RESUMO

Invasive fungal infections (IFIs) caused by Candida species are an emerging threat globally, given that patients at-risk and antifungal resistance are increasing. Antimicrobial peptides (AMPs) have shown good therapeutic capacity against different multidrug-resistant (MDR) microorganisms. This study evaluated the activity of the synthetic peptide, PNR20, against Candida albicans ATCC 10231 and a MDR Colombian clinical isolate of Candida auris. Perturbation of yeast cell surface was evaluated using scanning electron microscopy. Cell viability of Vero cells was determined to assess peptide toxicity. Additionally, survival, fungal burden, and histopathology of BALB/c mice infected intravenously with each Candida species and treated with PNR20 were analyzed. Morphological alterations were identified in both species, demonstrating the antifungal effect of PNR20. In vitro, Vero cells' viability was not affected by PNR20. All mice infected with either C. albicans or C. auris and treated with PNR20 survived and had a significant reduction in the fungal burden in the kidney compared to the control group. The histopathological analysis in mice infected and treated with PNR20 showed more preserved tissues, without the presence of yeast, compared to the control groups. This work shows that the utilization of PNR20 is a promising therapeutic alternative against disseminated candidiasis.

RESUMO

Candidiasis is an opportunistic infection affecting immunosuppressed and hospitalized patients, with mortality rates approaching 40% in Colombia. The growing pharmacological resistance of Candida species and the emergence of multidrug-resistant Candida auris are major public health problems. Therefore, different antimicrobial peptides (AMPs) are being investigated as therapeutic alternatives to control candidiasis effectively and safely. This work aimed to evaluate the in vitro antifungal activity of three synthetic AMPs, PNR20, PNR20-1, and 35409, against ATCC reference strains of Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, and Candida tropicalis, and clinical isolates of C. auris. Antifungal susceptibility testing, determined by broth microdilution, showed that the AMPs have antifungal activity against planktonic cells of all Candida species evaluated. In C. auris and C. albicans, the peptides had an effect on biofilm formation and cell viability, as determined by the XTT assay and flow cytometry, respectively. Also, morphological alterations in the membrane and at the intracellular level of these species were induced by the peptides, as observed by transmission electron microscopy. In vitro, the AMPs had no cytotoxicity against L929 murine fibroblasts. Our results showed that the evaluated AMPs are potential therapeutic alternatives against the most important Candida species in Colombia and the world.

RESUMO

Fungal infections have increased in recent decades with considerable morbidity and mortality, mainly in immunosuppressed or admitted-to-the-ICU patients. The fungal resistance to conventional antifungal treatments has become a public health problem, especially with Candida that presents resistance to several antifungals. Therefore, generating new alternatives of antifungal therapy is fundamental. One of these possibilities is the use of antimicrobial peptides, such as LL-37, which acts on the disruption of the microorganism membrane and promotes immunomodulatory effects in the host. In this study, we evaluated the in vitro antifungal activity of the LL-37 analogue peptides (AC-1, LL37-1, AC-2, and D) against different Candida spp. and clinical isolates obtained from patients with vulvovaginal candidiasis. Our results suggest that the peptides with the best ranges of MICs were LL37-1 and AC-2 (0.07 µM) against the strains studied. This inhibitory effect was confirmed by analyzing the yeast growth curves that evidenced a significant decrease in the fungal growth after exposure to LL-37 peptides. By the XTT technique we observed a significant reduction in the biofilm formation process when compared to yeasts untreated with the analogue peptides. In conclusion, we suggest that LL-37 analogue peptides may play an important antimicrobial role against Candida spp.

RESUMO

The repurposing strategy was applied herein to evaluate the effects of lopinavir, an aspartic protease inhibitor currently used in the treatment of HIV-infected individuals, on the globally widespread opportunistic human fungal pathogen Candida albicans by using in silico, in vitro and in vivo approaches in order to decipher its targets on fungal cells and its antifungal mechanisms of action. Secreted aspartic proteases (Saps) are the obviously main target of lopinavir. To confirm this hypothesis, molecular docking assays revealed that lopinavir bound to the Sap2 catalytic site of C. albicans as well as inhibited the Sap hydrolytic activity in a typically dose-dependent manner. The inhibition of Saps culminated in the inability of C. albicans yeasts to assimilate the unique nitrogen source (albumin) available in the culture medium, culminating with fungal growth inhibition (IC50 = 39.8 µM). The antifungal action of lopinavir was corroborated by distinct microscopy analyses, which evidenced drastic and irreversible changes in the morphology that justified the fungal death. Furthermore, our results revealed that lopinavir was able to (i) arrest the yeasts-into-hyphae transformation, (ii) disturb the synthesis of neutral lipids, including ergosterol, (iii) modulate the surface-located molecules, such as Saps and mannose-, sialic acid- and N-acetylglucosamine-containing glycoconjugates, (iv) diminish the secretion of hydrolytic enzymes, such as Saps and esterase, (v) negatively influence the biofilm formation on polystyrene surface, (vi) block the in vitro adhesion to epithelial cells, (vii) contain the in vivo infection in both immunocompetent and immunosuppressed mice and (viii) reduce the Sap production by yeasts recovered from kidneys of infected animals. Conclusively, the exposed results highlight that lopinavir may be used as a promising repurposing drug against C. albicans infection as well as may be used as a lead compound for the development of novel antifungal drugs.

RESUMO

BACKGROUND: Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene. OBJECTIVE: This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata. METHODS: Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection. FINDINGS: The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice. MAIN CONCLUSIONS: The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.

Assuntos

RESUMO

Commensal yeast from the genus Candida is part of the healthy human microbiota. In some cases, Candida spp. dysbiosis can result in candidiasis, the symptoms of which may vary from mild localized rashes to severe disseminated infections. The most prevalent treatments against candidiasis involve fluconazole, itraconazole, miconazole, and caspofungin. Moreover, amphotericin B associated with prolonged azole administration is utilized to control severe cases. Currently, numerous guidelines recommend echinocandins to treat invasive candidiasis. However, resistance to these antifungal drugs has increased dramatically over recent years. Considering this situation, new therapeutic alternatives should be studied to control candidiasis, which has become a major medical concern. Limonene belongs to the group of terpene molecules, known for their pharmacological properties. In this study, we evaluated in vitro the limonene concentration capable of inhibiting the growth of yeast from the genus Candida susceptible or resistant to antifungal drugs and its capacity to induce fungal damage. In addition, intravaginal fungal infection assays using a murine model infected by Candida albicans were carried out and the fungal burden, histopathology, and scanning electron microscopy were evaluated. All of our results suggest that limonene may play a protective role against the infection process by yeast from the genus Candida.

RESUMO

Candida auris and Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) are phylogenetically related species that share some physiological features and habits. In the present study, we compared the virulence of these yeast species using two different experimental models: (i) Galleria mellonella larvae to evaluate the survival rate, fungal burden, histopathology and phagocytosis index and (ii) BALB/c mice to evaluate the survival. In addition, the fungal capacity to form biofilm over an inert surface was analyzed. Our results showed that in both experimental models, the animal survival rate was lower when infected with C. auris strains than the C. haemulonii species complex. The hemocytes of G. mellonella showed a significantly reduced ability to phagocytize the most virulent strains forming the C. haemulonii species complex. Interestingly, for C. auris, it was impossible to measure the phagocytosis index due to a general lysis of the hemocytes. Moreover, it was observed a greater capability of biofilm formation by C. auris compared to C. haemulonii species complex. In conclusion, we observed that C. auris and C. haemulonii complex have different levels of pathogenicity in the experimental models employed in the present study.

RESUMO

Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America, with the highest prevalence in Brazil, Colombia, and Venezuela. Fungi of the Paracoccidioides genus are etiologic agents of the disease. The 15 amino acid peptide P10 is derived from gp43, the main diagnostic antigen of Paracoccidioides brasiliensis. We previously reported that P10-pulsed dendritic cells (DCs) induce a protective response against P. brasiliensis. Presently, dexamethasone-treated BALB/c mice were intratracheally infected with P. brasiliensis Pb18 to establish the therapeutic efficacy of P10-pulsed DCs. Immunosuppressed and infected animals that received DCs had a reduction in their fungal burden, and this result was most pronounced in mice receiving DCs primed with P10. The efficacy of therapeutic DCs was significantly augmented by concomitant treatment with trimethoprim-sulfamethoxazole. Additionally, primed-DCs with or without the antifungal drug induced a beneficial Th1-biased immune response and significantly reduced tissue damage. In conclusion, these studies with immunocompromised mice demonstrate that P10-pulsed DCs with or without concomitant antifungal drugs are potently effective in combating invasive PCM. These findings support further translational studies to validate the use of P10-primed DCs for PCM in immunocompetent and immunosuppressed hosts.

RESUMO

Vulvovaginal and invasive candidiasis are frequent conditions in immunosuppressed individuals caused by Candida albicans and non-albicans Candida spp. Fluconazole and Amphotericin B are the main drugs used to fight the infection. However, resistance to fluconazole and other azole antifungal drugs is an important clinical problem that encourages the search for new therapeutic alternatives. In this work, we evaluate the antifungal activity of the biphosphinic cyclopalladate C7a in the in vitro and in vivo model. Our results showed fungicidal activity, with low values of minimal inhibitory concentrations and minimum fungicidal concentrations, even for fluconazole and/or miconazole resistant Candida isolates. Fluorescence microscopy and transmission electron microscopy revealed that the compound was able to inhibit the formation of hyphae/pseudohyphae and, moreover, promoted morphological alterations in cellular organelles and structures, such as disruption of cell wall, apparent mitochondrial swelling, chromatin marginalization into the nuclei and increased numbers of electron-lucent vacuoles. C7a significantly decreased the biofilm formation and reduced the viability of yeast cells in mature biofilms when tested against a virulent C. albicans strain. In vivo assays demonstrated a significant decrease of fungal burden in local (vaginal canal) and disseminated (kidneys) infection. In addition, we observed a significant increase in the survival of the systemically infected animals treated with C7a. Our results suggest C7a as a novel therapeutic agent for vaginal and disseminated candidiasis, and an alternative for conventional drug-resistant Candida.

RESUMO

Paracoccidioidomycosis is a fungal disease endemic in Latin America. Polyclonal antibodies to acidic glycosphingolipids (GSLs) from Paracoccidioides brasiliensis opsonized yeast forms in vitro increasing phagocytosis and reduced the fungal burden of infected animals. Antibodies to GSL were active in both prophylactic and therapeutic protocols using a murine intratracheal infection model. Pathological examination of the lungs of animals treated with antibodies to GSL showed well-organized granulomas and minimally damaged parenchyma compared to the untreated control. Murine peritoneal macrophages activated by IFN-γ and incubated with antibodies against acidic GSLs more effectively phagocytosed and killed P. brasiliensis yeast cells as well as produced more nitric oxide compared to controls. The present work discloses a novel target of protective antibodies against P. brasiliensis adding to other well-studied mediators of the immune response to this fungus.