1. INTRODUCTION
1.1 The Candida genus
The Candida genus is composed of an extremely hetereogeneus group of microrganisms that normally grow as yeast in a wide range of pH and temperatures (Odds, 1988). Several species of the genus are also capable to produce a filamentous type of growth, represented by pseudohyphae and pseudomycelium, but only Candida
albicans and C. dubliniensis can grow as true hypahe (true mycelium) in addition to
pseudohyphae (pseudomycelium). Therefore, both species are considered to be polymorphic (Calderone, 2002).
Hyphal projections develop from an unbudded yeast and appear as long tubes with no constrictions, while pseudohyphae consist of chains of elongated cells with constrictions between adjacent cells (Sudbery, 2011). The ability to switch between yeast and filamentous forms is thought to be tightly linked to virulence.
Filamentous cells are more invasive and exhibit a better penetration of tissues; on the other hand, yeast cells are easily delivered and disseminated into the bloodstream (Sudbery, 2011). Reversible transition between yeast, pseudohyphae and hyphae can occur in response to alteration in the growth conditions (Silva et al., 2012). Both yeast-form and filamentous cells are found in infected tissues. (Odds, 1988).
The Candida genus contains over 150 heterogeneous species, but only a minority has been implicated in human candidosis, the exact number being subject of continual revision due to new identification and taxonomic reassignements. Only 35% of Candida species are able to grow at 37°C, which is a key feature of successful pathogens and/or commensal microorganisms of humans (Calderone, 2002).
The most prevalent species isolated from humans are: Candida albicans (53,5%),
C. tropicalis (12,8%), C. parapsilosis (12,8%), C. glabrata (12,4%), C. krusei (4,6%), C. kefyr (1%), C. guilliermondii (0,8%), C. dubliniensis, C. famata, C. rugosa, C. pelliculosa, and C. lipolytica (0,9%) (Pfaller et al., 2010).
The clinical relevance of fungal diseases has been recognised since the second half of the last century and it still represents a major threat to human health.
Candida species commonly reside in human body as commensal organisms, being
part of the normal microbiome in the oral cavity, gut, or vaginal enviroment in approximately 50% of the population (Moyes and Naglik, 2011). Although normally these fungi do not cause any pathology, changes in the local environment, such as alterations in normal microbiota or compromised immune defences, may determine pathogenic episodes.
In this respect, the increased number of patients affected by
immunocompromising illnesses, such as HIV infection, together with a growing percentage of individuals undergoing medical treatments, such as chemotherapy and transplantation, mainly contributes to the rising incidence of mucosal and systemic infections caused by Candida species (Moyes and Naglik, 2011).
In addition, the widespread use of broad spectrum antimycotic therapy led to the selection of drugs resistant strains.
Candida pathogenicity is mediated by a number of virulence factors: adherence to
host surfaces, biofilm formation and secretion of hydrolytic enzymes are among the most important traits that enable the pathogen to colonize and successfully infect the host (Silva et al., 2012).
In order to establish infection, an opportunistic pathogen has to evade the immune system, to survive, to reproduce in the host environment and, in the case of systemic infection, to disseminate into new tissues and organs.
The primary event that occurs in Candida infections is the adhesion of fungal cells to host surfaces, which is required to initiate colonization and to persist within the host.
Candida cell surface proteins involved in the adherence process are commonly
described as adhesins. The most studied proteins with this function are the agglutinin-like sequence proteins (ALS) of C. albicans, which enable fungal cell to attach to host tissues and concur to the initial development of biofilm formation (Hoyer et al., 2001).
Biofilm represents a surface-associated community of micro-organisms embedded within an extracellular matrix, and it is considered an important virulence factor, since it confers resistance to antifungal therapy by limiting the penetration of substances through the matrix and protecting cells from host immune responses (Donlan and Costerton, 2002; Mukherjee and Chandra, 2004). As a result, infection caused by
biofilm producing strains are more difficult to eradicate (Mukherjee and Chandra, 2004).
The destruction of host tissues by Candida species may be facilitated by the release of hydrolytic enzymes into the local environment. Secreted aspartyl proteinases (Saps), phospholipases, lipases (Lips) and haemolysins are the enzymes most frequently implicated in Candida species pathogenicity.
Saps facilitate invasion and colonization of the host tissues by disruption of the host mucosal membranes and by degrading important immunological and structural defence proteins (Pichová et al., 2001; Silva et al., 2012).
In addition, phospholipases can hydrolyze phospholipids into fatty acids, while lipases are involved in the hydrolysis of triacylglycerols; membrane damage induced by these enzymes seems to facilitate the adhesion of Candida cells, by exposing cryptic targets on host cell surface (Ghannoun et al., 2000; Katarciolu and Yucel, 2002).
Hemolysins are used by Candida species to degrade haemoglobin and facilitate iron recovery from host cells. Thus, haemolysins are considered key virulence factors enabling pathogen survival and persistence within the host (Manns et al., 1994; Watanabe et al., 1999; Luo et al., 2004).
An important role in tissue invasion is played by the ability of the fungus to switch between the filamentous and the yeast form, which is reported to facilitate mucosal and systemic infections, increasing resistance to phagocytosis (Gow et al., 2002; Pfaller et al., 2002; Trick et al., 2002).
Indeed, there is evidence that strains trapped in either the blastoconidial or filamentous state are both significantly less virulent than cells capable of undergoing morphogenesis (Saville et al., 2003).
Over the past decades, the incidence of infections caused by Candida species has increased considerably, with C. albicans being the main cause of candidosis (Moyes and Naglik, 2011).
However, the epidemiology of fungal infections has changed over in the last few years, with an increase in the frequency of isolation of non-Candida albicans (NCA) species, especially in haematological, transplanted and intensive care unit (ICU) patients, as reported by several authors (Nguyen et al., 1996; Krcmery and Barnes, 2002; Bassetti et al., 2006, 2011). This increased involvement of NCA species in human fungal infections could be related to improvements in diagnostic methods, but also to the higher level of resistance to certain antifungal drugs observed for these species, in
comparison with C. albicans, which could have led to the selection of resistant Candida species (Silva et. al, 2012).
Epidemiology studies reveal a different NCA species distribution worldwide, with the higher isolation frequency in South America, Asia, and South Europe (Falagas et al., 2010). Among these species, C. glabrata is commonly isolated in the USA and North/Central Europe, C. parapsilosis in South America, South Europe, and several parts of Asia, while C. tropicalis is frequently involved in fungal infections in South America and Asia (Falagas et al., 2010).
1.2 Candida glabrata
Historically, Candida glabrata has been considered a non-pathogenic saprophyte of the normal flora in healthy individuals (Stenderup and Pederson, 1962).
C. glabrata cells (1-4 µm) are noticeably smaller than C. albicans (4-6 µm), C. tropicalis (4-8 µm) and C. parapsilosis (2.5-4 µm) blastoconidia. On Sabouraud
dextrose agar, C. glabrata forms smooth and cream-coloured colonies, which are indistinguishable from those of other Candida species except for their relative size (Calderone, 2002).
The biochemical profile of C. glabrata is also quite distinct; in fact, this species ferments and assimilates only glucose and threhalose, in contrast with C. albicans, which ferments and/or assimilates a wider number of sugars, with the exception of sucrose (Odds, 1988).
Considering the increasing morbidity and mortality associated to C. glabrata infections and the capacity of this species to develop antifungal resistance, a rapid identification is mandatory for a successful clinical management of the infection.
In this regard, several differential agar media are commercially avaible for
Candida specie identification.
Among these, CHROM agar allows C. glabrata to grow pink or purple colonies, in contrast with C. albicans, C. parapsilosis and C. tropicalis colonies, which are characterized by blue-green, white and dark blue colonies respectively (Silva et al., 2012). Another differential agar medium is OCCA® (Oxoid Ltd, Basingstoke, UK), which is a new ready-to-use medium that contains chromogenic substrates for rapid detection and specific identification of C. albicans, C. tropicalis and C. krusei;
unfortunately this medium is not useful to unequivocally identify C. glabrata, since this species forms light to dark brown colonies, like others NCA species, such as C.
parapsilosis, C. metapsilosis and C. orthopsilosis (Ghelardi et al., 2008).
A main distinguishing genetic feature of C. glabrata is its haploid genome, in contrast to the diploid genome of C. albicans and several other clinically relevant
Candida species (Fidel et al., 1999).
In this respect, C. glabrata appears to be more closely related to Saccharomyces
cerevisiae than to C. albicans. When compared to S. cerevisiae genome, C. glabrata
shows a small number of genetic adaptations. Therefore, it could be speculated that C.
glabrata ability to infect humans emerged independently from other Candida species
(Roetzer et. al., 2010).
1.2.1 Candida glabrata epidemiology
Despite C. glabrata has historically been associated with reduced pathogenicity, the increased use of immunosuppressive therapies, together with broad-spectrum antimicrobial treatments, have induced a significant rise in the frequency of mucosal and systemic infections caused by this species (Hajjeh et al., 2004; Silva et al., 2012). In fact, nowadays C. glabrata is the second or third most common cause of candidiasis after C. albicans, depending on the site of infection (Fidel et al., 1999; Trama et al., 2005; Gygax et al., 2008). A global epidemiological study performed by Pfaller and colleagues, indicated that C. glabrata ranked second overall among 31 different species isolated, accounting for 11.6% of all isolates. Moreover, the global distribution of this species was not homogeneous; C. glabrata was most frequently isolated in North America (21.1% of all Candida isolates) and least frequently isolated in Latin America (7.4%). The frequency of isolation varied considerably within each of the geographic region considered, ranging from 2.1% (Indonesia) to 34.7% (Australia) in the Asia-Pacific region, from 3.1% (Turkey) to 27.9% (Germany) in Europe, from 7.2% (South Africa) to 14.0% (Saudi Arabia) in Africa and the Middle East, and from 3.4% (Mexico) to 11.3% (Brazil) in Latin America (Pfaller et al., 2010).
An independent study by Falagas and colleagues confirms that the highest C. glabrata distribution worldwide is in USA (18.8-24%) and United Kingdom (22.7%), while the lowest isolation frequency is found in Brazil and Kuwait (4.9% and 5.6%, respectively) (Falagas et al., 2010).
In a recent Italian study performed at San Martino University Hospital, Bassetti and co-workers found out that 50% of candidemia episodes occurred between January 2008 and December 2010 were caused by NCA species, with C. glabrata accounting the 9.5% of mycoses (Bassetti et al., 2011).
The increasing number of C. glabrata infections is particularly concerning due to the intrinsic resistance to azole antifungal agents observed in this species, together with the high mortality rate associated with C. glabrata fungemia (Gycax et al., 2008). In fact, numerous studies demonstrated that a considerable percentage of C. glabrata isolates are resistant to fluconazole and itraconazole (approximately 9% and 40% respectively) (Pfaller et al., 1998; Barchiesi et al., 1999; Moran et al., 2002) Notably, several investigators report that this intrinsic azole resistance can also further increase during fluconazole therapy (Bassetti et al., 2009; Pfaller et al., 2011).
Although C. albicans is still the species responsible for the majority of infections in HIV-positive and negative patients, there are an increasing number of evidences reporting the isolation of C. glabrata from the mucosal surfaces of immunocompromised patients (Fidel et al., 1999; Badiee et al., 2010). C. glabrata, in fact, is the NCA species most frequently associated with oropharyngeal candidiasis in HIV-positive patients (Lischewski et al., 1995). This species is also commonly found to be associated with urinay tract infection in hospitalized patients (Fidel et al., 1999). Finally, despite C. albicans still accounts for 90% of episodes of vaginal candidiasis, in the last two decades vaginal infections due to C. glabrata have increased and are difficult to eradicate, giving rise to recurrent episodes (Fidel, 1999; Trama et al., 2005; Sobel, 2007; Kennedy and Sobel, 2010).
1.2.2 Vulvovaginal candidiasis
Candida species are usually present in the vaginal environment as commensals, a
mechanisms of defence, such as i) physical barriers (e.g. vaginal epithelial cells and vaginal fluid) ii) vaginal resident bacterial flora iii) presence of innate immune system (e.g. antimicrobial peptides in vaginal secretions). Several factors might break this equilibrium, including immunodefinciency due to HIV infection, diabetes mellitus, pregnancy, use of contrapceptives or antibiotics or hormonal factors, leading to a symptomatic fungal infection (Sobel, 2007).
Clinical symptoms usually are vaginal soreness, irritation, vulvar burning, dyspareunia, and external dysuria. Odor if present, is slight and inoffensive. Others symptoms are pruritus and vaginal discharge that is usually described typically as cottage-cheese like (Sobel, 2007).
Vaginal infections caused by Candida species affect 70-75% of women at least once in a lifetime, representing the second cause of vaginitis following bacterial vaginosis (Sobel, 2007), with the 40-50% of women also experiencing recurrences (McCormack et al., 1994). In 5-8% of cases, the infection reoccurs following pharmacological treatment, often leading to a recurrent vulvovaginal candidosis, defined as four or more episodes every year (Hurley et al., 1979; Foxman et al., 1998; Mårdh et al., 2002). In the last, decade there have been increasing reports of vaginitis due to non-Candida albicans (NCA) species, which now accounts for the 20% of
Candida vaginal infections (Kennedy and Sobel, 2010). Among NCA species, C. glabrata is the prevalent species worldwide, being responsible for 10-20% of vaginitis
(Corselo et al., 2003; Buscemi et al., 2004). Although C. glabrata and others NCA species are considerably less pathogenic than C. albicans, the clinical manifestation of vulvovaginitis caused by these species are indistinguishable from C. albicans ones. Although the virulence factors leading to vaginal infection due to C. albicans are widely described, and it is now clear that they involve phenotype switching and secretion of proteolitic enzymes, in C. glabrata they are poorly characterized. In fact, in comparison with C. albicans, this species is not able to form hyphae and does not secrete proteolytic enzymes which are considered two major virulence factors (Kennedy and Sobel, 2010).
Despite these observations, C. glabrata infections are difficult to eradicate and often end up in recurrent episodes. Moreover, C. glabrata strains isolated from recurrent vulvovaginal candidosis often show reduced susceptibility to fluconazole compared with C. albicans ones (Richter et al., 2005). In a recent study performed on C. glabrata
vaginal isolates by Gycax and co-workers, the strains analyzed were found to be dose susceptible dependant (S-DD) and resistant (R) to fluconazole in 2.2% and 26.9%
respectively, confirming previous literature (51.8% S-DD and 15.2% R) (Richter et al.,
2005; Gycax et al., 2008).
Several risk factors have emerged for C. glabrata vaginitis. Among these, there is some evidences that women with type 2 diabetes are more prone to vaginal colonization due to C. glabrata (Fidel, 1999; de Leon, 2002; Goswami et al., 2006).
In addition C. glabrata is responsible for recurrent vulvovaginal candidiasis in HIV-infected women; in fact, despite C. albicans remains the dominant Candida species isolated in these patients, C. glabrata is second in frequency. In this respect, results from a two year project on the HIV Epidemiology Resarch Study (HERS), performed by Sobel and colleagues, confirmed that HIV-positive women are significantly more prone than HIV-negative women to develop oral and/or vaginal
Candida infection. Moreover, the isolation frequency of C. glabrata strains
characterized by reduced susceptibility to fluconazole was higher in HIV-positive women than the one found in the HIV-negative patients (Sobel et al., 2001).
Finally, vaginal infections due to C. glabrata are more frequent in older women (mean-age 44 years); this fact may be caused by hormonal variation and the change of the normal vaginal pH (4-4.5) which usually occur during menopause. It should also be noticed that C. glabrata vaginitis frequently coexists with bacterial vaginosis, and a higher pH may be indicative of a mixed infection (Fidel et al., 1999; Sobel, 2007).
1.2.3 Candida glabrata virulence
Very little is known about the virulence of C. glabrata and the mechanisms of the host defense directed against this microorganism, in contrast with what has been elucidated for C. albicans.
As mentioned above, the capacity to adhere to epithelial cells is the first step of infection. C. glabrata adhesion process is mediated by a group of GPI (Glycosyl Phosphatidyl Inositol)-anchored cell wall proteins, encoded by genes that are homolougus to C. albicans ALS (Aggluitin-like Sequence) genes.
Among these, EPA1 encodes for a calcium dependent lectin that binds to N-acetyl lactosamine-cointaining glycol conjugates (Cormack et al., 1999).
A study performed by Cormak and colleagues revealed that EPA1 is expressed at higher levels in vitro. However, the deletion of EPA1 produces no difference in the adherence ability in vivo, suggesting the presence of functional redundancy in the mechanism of adhesion of C. glabrata, as already hypothesized for others species (Cormak et al., 1999).
In C. glabrata the Epa proteins are also involved in biofilm formation, which is one of the main strategies to persist within the host, since it enhances the ability of the yeast to adhere to surfaces and cause infection, thereby conferering resistance to antifungal therapy and protecting cells by immune response (Ramage et al., 2006).
In comparison with others NCA species, C. glabrata has a lower capacity to form biofilm in Sabouraud dextrose broth, but an higher ability to produce biofilm on silicone surfaces, thus enabling the microorganism to substain medical devices associated infections (Shin et al., 2002; Ramage et al., 2006; Silva et al., 2009, 2012).
C. glabrata has been generally assumed to be less virulent in comparison with C. albicans, because its lack of morphogenesis. In fact, although C. glabrata isolates have
been proven to form pseudohyphae in response to nitrogen starvation, these forms have not yet been found in clinical specimens (Csank and Haynes, 2000; Kaur et al., 2005).
The lack of C. glabrata ability to undergo morphogenesis also affects the interaction of this microorganism with the host immune system; in fact, while C.
albicans can evade from macrophages by disrupting cells through hypha formation, C. glabrata gets trapped within the phagosome (Alvarez and Casadevall, 2006). However,
this species develops alternative strategies to escape from phagocytosis. Several transcriptomic studies showed that C. glabrata is able to switch on the expression of genes involved in the utilization of alternative carbon sources once engulfed by macropaheges (Lorenz et al. 2004; Kaur et al. 2007).
The expression profile induced by phagocytosis also includes the activation of the autophagy process, which allows the recycling of cellular constituents and organelles in order to survive during starvation (Klionsky, 2005; Xie and Klionsky, 2007; Roetzer et al., 2010). In addition, even if macrophages are known to cause oxidative damage to microorganisms, C. glabrata has been found to be relatively resistant to this stress (Roetzer et al., 2010).
Extracellular proteinases are divided into four classes: serine, cysteine, metallo and aspartyl proteinase (Saps), but only the last class is secreted by Candida species.
The presence of SAP genes has been identified in several Candida species, but not in C. glabrata, suggesting that this aspect of pathogenicity is yet to be fully characterized (Chakrabarti et al., 1991; Kaur et al., 2005; Li et al., 2007).
Another class of hydrolases which have been demonstrated to contribute to
Candida virulence are phospholipases. C. albicans is able to secrete four different types
of phospholipases: phospholipases A, B, C and D (Li et al., 2007). Compared with C.
albicans, the role of phospholipases in C. glabrata virulence appears to be
controversial. Screening experiments, carried out using the egg-yolk-based assay on different strains from oral and vaginal infections, evidenced no phospholipases activity in all C. glabrata isolates tested (Samaranayake et al., 1984; al-Rawi and Kavanagh, 1999). Moreover, in contrast with these results, Ghannoun and colleagues reported that 41% of C. glabrata strains isolated from blood stream infection (BSI) analyzed by egg yolk-based and colorimetric assays, were phospholipase producers (Ghannoun et al., 2000).
1.3 Antifungal drugs
In comparison with bacteria, only a few different classes of antimicrobial drugs are currently available to treat fungal infections. Indeed, the eukaryotic structure that fungi share with mammalian cells limits the number of drug targets which can be used to exert selective killing on fungal pathogens (Sanglard and Odds, 2002).
The currently available antifungal drugs have a selective mode of action (Sanglard and Odds, 2002). Among these, 5-flucytosine inhibits fungal replication since it is a base analogue that is converted into 5-fluorouracil by fungus-specific enzymes cytosine deaminase and uracil phosphoribosyltransferase, leading to the formation of non functional DNA and RNA (Morschhäuser, 2010).
The polyenes target ergosterol by binding it and creating a pore in the fungal membrane, determining cell death by lysis (Bolard et al., 1986). The azoles (triazoles and imidazoles) interrupt the conversion of lanosterol to ergosterol, resulting in a
sterol-depleted membrane that, together with the accumulation of toxic compounds, inhibits fungal replication (Pasko et al., 1990). The echinocandins (caspofungin, micafungin, anidulafungin) work by inhibiting β-1,3-glucan synthase, thus preventing the synthesis of glucan, an important component of Candida cell wall (Perlin, 2007; Morschhäuser, 2010).
1.3.1 Polyenes
The polyenes are a class of amphipatic compounds that target ergosterol, a specific fungal sterol related to cholesterol, which is an important component of Candida membranes. The polyenes include amphotericin B (AmB), nystatin and natamycin, with amphotericin B being the most used to treat systemic or oral fungal infections. Discovered in 1955, several amphotericin formulation were developed but only amphotericin B is used today due to its higher activity (Oura et al., 1955).
AmB interacts with ergosterol at the fungal cell membrane, forming trans-membrane pores and inducing leakage of cations, reduction in intracellular potassium levels and, eventually, cell death (Baginski et al., 2005). AmB has a strong affinity for ergosterol but, depending on its concentration, may also bind to the sterols present on mammalian cells, determining toxicity. Several preparations with reduced toxicity are available, such as liposomal formulations, which allow increased dosages and high tissue concentrations, but their use is hampered due to high production costs (Wong-Beringer et al, 1998). A drawback of AmB treatment is the lack of an oral preparation as it has to be administered intravenously, although oral administration is in development (Wasan et al., 2009).
Interestingly, very low levels of fungal resistance have been identified in clinical isolates, although the mechanism by which these isolates become resistant is still unknown. Notably, most of these isolates show a significant reduction of ergosterol in their plasma membrane, thus contributing to AmB resistance since the drug target is absent (Sanglard et al., 2003; Sanglard and White, 2006).
1.3.2 Flucytosine
5-Flucytosine (5FC) is an antifungal drug that targets nucleic acid synthesis. The drug is fungus specific, since fungi and plants have a cytosine deaminase that converts 5FC into 5-fluorouracil, which is incorporated into DNA and RNA and inhibits cellular function and division. Mammalian cells do not have cytosine deaminase, and thus 5FC is not effective in these cells. 5FC is usually used in combination with polyenes or other antifungal agents in the treatment of fungal infections since it has a high frequency of inducing drug resistance (Sanglard and White, 2006; White et al., 1998).
1.3.3 Echinocandins
This class of antifungal drugs is increasingly used as first-line agents for the treatment of patients with candidemia and invasive candidiasis. The echinocandins are generally well tolerated, have few clinically significant drug-drug interactions, and can be safely used in patients with renal and hepatic dysfunction (Denning et al., 2003).
Currently, three echinocandins have been approved by FDA for clinical use: caspofungin (CSF), micafungin (MCF), and anidulafungin (ANF), approved in 2002, 2005 and 2006 respectively (Denning et al., 2003).
The echinocandins are the newest category of antifungal drugs. These compounds work by noncompetitive inhibition of β-1,3-glucan synthase in the plasma membrane of the fungal cells; this enzyme is involved in the synthesis of glucan, an essential component of the fungal cell wall (Inoue et al., 1996; Kurtz et al., 1996; Qadota et al., 1996).
The 1,3-β-D-glucan synthase subunits are encoded by three genes called FKS1,
FKS2, and FKS3 in S. cerevisiae, C. glabrata, and C. albicans; moreover, the enzyme
requires a regulatory subunit encoded by RHO1 for its activity (Perlin, 2007; Cowen and Steinbach, 2008; Shapiro et al., 2011).
Early genetic studies by Myra Kurtz and Cameron Douglas (Merck Research Labs) on S. cerevisiae and C. albicans indicated that Fks1, the major subunit of glucan synthase, is the presumed target of the echinocandins (Douglas et al., 1994, 1997). Isolates of different Candida species which are resistant to echinocandins, in fact, have frequently acquired point mutations in the FKS1 or FKS2 target genes (Park et al., 2005;
Balashov et al., 2006; Walker et al., 2008; Perlin, 2011). Specific amino acid substitutions helped define two regions, termed “hot-spot” 1 and 2 (HS1/HS2), that confer reduced susceptibility to caspofungin (Park et al., 2005). These regions are highly conserved among the Fks family (Perlin, 2007). In C. glabrata, point mutations confering resistance have also been identified in FKS2, with two hot spot region including aminoacids between 659 to 667 and 1374 to 1381, respectively (Garcia-Effron et al., 2009).
1.3.4 Azoles
The azoles are the most important class of Ergosterol Biosynthesis Inhibitors (EBI). EBI are a diverse group of antifungal agents that include azoles, morpholines, thiocarbamates and allylamines. These drugs inhibit the biosynthesis of ergosterol; a lack in the ability to renew sterols in the plasma membrane results in loss of membrane function and loss of fluidity that prevents cell growth and division.
Based on chemical structure the azoles can be classified into two groups: imidazole (clormidazole, miconazole, econazole, chetoconazole) and triazoles (fluconazole, voriconazole, ketoconazole, itraconazole and posaconazole).
Fluconazole, as well as the other triazoles, acts by inhibiting the fungal cytochrome P-450 dependent enzyme lanosterol 14-α-demethylase, which is encoded by the gene ERG11. This enzyme converts lanosterol to ergosterol, and its inhibition interferes with fungal membrane synthesis, resulting in the replacement of ergosterol with methylated sterols in the plasma membrane (Sheehan et al., 1999).
Fungal pathogens have developed several mechanisms of resistance to azoles. These mechanisms involve at least four different types of alterations: i) alteration of the target enzyme, ii) alteration of the ergosterol biosynthetic pathway, iii) alteration of antifungal transport by enhancing ATP-dependent efflux and iv) alteration of antifungal transport by enhancing efflux dependent on the membrane proton gradient.
The alteration of the lanosterol 14-α-demethylase activity can result from either overexpression or mutation in ERG11 gene.
Indeed, several mutations in the encoding sequence of this gene cause amino acid substitutions in the enzyme, that reduce its affinity for fluconazole and thus contribute to a decrease in azole susceptibility in clinical yeast isolates (Sanglard et al., 1998).
In addition, the overexpression of the gene results in the production of high concentrations of the target protein, creating the necessity of higher intracellular fluconazole concentrations to inhibit the enzymatic activity present in the cell (White et al., 1998; Sanglard and Odds, 2002).
Azole resistance is also correlated with alterations in the ergosterol biosynthetic pathway. This has been linked with mutation or absence of sterol ∆5,6-desaturase activity encoded by ERG3 in certain Candida resistant strains. (Nolte et al., 2007). However, inactivation of this enzyme in C. glabrata results in an altered sterol composition of the membrane but not in fluconazole resistance (Geber et al., 1995).
As observed for others antimicrobial agents, azole resistance can be mediated by different types of efflux pumps such as: the ATP-binding cassette (ABC) transporters, which use ATP as the energy source to drive transport, and the major facilitator super family (MFS), which include transporters energized by the proton gradient across the membrane (Del Sorbo et al., 2000).
In C. albicans two efflux pumps have been identified which belong to MFS, both conferring resistance to azoles: CaMDR1, (Candida albicans multidrug resistance 1) and FLU1 (fluconazole resistance 1), (Morschhäuser, 2010).
Althought C. albicans genome contains many additional genes encoding ABC transporter and major facilitators, it is important to note that only two ABC transporters,
CDR1 and CDR2 (Candida drug resistance 1 and 2) have a clinical relevance. Due to
their high homology (84% amino acid identity), these two transporters confer resistance to azoles and to a similar but not identical spectrum of xenobiotics (Morschhäuser, 2010).
1.3.5 Candida glabrata azole resistance
Candida glabrata is characterized by an intrinsic reduced susceptibility to azole
derivates, in particular to fluconazole, but its resistance can also further increase during fluconazole therapy (Morschhäuser, 2010).
As described for C. albicans, the overexpression or mutation in ERG11 gene increase azole resistance.
Despite this observation, data available on C. glabrata, based on CgERG11 sequencing and expression analysis performed on clinical isolates, indicated no alteration or overproduction of Cgerg11 protein, favoring the hypothesis that CgERG11 is not involved in the azole resistance in this species (Nakayama et al., 2001; Sanguinetti et al., 2005).
In silico analysis performed on C. glabrata genome revealed the presence of 3 genes, termed CgCDR1, CgCDR2 and CgSNQ2, which are homologous to ABC transporters encoding genes in S. cerevisiae (Miyazaki et al., 1998; Sanglard et al., 1999, 2001).
These genes have a relevant role in the azole resistance of this species. In fact, knock-out experiments performed on a C. glabrata azole resistant strain have shown that CgCDR1 deletion resulted in increasing intracellular fluconazole accumulation and hyper susceptibility to other azoles; moreover, the additional deletion of CgCDR2 further enhanced the susceptibility of the double mutant (Sanglard et al., 1999, 2001). The CgSNQ2 deletion caused a similar phenotype, thus demonstrating that all of these ABC transporters mediate multidrug resistance in C. glabrata (Torelli et al., 2008).
Similarly to what has been described for C. albicans, efflux pumps expression in
C. glabrata is induced by several xenobiotics, including azoles (Vermitsky and Edlind,
2004; Sanguinetti et al., 2005).
In fact, expression analysis revealed that CgCDR1 and CgCDR2 genes are both induced by azole treatment in several clinical isolates. Moreover, some of the strains analyzed also overexpressed CgSNQ2. Notably, results from these studies elucidated that efflux pumps genes are up regulated simultaneously or in different combinations, depending on the strain considered (Vermitsky and Edlind, 2004; Sanguinetti et al., 2005; Torelli et al., 2008;).
The expression of such efflux pumps in C. glabrata is regulated by a single zinc Zn(2)-Cys(6) transcription factor, called CgPdr1p, homologous to S. cerevisiae Pdr1p/
Pdr3p proteins (Vermitsky and Edlind, 2004).
The deletion of CgPDR1 leads to a loss of CgCDR1, CgCDR2 and CgSNQ2 expression and increases susceptibility to azole and other drugs; on the contrary,
CgPDR1 over expression enhances drug resistance, by boosting efflux pump gene
transcription (Tsai et al., 2006; Vermitsky et al., 2006; Torelli et al., 2008).
The inducible activity of CgPdr1p relies on the presence of two distinct domains: a xenobiotic binding domain (XBD), which mediates its activation following azoles
binding, and a trans activation domain (TAD) that recruits a transcriptional co-activator, allowing the interaction with RNA polymerase II (Thakur et al., 2008).
CgPDR1 sequencing studies performed on a wide collection of fluconazole
resistant clinical isolates showed that, in most cases, mutations that determined an aminoacidic substitution in the transcriptional factor could be observed. Moreover, these mutations were found to be concentrated in the XBD and TAD regions (Ferrari et al., 2009, 2011).
Data available suggest that these mutations are responsible for CgPdr1p constitutive activation determining efflux pumps overexpression, which seems to be the main molecular mechanism of C. glabrata drug resistance (Ferrari et al., 2009, 2011).
In addition, C. glabrata shares with S. cerevisiae the ability to undergo loss of mithocondrial function, which is linked to the so called “petite” phenotype (Sanglard et al., 2001). In this particular phenotype the overexpression of CgCDR1 and CgCDR2 genes is involved in the increased azole resistance of such mutants (Sanglard et al., 2001).
Respiratory deficient mutants can be induced in vitro with ethidium or azole treatment, but these mutants have also been isolated from patients undergoing fluconazole therapy (Bouchara et al., 2000; Ferrari et al., 2009). A recent study by Ferrari and colleagues showed that a strain with mithocondrial dysfunction selected in vivo under azole therapy had an increased virulence in murine model, compared with the azole sensitive parental strain (Ferrari et al., 2011). Although these studies indicate a correlation between increased resistance to azoles and increased virulence in animal models, isolation of such C. glabrata mutants from patient receiving antifungal therapy or prophylaxis has rarely been reported. In fact, the majority of fluconazole resistant clinical isolates are not respiratory deficient (Vermitsky and Edlind, 2004).
1.4 Antimicrobial peptides
The management of infections caused by drug-resistant strains represents an important challenge; numerous authors agree in considering the wide use of broad spectrum antimicrobial agents as one of the main causes of such a concerning situation (Owens and Rice, 2006; Rauch et al., 2009; Yeung et al., 2011). Indeed, during the last decades it has become mandatory to individuate new classes of antimicrobial
compounds. In this regard, the therapeutic potential of natural anti-infective agents, such as antimicrobial peptides (AMPs), is an extremely promising approach. Structurally different AMPs have been isolated from a wide range of organisms, including protozoa, amphibians, plants and mammals, including humans (Hancock et al., 1998; Giuliani et al., 2008). It is now widely known that these molecules play an important role in innate immune system as a first line of defence, since they exhibit bactericidal, fungicidal and virucidal properties (Giuliani et al., 2008). Through evolutionary course AMPs have undergone minor modifications. From a structural point of view, AMPs have been divided into four major classes: β-sheet, α-helices, loop and extended peptides (Hancock et al., 1998). Most of them are less than 100 L-amino acid long and smaller than 10 kDa, and they present an overall positive charge (generally from +2 to +9) due to the presence of multiple lysine and arginine residues, together with a substantial portion (≥ 30% or more) of hydrophobic residues. These characteristics most likely represent the key of their antimicrobial properties (Bechinger et al., 2006). In fact, a common feature shared among AMPs is their ability to fold into amphipathic conformation, often induced by electrostatic interaction with microbe membranes, disrupting the bilayer integrity and leading to microbial cell death (Yeaman and Yount, 2003). Several differences exist between microbial and mammalian cells, such as membrane composition and transmembrane potential, and they appear to have a role in the selective toxicity towards pathogens (Powers et al., 2003). In fact, it is known that AMPs bind gram negative and gram positive bacteria by interacting with LPS and theiconic and theicuronic acids, respectively. In a similar way, the main interaction of these agents with the fungal surface occurs with mannan and glucans, two essential components of the fungal cell wall. The absence of these negatively charged molecules in the mammalian membrane makes these cells less prone to interact with AMPs (Larrick et al., 1995; Yeaman and Yount, 2003).
1.4.1 Human antimicrobial peptides
Several classes of AMPs have been isolated from humans, among which the most studied are: defensins, cathelecidins, histatins and lactoferrin (Lupetti et al., 2004; De Smet et al., 2005).
Human defensins are divided into two classes: α- defensins and β-defensins. These peptides share a similar structure, since they are cationic non-glycosilated proteins with arginine as prevalent positive charged residue, with molecular masses ranging from 3.5 to 6 KDa. Defensins also contain six cysteine residues that form intramolecular disulfide bridges (Lehrer et al., 2004; Ganz et al., 2005).
In humans, α-defensins can be divided into two group: neutrophil α-defensins and enteric α-defensins. Four human neutrophil defensins have been described, (HNP-1 to HNP-4) initially isolated from neutrophils but in later stage also found in B cells and natural killer cells (Ganz et al., 1985, 1990). Enteric α-defensins (HD5-and 6) have been found in small intestine and in the ephitelial cells of female urogenital tract (Jones and Bevins, 1992, 1993).
All the α- defensins are expressed as prepropeptides, with the C-terminal residues responsible for the antimicrobial activity against a wide range of microorganisms (such
as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus
cereus) (Lehner et al., 2004; De Smet et al., 2005).
Six β-defensins (hBDs) have been found in humans (hBD-1 to hBD-6). The first human β-defensin (hBD-1) has been isolated from the hemofiltrate of a patient undergoing dialysis treatment, and it is constitutively expressed in the epithelia (e.g. in the lung, salivary gland etc.), where it is directly exposed to environment or microbial flora (Bensch et al., 1995; Pazgier et al., 2006).
The second member of this family, named hBD-2, was first isolated in psoriatic skin and it is widely expressed in many epithelia, in leukocytes and in the bone marrow (Harder et al., 1997). In contrast to hDB-1, hBD-2 is not constitutively expressed, but it is upregulated following exposure of epithelial tissue to LPS or pro-inflammatory agents (THF-a or IL-1b) (Harder et al., 1997). The hBD-3 was isolated from psoriatic scales and from keratinocytes; this defensin is higher expressed in skin and tonsils (Harder et al., 2001). Like hBD-2, hBD-3 is also induced by inflammatory stimuli and by bacterial compounds (Harder et al., 2001). Beta defensins (hBD-4, 5 and 6) were found highly expressed in testis and epididimis (Yamaguchi et al., 2002).
All beta defensins are able to exert antimicrobial activity against a wide range of microorganisms. Notably, the hBD-1 and hBD-2 exert antimicrobial effect predominantly against Gram-negative bacteria, while hBD-3 shows a wide spectrum of antimicrobial activity against both gram-negative (P. aeruginosa) and positive bacteria
(S. aureus), and against the yeast C. albicans (Garcia et al., 2001; Harder et al., 2001; Joly et al., 2004; Feng et al., 2005; Maisetta et al., 2006).
In addition, defensins are able to exert other activities such as antitumor activity, stimulation of cell proliferation, interference with signal transduction pathways, chemoattraction of immune cells and stimulation of cytokine and adhesion molecule expression (Kamysz et al., 2003; Oppenheim et al., 2003).
Cathelecidins are present in a wide number of species, but only one has been characterized: LL-37 (Steinstraesser et al., 2008). This peptide is derived by proteolysis from the C-terminal end of the human CAP18 protein (hCAP18) (Gudmundsson et al., 1996). Regarding the molecular structure, LL-37 is composed of 37 amino acid residues, and it does not contain cysteine. Moreover, this peptide can adopt different conformations in response to different environmental conditions such as largely random coil and α-helical structure in hydrophilic and hydrophobic environment, respectively (Turner et al., 1998).
This AMP is widely expressed in leukocytes and many tissues such as skin, the gastrointestinal, respiratory and reproductive tracts (Gudmundsson et al., 1996; Agerberth et al., 2000; Steinstraesser et al., 2008).
LL-37 is induced by inflammatory or infectious stimuli and has antimicrobial activity against gram-positive and negative bacteria and fungi (Zanetti et al., 2004; Ciornei et al., 2005).
Furthermore, it is chemotatic for neutrophils, monocytes, and mast cells, it alters transcriptional responses in macrophages, and it is reported to stimulate wound vascularization and re-ephitelization of healing skin and it has antitumor activity (Okumura et al., 2004; Zanetti, 2004).
Histatins are a family of small (3-4 kDa), cationic, histidine-rich peptides present in human saliva. These peptides are part of the innate immune system and play an important role in maintaining oral health by limiting infections in the oral cavity.
The histatin family consists in several members: histatin 1, 3 and 5; these three peptides have a linear structure containing 38, 32 and 24 amino acid residues respectively, and each of them has seven histidine residues (Oppenheim et al., 1988; Castagnola et al., 2004).
Among the histatins, histatin 5 has the strongest antimicrobial activity and it has been shown to exert a potent antimicrobial activity against pathogenic fungi, such as C.
albicans (Oppenheim et al., 1988; Helmerhorst et al., 1999; Edgerton et al., 2000).
The fungicidal activity of histatin 5 resides in the 11-24 amino acids residues at C-terminal region (Helmerhorst et al., 1997). In addition, P-113, a 12 amino acid fragment of Histatin 5, was identified as the smallest fragment producing anticandidal effect compared with the whole compound (Rothstein et al., 2001).
Lactoferrin is an 80 kDa monomeric protein composed of 690 amino acids with different roles in physiological conditions, such as iron homeostasis maintenance, organ morphogenesis, host defense against infections and inflammation (Baker et al., 1998; Ward et al., 2005).
This peptide has been isolated from a wide range of biological fluids such as human milk, tears, seminal fluid, serum and saliva (Masson et al., 1971; Hennart et al., 1991; Adlerova et al., 2008;).
Lactoferrin has a broad spectrum of antimicrobial activity against bacteria, virus and fungi. It exerts its antimicrobial activity by iron sequestering and directly interacting with membrane surface, thus resulting in cell lysis (Yamauchi et al., 1993; Lupetti et al., 2004; Valenti and Antonini, 2005).
In addition it has a role in immune response; in fact, it is released by neutrophils on mucosal surface during inflammation (Venkatesh and Rong, 2008).
1.4.2 Antimicrobial peptide mode of action
The antimicrobial peptide mode of action is not yet fully understood. In general, these molecules are thought to exert their action with a membrane-perturbing or with intracellular mechanism, depending on the specific peptide considered.
In the last few years, many studies have been performed to highlight the molecular mechanisms on which relies the membrane-perturbing action. Currently, there are four main hypotheses: 1) the “aggregation” model 2) the “carpet-like” model 3) the “barrel-stave” model 4) the “toroid-pore” model (Oren and Shai, 1998; Yeaman and Yount, 2003; Jenssen et al., 2006).
In the “aggregation” model, the positively charged peptides may cover all the membrane or alternatively form local carpets on the membrane surface by electrostatic interaction, leading in both cases to the formation of transient holes in the bilayer, that cause membrane cracks, leakage of cytoplasmatic contents, disruption of the membrane potential and, eventually, disintegration of the lipidic bylayer (Oren and Shai, 1998).
According to the “carpet-like” model, peptide monomers accumulate on membrane surface, causing the reorientation of the hydrophobic residues and the formation of micelles which detach from the cell, thus inducing a detergent-like effect (Yeaman and Yount, 2003).
The `barrel-stave' mechanism has been described for Gramicidin S and Alameticin, two AMPs produced by Bacillus brevis and Trichiderma viridae respectively (Jenssen et al., 2006; Yeaman and Bayer, 2006).
Peptides that act this way usually form stable trans membrane pores in a way that their hydrophobic surfaces interact with the lipid core of the membrane and their hydrophilic surfaces point inward, producing an aqueous pore (Yeaman and Yount, 2003). These pores cause disruption of gradients and leakage of intracellular components, ultimately leading to cell death (Shai, 1999).
The “toroid-pore” or “wormhole mechanism” is a different mechanism of peptide-membrane interaction. A primary difference between toroid pore and barrel-stave models is that in the former, lipids are intercalated with peptide in a transmembrane channel. This mode of action has been described for various AMPs, including LL-37 and Magainin (Jenssen et al., 2006). According to this model, the peptide assumes an α-helical structure, and in this conformation it interacts with the charged and hydrophobic membrane; this interaction is necessary to penetrate thought the plasma membrane and finally to form a pore (Hara et al., 2001).
Notably, pore formation could be a transient event. In fact, following the disintegration of the pores, some peptides translocate into the cytoplasm, suggesting that disassembly may be a key mechanism by which peptides enter the microbial cytoplasm to access potential intracellular targets (Uematsu and Matsuzaki, 2000).
AMPs that translocate across the cytoplasmatic membrane can inhibit different internal targets including DNA\RNA synthesis, protein synthesis, cell wall synthesis, cell division, translation and protein folding. For example, cecropin PR-39 and buforin II inhibit cellular functions such as protein and nucleic acid synthesis in E. coli with a rapid mechanism of action, with no direct lysis effect on bacteria (Boman et al., 1993; Park et al., 1998).
A peculiar example of this mode of action is histatin 5, which is an unusual peptide; in fact, although it has an amphipathic structure, it does not induce pore formation (Brewer et al., 1998; Raj et al., 1998). Moreover, substitutions of some amminoacids in the histatin 5 structure, in order to obtain synthetic variants (dhvar4 and dhavar5) with reduced amphipathic features, do not modify fungicidal activity against
C. albicans (Situ et al., 2000; Ruissen et al., 2001).
These evidences suggest that histatin 5 exerts its fungicidal activity by a different mechanism; in this respect, in a recent study by Kumar and co-worker, it was demonstrated that this peptide is internalized in C. albicans cells by two polyamine transporters: Dur3 and Dur31 (Kumar et al., 2011). Once inside, histatin 5 induces a non-lytic form of cell death, accompanied by loss of cell volume, cell cycle arrest, and interference with mitochondrial respiration chain that causes an increase of intracellular levels of reactive oxygen species (ROS) (Helmerhorst et al., 1999; Helmerhorst et al., 2001).
1.4.3 Antimicrobial peptide role in immunity
As mentioned above, AMPs are a constitutive component of innate defense system in most multicellular organisms, forming the first line of defence against invading microbes (Yeung et al., 2011).
More recently, it has become evident that these peptides exhibit a wide range of biological activities from direct killing of invading microbes to modulation of innate immune response and other biological responses (Yeung et al., 2011).
In mammals AMPs are expressed in a wide variety of cell types including monocytes/macropages, neutrophils, epithelial cells, keratinocites and mast cells (Brown and Hancock, 2006; Mookhrjee et al., 2007; Guanì-Guerra et al., 2010).
Virtually all AMPs have a direct antimicrobial activities in vitro conditions, where the peptides are tested at very high concentrations or in dilute medium (10 mM phosphate buffer) (Lehrer et al., 1991).
However, activity of many cationic AMPs is often inhibited in biological fluids due to the lower concentrations of peptides and by physiological concentrations of monovalent and divalent cations, serum, and anionic macromulecules such as glycosaminoglycans (Bowdish et al., 2005). Therefore, several AMPs do not work primarly in host defences by direct antimicrobial action, but they rather enhance and/or modulate host defence mechanisms to contrast microbial infections at concentration much lower than those require for direct microbial killing (Yeung et al., 2011).
Despite the microbicidal activities of peptides are generally inhibited in the physiological environment of the host, AMPs are still able to exert their immunomodulatory action under these conditions (Nijnik et al., 2010).
Several immunomodulatory activities of AMPs are described and they include direct stimulation of chemotaxis through chemokine production, suppression of bacterial induced pro-inflammatory cytokine production, regulation of neutrophil and epithelial cell apoptosis, modulation of dendritic cell activation and differentiation, and promotion of angiogenesis and wound healing (Yeung et al., 2011).
Chathelicidins and defensins secreted at the site of infection enhance pro-inflammatory responses such as chemotaxis for effector cells, induction of transcription and secretion of chemockines and histamine release from mast cells (Befus et al., 1999; Niyonsaba et al., 2001, 2002).
AMPs induced or released by degranulation of phagocytes at the site of infection can, at higher concentrations (micromolar), act as direct chemoattractants for cells of innate and adaptive immunity, and at lower concentrations (sub-micromolar), stimulate the production and release of more potent chemokines (Brown and Hancock, 2006; Mookherjee and Hancock, 2007; Jenssen and Hancock, 2010).
Following pathogenic invasion of tissues, α-defensins and hBD-2, 3 and 4 are chemotactic for monocytes, T cells and dendritic cells (Territo et al., 1989; Garcia et al., 2001), whereas human chathelicidin LL-37 promotes histamine and IL-8 release, which in turn promotes the chemotaxis of neutrophils (Yang et al., 2000).
Although cationic AMPs have been demonstrated to induce expression of certain pro-inflammatory responses to boost host innate immunity, they have also been shown to offer host protection against endotoxemia by selectively suppressing certain pro-inflammatory responses and stimulating the expression of particular anti pro-inflammatory genes. In this respect, LL-37 can selectively suppress the induction of proinflammatory mediators by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) of gram negative and positive bacteria respectively, and the induction of the transcription/production of the potent cytokines like TNF-α and IL-6 (Nijnik and Hancock, 2009).
Owing their wide immunomodulatory abilities and direct microorganism killing, AMPs appear to be promising candidates for new therapeutic approaches also in wound healing.
The role of AMPs in wound healing is supported by the observation on the activity of cathelicidin hCAP18/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 enhanced re-epithelialization of wounds in an animal model (Hirsch et al., 2009). Moreover, several growth factors are able to induce the expression of hCAP/LL-37. This cathelicidin has a direct activity on epidermal cells and fibroblasts and serves as chemoattractant for wound healing macrophages and fibroblasts (Sorensen et al., 2003; Yeung et al., 2011). Overall, understanding the role of AMPs in the immunity could provide important evidence for developing new antimicrobial drugs.
1.4.4 Advantages and disadvantages in the use of antimicrobial peptides
There are several potential advantages in the use of AMPs as antimicrobial drugs over conventional antibiotics. These peptides can be used alone due to their rapid mechanism of action, or in combination with other antimicrobials, as immunomodulatory\anti-infiammatory compounds, or giving rise to a synergistic effect (Yeung et al., 2011). Moreover, in comparison with conventional antibiotics, the acquisition of resistance by a sensitive microbial strain against AMPs is highly improbable due to the deep changes in the membrane structure that would be needed to confer an effective resistance (Yeung et al., 2011).
Currently, the interest on AMPs is rising due to increasing resistance of pathogens against conventional therapy, and the fact that they have the potential to overcome microbial resistance makes them promising candidates for novel therapeutic drugs.
In this respect AMPs could represent a novel therapeutic strategy against C.
glabrata, which is widely reported as a yeast characterized by a reduced susceptibility
to azole derivates, in particular to fluconazole (Morschhäuser et al., 2010).
Unfortunately, C. glabrata results to be scarcely susceptible to a wide spectrum of AMPs from different derivation; among these, histatin, catelecidins, defensins and magainin have been proven to exert antifungal activity versus clinically relevant
Candida species but not against C. glabrata (Joly et al., 2004; Helmerhorst et al., 2005,
2006; Benincasa et al., 2006).
The mechanism by which C. glabrata results resistant to these AMPs is still unknown; the differences exisisting between C. glabrata and others susceptible
Candida species most likely rely in membrane composition (Feng et al., 2005).
Despite many advantages of AMPs, clinical trials performed so far have been restricted to topical applications. The high cost of production, poor pharmacokinetics due to their susceptibility to host proteases and perhaps other clearance mechanisms, and unknown toxicity profiles have limited potential systemic application of AMPs (Yeung et al., 2011).
Several studies are focused on reducing these disadvantages by using several chemical modifications on original structures, such as D-aminoacids for improving stability and pharmacokinetics (Bradshaw et al, 2003, Papo and Shai et al., 2004; Svenson et al., 2008).
1.4.5 Hepcidins
In the last decade, hepcidin, a small peptide hormone, has emerged as key regulator of systemic iron homeostasis (Ganz and Nemeth, 2011). Hepcidin 25 is mainly produced by the liver, but can also be found in heart, brain, kidney and muscles (Kemna et al., 2008).
In the hepatic cells, hepcidin is found as an 84-amino acid precursor that subsequently undergoes proteolytic cleavages to generate the mature form, which is secreted in bloodstream (Kemna et al., 2008; Valore et al., 2008). Moreover, during
maturation Hep-25 generates two amino-terminal truncated isoforms, Hepcidin-22 (Hep-22) and Hepcidin (Hep-20), whose physiological role is still unclear (Park et al., 2001). These peptides have been initially isolated from urine and plasma, but they have been also found in bile, pleural and cerebrospinal fluid (Kemma et al., 2008; Marques et al., 2009; Arnold et al., 2010; Jayanthaet et al., 2010).
A study performed by Melino and colleagues demonstrated that the first 5 aminoacis in the amino terminal region of the Hepcidin-25 (Hep-25) play a fundamental role in the explication of its biological activity (Melino et. al, 2005). In fact, the N-terminal region contains three amminoacids, NH2-Asp-Thr-His, called ACTUN motif (Amino-Terminus-Copper-Nikel binding site); the presence of this metal-binding motif in the N-terminal region of this peptide is purposed to be responsible for the interaction with the ferroportin (Melino et. al, 2005). This hypothesis was reinforced by functional studies demonstrating that the two truncated isoforms (Hep-20 and Hep-22) almost completely lose the ability to interact with ferroportin (Nemeth et al., 2006).
An experiment performed on Hep-25 stability in biological fluids, such as serum and urine, indicated that the peptide is very stable and not converted into its smaller isoforms. This provides the first evidence that Hep-20 is formed intracellularly (Kartikasari et al., 2008).
Studies on Hep-20 and Hep-25 molecular structure revealed that both peptides
exhibit a net positive charge, and an overall amphipathic β-sheet structure stabilized by
disulfide and hydrogen bonding. In addition, this structure is highly homologue to defensins (Hunter et al., 2002; Jordan et al., 2009).
The role of hepcidins as antimicrobial peptides was first described by Park and colleagues, who demonstrated that Hep-20 and Hep-25 inhibit Aspergillus fumigatus and Aspergillus niger growth as well as Escherichia coli and Staphylococcus
epidermidis (Park et al., 2001).
In addition, recent studies have suggested that Hep-20 may retain greater antimicrobial and antifungal activity than Hep-25, particularly at acidic pH against clinically relevant bacterial strains such as E. coli, P. aeruginosa, S. aureus and S.
epidermidis (Park et al., 2001; Maisetta et al., 2010). As described for others AMPs,
hepcidins have a role during inflammation trough IL-6: Hep-25 improves the host response to pathogens with a direct action against microbial invasion and through limiting the iron availability. In this regard patients suffering from hemochromatosis
exhibit a low hepcidin levels and present an increased rate of bacterial infections (Verga Falzacappa and Muckenthaler 2005; Wessling-Resnick et al., 2010).
1.5 Aims of the study
Candida glabrata infections are often difficult to eradicate due to the intrinsically
low susceptibility to azoles of this species. In addition, C. glabrata has also been shown to be insensitive to several cationic peptides, which have been indicated as promising novel therapeutic candidates for the treatment of fungal infections. The present study was aimed to evaluate the in vitro fungicidal activity of the human cationic peptide hepcidin 20 (Hep-20) against clinical isolates of C. glabrata with different levels of fluconazole susceptibility. A potential synergistic effect between Hep-20 and commonly used antifungals was also investigated.
Since the peptide was shown to be active versus all C. glabrata isolates at neutral pH 7.4, and its activity was increased under acidic conditions, we hypothesized a potential application of the peptide in body districts characterized by an acidic pH, such as the vaginal ones. To this end, the fungicidal activity of the peptide was evaluated in a vaginal fluid simulant, resembling human secretion and then experiments were repeated in human vaginal fluids, collected from three healthy donors. Finally, even if Hep-20 is a human derived peptide, therapeutically concentrations are usually higher than physiological one. For this reason, Hep-20 cytotoxicity versus red blood cells, A549 epithelial cells, and peripheral blood mononuclear cells (PBMCs) was evaluated by hemolytic assay, XTT reduction assay and PI staining. Co-incubation of the peptide in the presence/absence of EDTA, was also carried out at different time points to obtain indirect evidence on Hep-20 stability in vaginal fluid.
The present PhD project was carried out at the Department of Biology of the University of Pisa, Via San Zeno, 37, under the supervision of Dr. Arianna Tavanti.
