NaD1 in combinations with LL-37 or HBD2 can reinforce each other by enhancing membrane disruption. Using the Caco-2 cell monolayer model, we demonstrated that NaD1 impaired the adhesion of C. albicans cells to the human epithelium. Moreover, NaD1 inhibited the formation of fungal biofilms in Sabouraud broth and less markedly in nutrient-rich RPMI-1640 medium, and enhanced the antibiofilm activity of caspofungin. Thus, we hypothesized that NaD1 might affect the development of candidiasis in vivo, including that caused by resistant fungal strains.

Background/Objectives: The growing resistance development among fungi, including those of Candida species, poses significant challenges to public health, emphasizing the need for the implementation of innovative therapeutic approaches. The tobacco defensin NaD1 exhibits a pronounced activity against C. albicans, but its relatively high cytotoxicity toward mammalian cells limits its potential application. Here, we investigated anticandidal activity and cytotoxicity of four modified analogues of NaD1 (NaD1-1 T44R/K45R, NaD1-2 L38R, NaD1-3 K36R/L38R, NaD1-4 L38R/T44R/K45R). Methods: These peptides contained substitutions with arginine of some amino acid residues in the C-terminal region of NaD1 and in its L5 loop (S35KILRR40), responsible for the “cationic grip” and binding to phosphatidylinositol 4,5-bisphosphate (PIP4,5), one of the primary targets of tobacco defensin action. Results: We showed that the modified NaD1 analogues effectively inhibited the growth of C. albicans cells but had a less fungicidal action than NaD1. As compared to NaD1, its modified analogues differed in their sensitivity to the presence of various salts; antifungal activities of NaD1-3 and NaD1-4 were more tolerant to the presence of NaCl and CaCl2, respectively. All modified analogues except NaD1-1 did not exhibit hemolytic activity and showed significantly less cytotoxicity towards human immune and epithelial cells compared to NaD1. All modified analogues enhanced the permeability of PIP4,5-containing liposomes, although less effectively than NaD1. Differences in their properties were also demonstrated through experiments on oligomerization and zymosan binding. Conclusions: Thus, we proposed that the modified NaD1 analogues NaD1-2, NaD1-3, and NaD1-4 appear to be promising candidate antifungals. However, further in vitro and in vivo studies are required to evaluate their therapeutic potential against critical fungal pathogens.

Background/Objectives: Defensins are important components of the innate plant immune system, exhibiting antimicrobial activity against phytopathogens, as well as against fungi pathogenic to humans. Along with antifungal activity, plant defensins are also capable of influencing various immune processes, but not much is known about these effects. In this study, we investigated the immunomodulatory effects of the tobacco defensin NaD1, which possesses a pronounced antifungal activity. Methods and Results: We showed that NaD1 could penetrate the Caco-2 polarized monolayer. Using a multiplex assay with a panel of 48 cytokines, chemokines and growth factors, we demonstrated that NaD1 at a concentration of 2 μM had immunomodulatory effects on human dendritic cells and blood monocytes, mainly inhibiting the production of various immune factors. Using the sandwich ELISA method, we demonstrated that NaD1 at the same concentration had a pronounced immunomodulatory effect on unstimulated THP-1-derived macrophages and those stimulated by bacterial LPS or fungal zymosan. NaD1 had a dual effect and induced the production of both pro-inflammatory cytokine IL-1β as well as anti-inflammatory IL-10 on resting and pro-inflammatory THP-1-derived macrophages. We also found that the immunomodulatory effects of the tobacco defensin NaD1 and the pea defensin Psd1 differed from each other, indicating nonuniformity in the modes of action of plant defensins. Conclusions: Thus, our data demonstrated that the tobacco defensin NaD1 exhibits different immunomodulatory effects on various immune cells. We hypothesized that influence on human immune system along with antifungal activity, could determine the effectiveness of this peptide under infection in vivo.

Antimicrobial peptides (AMPs) play a pivotal role in plant immune responses to diverse stresses, and hence, have become the novel molecules for studying plant responses to environmental harsh conditions. For the first time, the effect of the antimicrobial peptide defensin NaD1 on drought stress was investigated in tobacco by generating the NaD1 transgenic lines overexpressing the coding sequence of NaD1. The NaD1 expression was confirmed by RT-PCR, and the NaD1 peptide presence was verified using ELISA and western blot analysis in the tobacco transgenic lines. In silico bioinformatic analysis revealed that the most abundant components in Cis-regulatory elements in NaD1 homologs in Nicotiana attenuate (NaDEF genes) are MYB, MYC and ABRE elements suggesting that the NaD1 promoter is involved in the abiotic stress regulation. The chlorophyll a, b, and total chlorophyll contents, and correspondingly, the index of chlorophyll stability (ICS) significantly (P ≤ 0.01) increased in all 3 NaD1 transgenic lines under drought stress. Moreover, activities of Catalase (CAT), Peroxidase (POD), Ascorbate peroxidase (APX) and Superoxide dismutase (SOD) were significantly enhanced in response to drought stress in the transgenic lines. Among the three transgenic lines, line 1 showed more tolerance to the drought stress. The data together suggest that the expression of NaD1 increases the antioxidant activity of the enzymes presumably leading to elimination of ROS levels and maintenance of the chlorophyll content and stability, resulting in enhanced drought tolerance in the transgenic tobacco lines. Therefore, the defensin NaD1 is an essential factor in regulation of plant responses to drought stress.