CTP chemical stability versus anti-Candida activity
Firstly, we conducted a new round of MIC determination using a freshly synthesized batch and solution of CTP. The results mirrored the previously published data by our research group (Frota et al. 2023), showing consistent MIC values for C. auris, C. glabrata, C. haemulonii, C. krusei, C. parapsilosis and C. tropicalis (Table 1). Subsequently, we aimed to assess the chemical stability of CTP concerning its antifungal potency when stored at room temperature for up to four months. To achieve this, the compound was monthly subjected to susceptibility tests with all NACs. Impressively, after four months, CTP exhibited consistent potency in inhibiting fungal growth, reflected by similar MIC values against all NACs (Table 1).
Table 1. Antifungal susceptibility profiles of NACs to standard antifungal drugs and CTP.
Candida species (strain)
|
Fluconazole
|
Amphotericin B
|
CTP, [µM]
|
Fresh solution
|
Four-month-old solution
|
C. auris (885)
|
Resistant
|
Resistant
|
31.25
|
31.25
|
C. glabrata (M)
|
Resistant
|
Susceptible
|
31.25
|
31.25
|
C. haemulonii (LIP 2)
|
Resistant
|
Resistant
|
15.62
|
15.62
|
C. krusei (MG 26)
|
Resistant
|
Resistant
|
7.81
|
7.81
|
C. parapsilosis (LIP 45)
|
Resistant
|
Susceptible
|
31.25
|
31.25
|
C. tropicalis (MG 6)
|
Resistant
|
Susceptible
|
62.50
|
62.50
|
CTP perturbs the mitochondrial metabolic activity
Mitochondria play a crucial role in oxidative phosphorylation, resulting in the generation of a substantial amount of ATP for metabolic reactions that govern various biological activities, such as growth and proliferation (Mahl et al. 2015). Consequently, the impact of CTP on mitochondrial metabolic activity was assessed using the classical XTT reduction assay. Our findings revealed that CTP, at different extensions, inhibited the activity of mitochondrial dehydrogenases, with the exception of C. glabrata (Fig. 2). The dose-dependent mode of action of CTP was seen in C. auris, which was the most prominently affected Candida species in our study, as well as in C. haemulonii, C. krusei and C. tropicalis (Fig. 2). For instance, treatment with CTP concentrations of 31.25 and 62.5 µg/mL resulted in approximately 86% and 93% reduction, respectively, in the mitochondrial metabolic activity of C. auris (Fig. 2). Similarly, previous research has demonstrated the high potential of copper-based and other metal-based complexes, as well as theophylline molecule, against the metabolism of C. albicans, resulting in alterations in the cellular respiration process (Ramage et al. 2001; Ashrafi et al. 2020; Singh et al. 2020; Yang et al. 2022)
CTP induces ROS generation
Mitochondrial damage can act as both a cause and a consequence of uncontrolled ROS production. An imbalance resulting from excessive ROS generation can lead to cellular damage and, in turn, trigger cell death (Mesa-Arango et al. 2014). Following this premise, we examined the post-treatment of NACs with CTP to assess the production of ROS as a marker of oxidative stress within the cells (Fig. 3). CTP induced the production of ROS in all tested Candida species in a classical dose-dependent manner, with varying degrees, except for C. glabrata, as judged by the observable increase in H2DCFDA-labeled fluorescent cells (Fig. 3). Remarkably, CTP was notably effective in inducing ROS formation in C. haemulonii, followed by C. parapsilosis. In the case of C. haemulonii, the treatment led to an escalation in fluorescent cells from 20–55% as concentrations increased from 1×MIC to 4×MIC. These results are in line with those obtained by Savić and colleagues (Savić et al. 2018), who demonstrated that a silver(I) coordination compound with 1,7-phenanthroline increased oxidative stress levels in C. albicans strains. Furthermore, research groups have shown the antimicrobial action associated with the induction of oxidative stress by metal ion molecules complexed with 'phenanthroline-like' ligands against different clinically relevant pathogens, such as P. aeruginosa, E. coli, C. albicans, C. parapsilosis and C. glabrata (Savić et al. 2018; Galdino et al. 2022; Sheng et al. 2019). Studies have demonstrated that copper nanoparticles exert different mechanisms in eukaryotic cells, such as oxidative stress, coordination effects and homeostasis imbalance. Nanoparticles can penetrate the cell membrane and, within the cell, interact directly with oxidative organelles such as mitochondria, inducing the production of ROS, which can promote DNA chain breaks. Additionally, Cu2+ ions have the ability to promote functional inactivation of proteins due to their ability to move metal ions into specific metalloproteins or form chelates with biomolecules (Chang et al. 2012; Ingle et al. 2014).
CTP induces DNA fragmentation
An imbalance in ROS production can be detrimental to cells, leading to oxidative damage in crucial biomolecules, including lipids, proteins and DNA (Li et al. 2020). In this sense, DNA injury can be irreversible and result in cell death. To investigate this, DNA fragmentation was assessed in NACs treated with CTP using the TUNEL assay, which is a technique based on the incorporation of nucleotides labeled with fluorescent dye at the free 3'-OH end (Dutta, Chowdhury and Gates, 2007). The treatment with CTP significantly elevated the percentage of TUNEL-positive cells when compared to untreated cells across all studied NACs, indicating the occurrence of DNA fragmentation (Fig. 4). Interestingly, treatment of the multidrug-resistant microorganism P. aeruginosa with bactericidal concentrations of copper(II)-1,10-phenanthroline-5,6-dione (Cu-phendione; 15 µM) induced DNA fragmentation. Treatment with Cu-phendione was able to bind to double-stranded DNA through hydrogen bonding, hydrophobic and electrostatic interactions. Additionally, Cu-phendione was able to induce DNA relaxation mediated by topoisomerase I of supercoiled plasmid DNA, as well as induce oxidative lesions in the DNA (Galdino et al. 2022). In another study conducted by our group, treatment of Leishmania with the Cu-phendione complex at concentrations ranging from 7.5 to 40 nM also induced DNA cleavage (Oliveira et al. 2023).
CTP causes damage in plasma membrane
Excessive production of ROS can induce damage to cell membranes by oxidizing phospholipids. As a result, lipid peroxidation by ROS can compromise the integrity, functionality and fluidity of the plasma membrane (Li et al. 2020). Hence, to determine whether the antifungal mechanism of CTP is associated with damage of the plasma membrane, a vital cellular structure, the passive incorporation PI assay was performed. The results stressed that in all the clinical isolates of NACs studied, a concentration-dependent increase in PI incorporation was notably observed after treatment with CTP (Fig. 5). Remarkably, C. glabrata, which showed no mitochondrial effects from CTP, exhibited significant plasma membrane injuries, as evidenced by a high number of PI-positive cells (~ 90%) when treated with 4×MIC (Fig. 5). A study conducted by Singh and colleagues (Singh et al. 2020) demonstrated that treatment with theophylline (1.4 to 1.8 mg/mL) against C. albicans species acts on the fungal plasma membrane as one of its main targets, promoting variations in its permeability, alterations in ergosterol composition, and imbalances in the transport of sodium and potassium ions, capable of regulating the potential of the plasma membrane (Singh et al. 2020). Furthermore, a study has demonstrated that coordination compounds containing copper metal and/or derivatives of phenanthrene exhibit antifungal activity against species such as C. albicans, C. tropicalis, C. glabrata and C. krusei, through varied mechanisms of action, including compromise of cellular plasma membrane integrity, as well as induction of morphological alterations in its structure (Dar et al. 2019).
CTP promotes alteration in ultrastructure
After establishing that CTP causes injuries to the plasma membrane, we evaluated possible ultrastructural alterations corroborating this phenomenon in Candida isolates. In the untreated cell system, spherical to oval yeasts, without morphological deformity, were observed (Fig. 6, control). Conversely, cells treated with CTP resulted in severe ultrastructural alterations, including irregularities and/or deformations across the fungal surface, such as: reduced cell diameter, concavities/invaginations, intensely rough cell surface and perforations (Fig. 6). These observations suggest that the fungal surface was affected by the treatment with the test compound. Similar to the findings in the present study, investigations conducted by Dar and colleagues (Dar et al. 2019) and Malik and colleagues (Malik et al. 2020) revealed that the utilization of Cu2+-based complexes containing S-benzyl dithiocarbazate Schiff base ligands exhibit remarkable antifungal activity against a broad spectrum of fluconazole-susceptible and resistant C. albicans isolates. Significantly, these complexes are up to 1000× more active than their corresponding Schiff base ligands and induce apoptosis through inhibition of ergosterol biosynthesis and membrane disruption (Dar et al. 2019; Malik et al. 2020)
CTP inhibits adhesion to inert substrate
The initial crucial step for colonization and establishment of infection by Candida species and other fungal pathogens is adhesion to aa substrate, whether biotic or abiotic in origin (Sardi et al. 2013). Considering that CTP induced morphological alterations on the surface of planktonic cells (closely related to the adhesion process), its effect on the adhesion capacity of NACs to polystyrene substrate was also assessed. Remarkably, CTP significantly reduced the ability of NACs cells to adhere to polystyrene, particularly at higher tested concentrations (Fig. 7). Specifically, the adhesion of C. auris and C. haemulonii was notably affected by CTP treatment, resulting in a reduction in the number of adhered cells from 30–60% and from 20–50%, respectively, as the concentration increased from 1× to 4×MIC value (Fig. 7). A similar effect of CTP on the adhesion capacity to both substrates was described in C. parapsilosis (Gandra et al., 2017). Furthermore, it is known that complexes based on phen and theophylline act as potent inhibitors of proliferation and cell growth processes of Candida yeasts, such as C. albicans, C. haemulonii, C. parapsilosis and C. glabrata (Gandra et al. 2017; Savić et al. 2018; Gandra et al. 2020; Singh et al. 2020).
CTP blocks biofilm formation
Biofilm represents a dual structure that significantly contributes to microbial virulence and resistance, formed through a multistep process. The initial stage in the biofilm formation cycle is adhesion (Ramos et al. 2017). Having observed that CTP blocked the adhesion process, we subsequently conducted tests to evaluate the impact of CTP on controlling biofilm formation on a polystyrene surface. The treatment with CTP affected the capacity of fungal cells to generate a robust biofilm, as evidenced by a considerable and dose-dependent reduction observed in both biomass and viability parameters when compared to untreated cells (Fig. 8). In general, CTP treatment elicited a more significant impact on the viability rather than the biomass of biofilms formed by the tested NACs. Among the classical biofilm parameters analyzed, C. auris and C. haemulonii displayed the highest susceptibility to CTP treatment. Specifically, the treatment with CTP at a concentration corresponding to 4×MIC resulted in a substantial decrease in biofilm viability, with reductions of 78% for C. auris and 73% for C. haemulonii (Fig. 8). Research conducted by Viganor and colleagues (Viganor et al. 2016) demonstrated the efficacy of Cu(II)-phendione metal complexes in inhibiting biofilm formation. These complexes exhibited significant reductions in both biomass (48% and 44%, respectively) and viability (78% and 77%, respectively) and effectively disrupted the mature biofilms of P. aeruginosa clinical isolates in a dose-dependent fashion (Viganor et al. 2016).
CTP provokes biofilm disarticulation
Finally, the efficacy of CTP in disrupting mature biofilms formed by NACs was investigated. The results demonstrated a dose-dependent adverse effect of CTP on both biofilm viability (Fig. 9A) and biomass parameters (Fig. 9B), highlighting its potential for disintegrating established biofilms, particularly in C. auris and C. haemulonii. Similar effects of this and other compounds containing copper complexes, phen-derived ligands, and theophylline molecules have been described in the literature across different Candida species, against both formation and disruption of mature biofilms formed by C. albicans, C. glabrata, C. krusei, C. haemulonii, C. auris and C. tropicalis, also highlighting the potential for complexation of these molecules to enhance their antifungal properties (Viganor et al. 2016; Gandra et al. 2017; Savić et al. 2018; Gandra et al. 2020; Singh et al. 2020; Frei et al. 2022). Borowiecki and colleagues (Borowiecki et al. 2018) showed that two derivatives of 1,3-dimethylxanthine exerted dual activity against fungal adhesion and damage to the mature biofilm at concentrations of 20 and 248 µM, in a dose-dependent way (Borowiecki et al., 2018). Furthermore, it is important to note that the tested coordination compound acted more effectively on reducing the cell viability of yeast biofilms compared to biomass quantification analyses, and this pattern was also observed in the disruption of mature biofilms. This observation suggests the hypothesis that CTP alters the cellular metabolism of Candida species as one of its mechanisms of action, an effect observed in both planktonic growth and biofilm processes in the present study. However, further tests are required to confirm the validity of this potential hypothetical mechanism of action.