Antifungal and anti-biofilm activity of designed derivatives from kyotorphin

© 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous analgesic neuropeptide first isolated from bovine brain in 1979. Previous studies have shown that kyotorphins possess anti-inflammatory and antimicrobial activity. Six kyotorphins—KTP-NH2, KTP–NH2–DL, ibuprofen-conjugated KTP (IbKTP), IbKTP-NH2, N-methyl-D-Tyr-L-Arg, and N-methyl-L-Tyr-D-Arg—were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. This study assessed the antimicrobial and antibiofilm activity of these peptides. The antifungal activity of kyotorphins was determined in representative strains of Candida species, including Candida albicans ATCC 10231, Candida krusei ATCC 6258, and six clinical isolates—Candida dubliniensis 19-S, Candida glabrata 217-S, Candida lusitaniae 14-S, Candida novergensis 51-S, Candida parapsilosis 63, and Candida tropicalis 140-S—obtained from the oral cavity of HIV-positive patients. The peptides were synthesized by standard solution or solid-phase synthesis, purified by RP-HPLC (purity >95 %), and characterized by nuclear magnetic resonance. The results of the broth microdilution assay and scanning electron microscopy showed that IbKTP-NH2 presented significant antifungal activity against Candida strains and antibiofilm activity against the clinical isolates. The absence of toxic activity and survival after infection was assessed after injecting the peptide in larvae of Galleria mellonella as experimental infection model. Furthermore, IbKTP-NH2 had strong antimicrobial activity against multidrug-resistant bacteria and fungi and was not toxic to G. mellonella larvae up to a concentration of 500 mM. These results suggest that IbKTP-NH2, in addition to its known effect on cell membranes, can elicit a cellular immune response and, therefore, is promising for biomedical application.This research was supported by FAPESP (Grant No. 2017/00032-0). This article is also part of the Fungal Adaptation to Hostile Challenges special issue for the third International Symposium on Fungal Stress (ISFUS), which is supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant No. 2018/20571-6) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Grant No. 88881.289327/2018-01).info:eu-repo/semantics/publishedVersio

Mixed biofilms formed by C. albicans and non-albicans species: a study of microbial interactions

Abstract Most Candida infections are related to microbial biofilms often formed by the association of different species. The objective of this study was to evaluate the interactions between Candida albicans and non-albicans species in biofilms formed in vitro. The non-albicans species studied were:Candida tropicalis, Candida glabrata andCandida krusei. Single and mixed biofilms (formed by clinical isolates of C. albicans and non-albicans species) were developed from standardized suspensions of each strain (107 cells/mL), on flat-bottom 96-well microtiter plates for 48 hour. These biofilms were analyzed by counting colony-forming units (CFU/mL) in Candida HiChrome agar and by determining cell viability, using the XTT 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay. The results for both the CFU/mL count and the XTT colorimetric assay showed that all the species studied were capable of forming high levels of in vitro biofilm. The number of CFU/mL and the metabolic activity of C. albicans were reduced in mixed biofilms with non-albicans species, as compared with a singleC. albicans biofilm. Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans. In conclusion, C. albicans established antagonistic interactions with non-albicans Candida species in mixed biofilms

Correlation of phospholipase and proteinase production of Candida with in vivo pathogenicity in Galleria mellonella

An essential factor to the virulence of the genus Candida is the ability to produce enzymes and this may be crucial in the establishment of fungal infections. AIM:This study investigated in vitro enzymatic activities of Candida species and their virulence in an in vivo Galleria mellonella experimental model. METHODS: Twenty-four clinical strains of Candida spp. isolated from the human oral cavity were evaluated, including the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. norvegensis, C. lusitaniae and C. guilliermondii. All Candida strains were tested in vitro for production of proteinase and phospholipase. The Candida strains were also injected into Galleria mellonella larvae to induce experimental candidiasis, and after 24 hours, the survival rate was assessed. RESULTS: Phospholipase and proteinase activity were observed in 100% of the C. albicans strains. In the non-albicans species, proteinase and phospholipase activity were observed in 25 and 43% of the studied strains, respectively. The most pathogenic Candida species in G. mellonella were C. albicans, C. dubliniensis and C. lusitaniae, whereas C. glabrata was the least virulent species. Furthermore, a positive significant correlation was found between both enzymatic activities with virulence in G. mellonella. CONCLUSIONS: The virulence of Candida strains in G. mellonella is related to the quantity of proteinases and phospholipases production of each strain

Correlation of phospholipase and proteinase production of Candida with in vivo pathogenicity in Galleria mellonella

ntial factor to the virulence of the genus Candida is the ability to produce enzymes and this may be crucial in the establishment of fungal infections. Aim: This study investigated in vitro enzymatic activities of Candida species and their virulence in an in vivo Galleria mellonella experimental model. Methods: Twenty-four clinical strains of Candida spp. isolated from the human oral cavity were evaluated, including the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. norvegensis, C. lusitaniae and C. guilliermondii. All Candida strains were tested in vitro for production of proteinase and phospholipase. The Candida strains were also injected into Galleria mellonella larvae to induce experimental candidiasis, and after 24 hours, the survival rate was assessed. Results: Phospholipase and proteinase activity were observed in 100% of the C. albicans strains. In the non-albicans species, proteinase and phospholipase activity were observed in 25 and 43% of the studied strains, respectively. The most pathogenic Candida species in G. mellonella were C. albicans, C. dubliniensis and C. lusitaniae, whereas C. glabrata was the least virulent species. Furthermore, a positive significant correlation was found between both enzymatic activities with virulence in G. mellonella. Conclusions: The virulence of Candida strains in G. mellonella is related to the quantity of proteinases and phospholipases production of each strain

Competitive interactions between C. albicans, C. glabrata and C. krusei during biofilm formation and development of experimental Candidiasis

In this study, we evaluated the interactions between Candida albicans, Candida krusei and Candida glabrata in mixed infections. Initially, these interactions were studied in biofilms formed in vitro. CFU/mL values of C. albicans were lower in mixed biofilms when compared to the single biofilms, verifying 77% and 89% of C. albicans reduction when this species was associated with C. glabrata and C. krusei, respectively. After that, we expanded this study for in vivo host models of experimental candidiasis. G. mellonella larvae were inoculated with monotypic and heterotypic Candida suspensions for analysis of survival rate and quantification of fungal cells in the haemolymph. In the groups with single infections, 100% of the larvae died within 18 h after infection with C. albicans. However, interaction groups achieved 100% mortality after 72 h of infection by C. albicans-C. glabrata and 96 h of infection by C. albicans-C. krusei. C. albicans CFU/mL values from larvae hemolymph were lower in the interacting groups compared with the monoespecies group after 12 h of infection. In addition, immunosuppressed mice were also inoculated with monotypic and heterotypic microbial suspensions to induce oral candidiasis. C. albicans CFU/mL values recovered from oral cavity of mice were higher in the group with single infection by C. albicans than the groups with mixed infections by C. albicans-C. glabrata and C. albicans-C. krusei. Moreover, the group with single infection by C. albicans had a higher degree of hyphae and epithelial changes in the tongue dorsum than the groups with mixed infections. We concluded that single infections by C. albicans were more harmful for animal models than mixed infections with non-albicans species, suggesting that C. albicans establish competitive interactions with C. krusei and C. glabrata during biofilm formation and development of experimental candidiasis

Quantification of fungal cells recovered from the buccal cavity of mice.

<p>(A) Mean and standard deviation of the CFU/mL (Log) of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>krusei</i> recovered from the buccal cavity of immunosuppressed mice with single and mixed infections. Student <i>t</i> test. *CFU/mL of <i>C</i>. <i>albicans</i>: comparison between single infection by <i>C</i>. <i>albicans</i> and mixed infection by <i>C</i>. <i>albicans</i>-<i>C</i>. <i>krusei</i> (<i>p</i> = 0.012); **CFU/mL of <i>C</i>. <i>krusei</i>: comparison between single infection by <i>C</i>. <i>krusei</i> and mixed infection by <i>C</i>. <i>albicans</i>-<i>C</i>. <i>krusei</i> (<i>p</i> = 0.004). (B) Mean and standard deviation of the CFU/mL (Log) of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>glabrata</i> recovered from the buccal cavity of immunosuppressed mice with monotypic and heterotypic infections. Student <i>t</i> test. *CFU/mL of <i>C</i>. <i>albicans</i>: comparison between single infection by <i>C</i>. <i>albicans</i> and mixed infection by <i>C</i>. <i>albicans</i>-<i>C</i>. <i>glabrata</i> (<i>p</i> = 0.079); **CFU/mL of <i>C</i>. <i>glabrata</i>: comparison between single infection by <i>C</i>. <i>glabrata</i> and mixed infection by <i>C</i>. <i>albicans</i>-<i>C</i>. <i>glabrata</i> (<i>p</i> = 0.001).</p

Quantification of hyphae, yeast and epithelial changes.

<p>(A) Scores and medians of the number of hyphae and yeast derived from single and mixed infection. Kruskal-Wallis and Dunn test: significant statistical differences were confirmed between the groups (<i>p</i> = 0.0009), with variations between <i>C</i>. <i>albicans</i> monospecies and multi-species groups, and similarities amid the latter. (B) Number of epithelial changes and medians observed in candidosis microscopic lesions on the tongue dorsum of mice inoculated with <i>Candida</i> monospecies and multi-species suspensions. Kruskal-Wallis and Dunn test: significant statistical differences were confirmed between the groups (<i>p</i> = 0.0013), with variations between <i>C</i>. <i>albicans</i> monospecies and multi-species groups, and similarities amid the latter.</p

Macroscopic analysis of candidiasis lesions.

<p>Scores and medians obtained from the macroscopic examination of the tongue dorsum of groups infected with <i>C</i>. <i>albicans</i> monospecies, <i>C</i>. <i>krusei</i> monospecies, <i>C</i>. <i>glabrata</i> monospecies, <i>C</i>. <i>albicans</i>-<i>C</i>. <i>krusei</i> multi-species and <i>C</i>. <i>albicans</i>-<i>C</i>. <i>glabrata</i> multi-species. Kruskal-Wallis and Dunn test: significant statistical differences were confirmed between the groups (<i>p</i> = 0.0001), with similarities between <i>C</i>. <i>albicans</i> monospecies and multi-species groups, and variations when compared with <i>C</i>. <i>krusei</i> and <i>C</i>. <i>glabrata</i> monospecies groups.</p