Gomesin is an antimicrobial peptide isolated from haemocytes of the spider Acanthoscurria gomesiana and has a broad-spectrum of activity against bacteria, fungi, protozoa and tumour cells [4, 7, 9, 17, 18]. The antifungal activity of gomesin in vitro has previously been reported [4, 7]. However, the antifungal activity against clinical isolates of Candida albicans resistant to antifungal drugs has not been studied. In this paper, we analysed the antifungal activity of gomesin in vitro and in vivo against a clinical strain of C. albicans (isolate 78), as well as its biodistribution and toxicity in mice.
Our data showed that C. albicans (isolate 78) is resistant to fluconazole up to 1.5 mM, but gomesin is effective against this strain at a lower concentration (MIC = 5.5 μM). This resistance to fluconazole is a common cause of treatment failure . A synergism between gomesin and fluconazole against two isolates of Candida albicans (78 and ATCC 90028) was demonstrated using the FICI calculation method. The synergistic mechanism of gomesin and fluconazole is not completely understood, but studies with Cryptococcus neoformans suggested that gomesin, through membrane permeabilisation, promotes an increased entry of fluconazole into the fungal cytoplasm, which results in a better inhibition of the ergosterol synthesis. In this way, fluconazole is effective against C. neoformans at lower doses when applied in combination with gomesin . A similar phenomenon was observed in murine melanoma cells (B16F10-Nex2) treated with gomesin and the monoclonal Mab A4M in vitro. The cytotoxicity of Mab A4M was only detected in the presence of gomesin, after permeabilisation of the cell membrane allowed the entry and action of the monoclonal antibody . From these studies, we hypothesised that gomesin facilitates the entry of fluconazole in Candida albicans through membrane permeabilisation.
The literature on the use of antimicrobial peptides in the treatment of disseminated candidiasis is rather scarce. A study of the HLF peptide (1-11) originated from lactoferrin in immunosuppressed mice with disseminated candidiasis showed that a single dose of 0.4 ng/kg, 24 h after infection, was able to significantly reduce CFU in the kidneys . ETD-151, an analogue of heliomicin also has been shown to be particularly effective against systemic candidiasis in comparison with amphotericin B and several azoles . Likewise, treatment with gomesin proved to be effective against disseminated candidiasis. The peptide effectively reduced the fungal burden in the kidneys, which is the highest tropism organ for Candida. A similar effect was observed with fluconazole; however, this drug has some toxic effects and has selected resistance in Candida albicans . Therefore, the use of gomesin as a therapeutic may be an alternative treatment for candidiasis because our results show that it is non-toxic in mice. Unlike in vitro treatment with gomesin and fluconazole, we have not detected any the synergistic effect of treatment with both drugs in vivo.
The treatment and prevention of recurrent vaginal candidiasis includes the use of imidazoles and triazoles as a first-line treatment, unless it is caused by a confirmed or suspected azole-resistant Candida strain. The efficacy of both oral and local therapy is similar, but, the local treatment presents several advantages, including a reduction of adverse effects; however, local treatment is contraindicated during pregnancy and breast feeding . In recent years, there has been a focus on both understanding drug resistance to antifungal agents and optimising therapy of Candida infections . There are no reports of topical treatment with antimicrobial peptides against vaginal candidiasis. In this paper, we are the first to describe an effective topical formulation of an antimicrobial peptide that is able to reduce CFUs count in an experimental vaginal candidiasis model. We found that 0.2% and 0.5% gomesin cream reduced the CFU on vaginas of the animals by 10 fold when compared to control animals. Minor changes in the treatment protocol with gomesin, either by increasing the frequency or changing the doses, may potentially produce better results. Treatment with 2% miconazole cream was also effective in controlling the CFUs of the vaginas of the animals. However, it was necessary to use a dose of miconazole that was at least four times higher than the dose of gomesin to produce a similar effect. No synergistic effect was observed after treatment with a combination of gomesin and miconazole.
In addition to the direct action of AMPs on microorganisms, either through membrane permeabilisation or internal target interference , it has been reported that some AMPs may possess an immunomodulatory function . In order to verify if gomesin has such activity, the concentrations of IFN-γ, TNF-α and IL-6 were evaluated in the kidneys of mice that had been infected with C. albicans and treated with this peptide. These cytokines, especially IL-6, activate neutrophils, which play an essential role in the defence mechanism against Candida. We observed that treatment with 5 mg/kg gomesin significantly increased the concentration of the three cytokines analysed. A similar effect was also observed with fluconazole treatment. The increase of cytokine levels in the kidneys might help to control candidiasis through the activation of the host immune system. This action appears to be similar to that observed with another AMP, murine β defensin-2, which acts via TLR4 and leads to the production of various cytokines, such as IL-12 and IL-6, as well as chemokines . However, we cannot dismiss the hypothesis that the direct action of gomesin can trigger the release of pathogen-associated molecular patterns, or PAMPs, which would exacerbate the immune response of animals. This has been previously reported for the antimicrobial peptide human β defensin-2 . The use of antimicrobial peptides as immunomodulatory agents for therapeutic application is an effervescent field in progress .
After verifying that the gomesin treatment was effective against disseminated candidiasis in healthy mice, we decided to evaluate the activity of gomesin in immunosuppressed animals, as candidiasis is typically observed in immunocompromised hosts . Treatment with gomesin (5 mg/kg) showed no significant increase in survival compared to control animals. This suggests that the direct action of gomesin was not sufficient to control the infection and that immunomodulatory action is required to suppress the candidiasis. Treatment with fluconazole (20 mg/kg) also did not result in a significant increase in the survival of treated animals as compared to control animals. However, the combined treatment of 5 mg/kg gomesin and 20 mg/kg of fluconazole resulted in 23% survival of mice 30 days after infection. This could be due to gomesin facilitating the entry of fluconazole into the yeast, thus leading to the survival of animals. Another hypothesis is that treatment with fluconazole, being fungistatic, would allow time for gomesin to act.
To evaluate whether gomesin could be used as a therapeutic treatment for C. albicans infection, we performed blood analyses to determine the toxicity of gomesin in mice. No difference in the total number of leukocytes was observed in animals treated with gomesin. However, the number of eosinophils in mice not infected with Candida albicans but treated with gomesin was higher than the control group. The eosinophilia caused by gomesin may be due to the induction of an allergic response. Further experiments are needed in order to evaluate this effect. We have also noticed that gomesin treatment leads to a higher number of neutrophils. This effect might be a consequence of the induction of the pro-inflammatory response by gomesin, which would stimulate the bone marrow to recruit neutrophils. However it is not currently known if these cells are being recruited to the site of infection.
In addition, gomesin did not change the haemoglobin levels, which suggests that this peptide was not toxic to erythrocytes. However, the quantity of reticulocytes is greater in treated animals, suggesting that the peptide provokes an erythropoiesis compared to control animals (non-gomesin treated). Perhaps treatment with gomesin causes hypoxia in animals, thus increasing erythropoietin . Furthermore, gomesin was not nephrotoxic or hepatotoxic, as the bilirubin, creatinine, and Gamma GT levels from treated animals are similar to the control group. Therefore, gomesin seems to be non-toxic to mice.
In addition to the evaluation of toxicity, the biodistribution of gomesin was performed to understand its pharmacokinetics and therefore its therapeutic potential. The biodistribution data revealed that the peptide mainly accumulates in the liver, although it also accumulates in the kidneys and spleen, within the first several minutes after administration. This suggests a rapid clearance from the circulation. The presence of gomesin in the sites of infection might explain the reduction of Candida albicans observed in our experiments. However, other studies are needed to monitor the excretion of the peptide.