| Congresso Brasileiro de Microbiologia 2023 | Resumo: 638-1 | ||||
Resumo:Candida is a genus of fungi that can cause infections in humans, with C. albicans
historically being primarily responsible for such infections. However, other non-
albicans Candida (NAC) species, such as C. glabrata and C. krusei, have emerged as
significant pathogens in hospitalized and immunocompromised patients, and are also
becoming more common in patients without risk factors. It is noteworthy that the
increasing prevalence of NAC species infections is associated with greater resistance to
antifungal agents and higher mortality rates. This underscores the importance of
discovering new molecules with antimicrobial potential. The objective of this study was
to evaluate the antifungal activity of the peptide CaDef2.1 G27-K44 , inspired by a defensin
found in Capsicum annuum fruits, against Candida yeasts. The antifungal activity was
assessed both in vitro and in vivo, and the mechanism of action of the peptide was also
investigated. Assays were performed to evaluate growth inhibition, cell viability, time of
cell death, membrane permeabilization, oxidative stress, mitochondrial potential,
vacuolar alteration, and intracellular localization of the peptide. Additionally,
cytotoxicity assessments and the therapeutic potential of the peptide were conducted
using Galleria mellonella larvae. CaDef2.1 G27-K44 exhibited equivalent minimum
inhibitory concentration (MIC 100 ) and minimum fungicidal concentration (MFC 100 )
values against C. bracarensis (100 μM), C. glabrata (200 μM), and C. guilliermondi (50
μM) cells. For C. krusei and C. nivariensis, the MFC 100 was 50 μM and 200 μM,
respectively, which corresponded to twice the MIC 100 value. The kinetics of C. krusei
cell death revealed that after 1 hour of incubation with the peptide, all cells were dead.
CaDef2.1 G27-K44 caused plasma membrane permeabilization, oxidative stress, reduction in
mitochondrial potential, and vacuolar fragmentation in C. krusei. Additionally, we
utilized fluorescence confocal microscopy with the peptide labeled with 5-
carboxyfluorescein to investigate the interaction of CaDef2.1 G27-K44 with C. krusei cells
and its ability to penetrate the intracellular space. We observed that, after 3.5 minutes of
incubation, the peptide started to accumulate in the intracellular space of C. krusei,
increasing progressively with the incubation time. The peak fluorescence intensity was
reached at 20.5 minutes. Furthermore, CaDef2.1 G27-K44 did not show cytotoxicity at the
concentrations used (1000, 500, and 250 µM) in G. mellonella larvae. Treatment with
100 μM of the peptide resulted in survival rates of 73.3% and 46.6% in larvae
inoculated with lethal doses (1x10 6 cells/ml) of C. krusei and C. albicans, respectively.
In conclusion, CaDef2.1 G27-K44 demonstrated significant in vitro and in vivo antifungal
activity against different species of the Candida genus. Moreover, this study revealed
that CaDef2.1 G27-K44 is capable of inducing a series of deleterious cellular alterations that
collectively result in the death of C. krusei cells. These findings pave the way for further
investigation of this class of peptides, aiming at potential pharmacological applications
in the treatment of Candida genus caused infections. Palavras-chave: Antimicrobial peptide, Anti-Candida, mechanism of action, therapeutic potential. Agência de fomento:FAPERJ, UENF, CNPQ | |||||