4. DISCUSSION
The epidemiology of infections caused by Candida species has changed over the last decade, with an increase in the proportion of non-Candida albicans (NCA) species.
Among these, C. glabrata, previously considered a low pathogenic species, has emerged as the most common Candida species responsible for both systemic and mucosal infections, second in frequency only to C. albicans (Hajjeh et al., 2004; Silva et al., 2012). In particular, vaginal infections due to C. glabrata have increased and are difficult to eradicate, giving rise to recurrent episodes (Fidel, 1999; Sobel, 2007;
Kennedy and Sobel, 2010; Trama et al., 2005). In fact, C. glabrata vaginitis is difficult to treat due to an intrinsic reduced susceptibility to azoles, in particular to fluconazole, which is widely used for the topical treatment of this mycosis (Richter et al., 2005;
Morschhäuser, 2010).
For this reason, there is an urgent need to individuate novel strategies for the treatment of C. glabrata infections. In this regard, the therapeutic potential of natural anti-infective agents, such as antimicrobial peptides (AMPs), has generated much interest due to the potential therapeutic approaches that these molecules offer (Yeung et al., 2011).
The present study was focused on human hepcidin 20 (Hep-20), a small peptide involved in the regulation of iron homeostasis (Ganz and Nemeth, 2011). Hep-20 has been recently demonstrated to exert antimicrobial activity against clinically relevant gram positive and gram negative bacteria (Maisetta et al., 2010); however, no data on Hep-20 anticandidal activity is currently available.
In this study the in vitro fungicidal activity of Hep-20 was evaluated against a panel of C. glabrata clinical isolates with different levels of fluconazole susceptibility.
Experiments were performed at different pH conditions in a sodium phosphate buffer (SPB), containing a reduced salt concentration, since it is reported in literature that high concentration of salts inhibits the activity of cationic antimicrobial peptides (Lehner et al., 1991; Maisetta et al., 2010).
At neutral pH, all C. glabrata isolates were found to be susceptible to Hep-20, following 90 minutes of incubation at 30°C with minimal fungicidal concentration
(MFC) ranging from 100 to 200 µg/ml. This finding was of particular relevance, since C. glabrata has been associated with a remarkable resistance to several antimicrobial peptides, such as histatins, magainins, cathelicidins and lactoferrin derived peptides (Helmerhorst et al., 2005, 2006; Benincasa et al., 2006; Kondori et al., 2011). Despite the fact that Hep-20 fungicidal concentrations were higher than those observed for bacterial species, they were comparable to MFCs reported for other peptides, which were considered active versus different Candida species (concentration ranging from 50 to 225 µg/ml) (Helmerhorst et al., 2006; Maisetta et al., 2010; Kondori et al., 2011).
In addition, the activity exerted by Hep-20 on C. glabrata isolates with different levels of fluconazole susceptibility, suggests that the molecular mechanisms underlying fluconazole resistance do not affect the antifungal activity of the peptide.
Notably, at acidic pH (5.0) the fungicidal effect exerted by Hep-20 on C.
glabrata isolates with different fluconazole susceptibility was potentiated (MFC ranging
from 50 to 100 µg/ml), in comparison with the one obtained at neutral pH (MFC ranging from 100 to 200 µg/ml). Indeed, acidic pH not only reduced the fungicidal concentration of Hep-20 to half the one observed at neutral pH, but the peptide exerted its antifungal activity earlier (30 minutes), as compared to kinetics obtained at pH 7.4 (60 minutes). This finding is consistent with a proposed mechanism by which the protonated basic residues of Hep-20 at acidic pH may facilitate the interaction with negatively charged fungal surfaces, as also observed for other cationic peptides (Kacprzyk et al., 2007; Maisetta et al., 2010). In addition, it is likely that at low pH values, hepcidins reduce their aggregation tendency, thus resulting in correctly folded molecules, which are available to express their antimicrobial activity (Zhang et al., 2010). This hypothesis is supported by nuclear magnetic resonance (NMR) diffusion experiments demonstrating the monomeric nature of Hep-20 in acidic pH conditions (Hunter et al., 2002). In this regard, previous studies showed a low tendency of Hep-20 to aggregate, as compared to its 25 aminoacid full length isoform hepcidin- 25 (Hep-25) (Melino et al., 2005). This is most likely due to the absence of the first five amino terminal residues, which play an important role in the aggregation process (Melino et al., 2005).
One of the potential advantages of using AMPs as novel therapeutic strategy is the possibility to perform combination experiments with common antibiotics (Hancock and Lehner, 1998; Harris and Coote, 2010). The benefits of this approach are: improved
efficacy over the use of single drugs, reduced cost of each individual drug, reduced toxicity and improved tolerance, broader spectrum of efficacy, and, finally, limited selection of resistant microorganism, in comparison with those resulting from monotherapy (Harris and Coote, 2010).
In this respect, we evaluated the synergistic effect of Hep-20 in combination with common antifungal drugs in SPB at pH 5.0. In particular, the synergistic effect of Hep- 20 was evaluated in combination with amphotericin B (AmB), caspofungin and fluconazole, three common antifungal drugs representative of a class of polyenes, echinocandins and azoles respectively.
Due to the different mechanisms of action of these antifungal drugs we evaluated the synergistic effect with two different methods: by killing assay and by the fractional inhibitory concentration index method (FICI) (Odds, 2003).
The killing assay was performed to evaluate the combination Hep-20/AmB and Hep-20/caspofungin, using sub-fungicidal concentration of each drug. The results obtained indicated a synergistic effect of the Hep-20/AmB combination following 90 minute incubation at 30°C, and a significantly enhanced effect of the combination in comparison with the most active component alone following 24 hour incubation.
Considering that AmB exerts its antifungal activity by interacting with ergosterol at the fungal cell membrane and that it forms trans-membrane pores that eventually lead to cell death (Baginski et al., 2005), the synergism between Hep-20 and AmB could be explained by an enhanced disruption of C. glabrata membrane.
A synergistic effect was also observed for Hep-20/caspofungin combination following 90 minute incubation in SPB, pH 5.0, and it was also confirmed at later time points. Echinocandins work by a noncompetitive inhibition of β-1,3-glucan synthase in the plasma membrane of the fungal cells; this enzyme is involved in the production of β-glucan, an essential component of the fungal cell wall (Inoue et al., 1996; Kurtz and
Duglas, 1996; Qadota et al., 1996). An intact cell wall represents a potential barrier that cationic peptides must pass through before interacting with plasma membrane.
Supporting this hypothesis, Harris and co-workers demonstrated that disruption of the outer cell wall phosphomannan content has a significant influence on the degree of susceptibility of C. albicans to cationic peptides (Harris et al., 2009). In addition, the same group studied the nature of synergistic combination of echinocandins with different cationic peptides against C. albicans and C. glabrata. They demonstrated, by fluorescence microscopy, that exposure of yeast to these combinations enhanced the
uptake of the cationic peptide into the cell, resulting in a potentiated killing effect (Harris and Coote, 2010). Based on this evidence, it could be speculated that synergistic effect of Hep-20/caspofungin combination has a similar mode of action.
Due to the fungistatic nature of fluconazole, the activity of the peptide in combination with this antifungal was evaluated by FICI and a synergistic effect of the combination was observed for the fluconazole resistant isolate.
This finding was particularly relevant, since C. glabrata isolates are commonly associated with a reduced susceptibility to fluconazole which, in this species, is mainly due to the overexpression of efflux pumps (Sanguinetti et al., 2005; Ferrari et al., 2009).
The destabilization of plasma membrane due to Hep-20 could reduce the activity of efflux pumps and consequently increase the accumulation of fluconazole inside the cells, even though the exact mechanism leading this phenotype is still not fully understood.
The enhanced fungicidal activity of Hep-20 observed under acidic conditions suggested that this peptide could be used as potential therapeutic agent of C. glabrata related infections in body district characterized by low pH, such as vaginal district. To verify this hypothesis, an artificial vaginal fluid resembling human vaginal secretions (VFS) was used in order to evaluate whether the activity of Hep-20 alone and in combination with common antifungal drugs was maintained in a biological fluid simulant.
Considering that the average volume of vaginal fluid ranges from 0.5 to 0.75 ml, in our experiments VFS was diluted 4-fold to mimic fluid dilution by a vaginal suppository (Owen and Katz 1999; Lai et al., 2008).
In this experimental condition, the Hep-20 fungicidal effect observed in SPB was not confirmed for any of the concentrations tested (50 to 400 µg/ml). This finding suggested that Hep-20 activity could be inhibited by the presence in the medium of BSA and mono and divalent cations such as Ca2+, K+ and Na+ (Owen and Katz 1999).
Indeed, the antimicrobial activity of AMPs was shown to be strongly reduced in biological fluids due to the presence of physiological concentration of salts, such as K+, Na+, Mg2+ and Ca2+ (Wei and Bobek, 2005; Batoni et al., 2006; Wei et al., 2007;
Maisetta et al., 2008)
Among the different mechanisms proposed to explain the loss of antimicrobial activity of the peptide in biological fluids, it has been hypothesized that the presence of divalent cations such as calcium and magnesium mediates an electrostatic interaction
with the negatively charged membrane of microorganisms and it creates a barrier preventing the interaction between AMPs and microbial plasma membrane. For this reason, in the attempt to restore the activity of the peptide in VFS, experiments were repeated in the presence of 1 mM EDTA.
In fact, EDTA is a synthetic chelanting agent that forms strong complexes with cations and it is widely used as stabilizer in food and in pharmaceutical vaginal applications (Garg et al., 2001; Kubo et al., 2005). Furthermore, EDTA has been shown to limit the availability of essential cations for growth and/or to destabilize cell membrane of bacteria (Banin et al., 2006). It has been also demonstrated that EDTA inhibits hyphal development in C. albicans and that it exerts fungicidal or fungistatic activity against S. cerevisiae (Gil et al., 1994; Kubo et al., 2005).
In a study performed on human saliva by Wei and colleagues, the addition of EDTA enhanced the antifungal activity of different peptides such as MUC7, histatin 5 and magainin against C. albicans, in comparison with saliva alone (Wei and Bobek, 2005).
In our case, the addition of EDTA to the VFS resulted in a statistically significant enhancement of Hep-20 antifungal activity against C. glabrata BPY44 after 90 minutes of incubation and at end point (24 hours).
This finding reinforced the view that divalent cations inhibit the activity of Hep- 20: the presence of EDTA most likely enhanced Hep-20 activity by removing cations that protect cell membrane from interaction with the peptide or by sequestering ions which are required by intracellular enzymes (Kubo et al., 2005; Wei and Bobek, 2005).
In order to confirm the Hep-20 synergic effect observed in SPB in combination with caspofungin, amphotericin B and fluconazole, the killing assays were repeated in 4-fold diluted VFS supplemented with 1 mM EDTA. In contrast with was previously observed in SPB, no significant enhancement of antifungal activity was observed for all the Hep-20/amphotericin B combinations tested, while the combination Hep- 20/caspofungin produced a significantly enhanced effect in VFS against BPY44 following 90 minutes of incubation at 30°C.
Most importantly, an enhanced effect of Hep-20 was observed for the Hep- 20/fluconazole combination on the C. glabrata fluconazole resistant isolate BPY44 in the killing assay performed in 4-fold diluted VFS supplemented with 1 mM EDTA.
This finding was particularly interesting in view of a potential application of this peptide in the topical treatment of fungal vaginitis. To further pursue such a possibility
the fungicidal activity of Hep-20 was evaluated in human vaginal fluid (HVF) collected from 3 healthy donors (D1-D3) and diluted to final concentrations of 2 g/l and 0.018 g/l to consider HVF biological variability (Tomás and Nader-Macías, 2007). All experiments were performed in 4-fold diluted HVF to consider the average dilution of fluid by vaginal suppositories.
Killing experiments in HVF indicated that a biological variability occurred among vaginal fluid collected from three different donors, since for two of them a significant reduction in the number of viable fungal cells was induced by Hep-20 following 90 minutes of incubation at 37°C without EDTA. However, in the case of donor 1, the antifungal activity was almost completely abolished in HVF in the absence of EDTA.
This finding points out that vaginal fluid composition may differentially affect the peptide activity. Indeed, it is recognised that HVF contains several antimicrobial peptides such as calcoprotectin, lysozime, SLPI, HBD1-2, HNP1-3 and LL-37 which could act synergistically with Hep-20, thus overcoming the natural inhibitory effect caused by ions or other fluids components (Singh et al., 2000; Valore et al., 2002; Shaw et al., 2007). Notably, when 1.5 mM EDTA was added to the HVF, Hep-20 exerted a complete fungicidal effect versus C. glabrata BPY44 following 90 minutes of incubation, and it was maintained after 24 hour incubation. Interestingly, Hep-20 MFC that produced a fungicidal effect was 50 µg/ml for all donors, a quarter of the peptide concentration that significantly reduced fungal cell number in the vaginal fluid simulant. When Hep-20 activity was evaluated in human vaginal fluid diluted to a lower protein concentration, similar to the one of VFS (0.018 g/l), the trend of antifungal activity was analogous to the one observed in HVF with a higher protein content.
Overall, the results obtained on Hep-20 fungicidal activity in VFS, and, most importantly, in HVF, reinforced the view that this peptide is a promising candidate for the topical treatments of C. glabrata vaginal infections.
Even if Hep-20 is a human derived peptide, therapeutically active concentrations can be higher than the physiological ones. Therefore, it is mandatory to investigate the potential cytotoxic effects exerted by this peptide on human cells.
In this respect, we evaluated the cytotoxic activity of Hep-20 on human red blood cells (RBCs), peripheral blood mononuclear cells (PBMCs) and a human derived epithelial cell line (A549) by hemolysis assay, propidium iodide staining and XTT reduction assay. The results of the hemolysis assay showed that fungicidal Hep-20 concentrations in HVF produced no hemolysis on human erythrocytes. Moreover, all
Hep-20 concentrations tested (12.5 to 200 µ g/ml) did not show any cytotoxic effect on PBMCs and A549 cells, as demonstrated by XTT and PI staining. These results are in agreement with a study by Park and co-workers in which both hepcidins (Hep-20 and Hep-25) have been tested for cytotoxicity on erythroleukemia type K562 human cells, which share similar traits with both undifferentiated granulocytes and erythrocytes, at concentrations up to 66 µg/ml (about 3000 fold higher than that found in urine), with no significant effect on human cells (88% viable cells post treatment) (Park et al., 2001).
Notably, Hep-20 produced a significant increase in A549 cell proliferation at the highest concentration tested (200 µg/ml, P=0.0033). This result is not surprising, since it is reported in literature that some antimicrobial peptides, such as hBD-2, are able to induced A549 proliferation in a dose dependent manner (Zhuravel et al., 2011). In addition, the role of AMPs in wound healing is supported by data on the activity of cathelicidin LL-37 and hBD-2 and -3. These peptides are highly expressed in epidermal keratinocytes in response to injury or infection of the skin (Dorschner et al., 2001;
Sorensen et al., 2003). In addition, treatment with exogenous hBD-3 leads to an enhanced re-epithelialization of wounds in an animal models (Hirsch et al., 2009). Hep- 20 shares a high structural homology with defensins, and it could be hypothesized to exert a similar proliferative stimulus on A549 cells (Hunter et al., 2002; Jordan et al., 2009).
In order to assess the potential of Hep-20 as a therapeutic agent against C.
glabrata vaginal infections, it is necessary to evaluate if the peptide retains its activity
in physiological conditions. The results obtained in this study demonstrated that Hep-20 maintains its antifungal activity in the presence of EDTA at pH condition, salt concentration and protein content similar to those found in vivo. Moreover, this finding was also confirmed when the peptide activity was evaluated in human vaginal fluid. At present, nothing is known with regard to the stability of the peptide in human vaginal fluid, or on Hep-20 half-life in humans. Data on Hep-25 clearance from human plasma indicated that peptide levels decrease with time, with a mean plasma elimination half- life ranging between 5 and 8 h (Zaritsky et al., 2010).
An indirect evidence of Hep-20 stability was obtained in this study from co- incubation experiments of the peptide with HVF (protein content 2 g/l) at 37°C at two different time points, 90 minutes and 24 hours. SDS-PAGE gels indicated that the peptide is not naturally present in the human vaginal fluid, and when it was co- incubated with HVF alone, Hep-20 was not degraded. Surprisingly, when the peptide
was co-incubated with HVF in the presence of 1.5 mM EDTA, Hep-20 resulted partially degraded, and this results was even more evident after 24 hours of co-incubation. This finding was unexpected, since it is widely reported that EDTA inhibits metal-dependent proteases (serine, cysteine, aspartic, and metalloproteases) in a specificity manner (Wei and Bobek, 2005). Indeed, a proteomic analysis on cervico vaginal fluids identified several proteases, such as serine (e.g., kallikreins) and cysteine (e.g., cathepsins) ones, which could degrade Hep-20 (Shaw et al., 2007; Moncla et al., 2011). Further experiments will be required to understand the exact mechanism leading to this partial peptide degradation following 90 minutes and 24 hours of incubation in HFV.
Nevertheless, killing experiments performed in HVF clearly demonstrated that, even if partially degraded, the peptide is still able to exert fungicidal activity against C.
glabrata at both 90 minutes and within 24 hours.
The overall results demonstrated that Hep-20 exerts antifungal activity against C.
glabrata fluconazole resistant isolate, alone and in combination with commonly used
antifungals, including fluconazole in SPB under acidic conditions. When assessed in the vaginal fluid simulant, the peptide showed a significant reduction of fungal viable cells, in the presence of EDTA. Interestingly, the fungicidal activity of Hep-20 against C.
glabrata resulted potentiated in human vaginal fluid supplemented with EDTA,
confirming that this peptide is a promising candidate for the topical treatment of vaginitis caused by fluconazole resistant C. glabrata strains. This is also supported by the absence of any cytotoxix activity exerted by Hep-20 on human (or human derived) cells, which was demonstrated for all the active peptide concentrations used in HVF experiments.
Therefore, despite the fact that further studies are still needed to characterize the peptide mode of action and to optimize Hep-20 fungicidal activity, our results indicate that this peptide or its derivatives should be further studied as novel therapeutic agents for the control of vaginal C. glabrata infections.
