R03014 1..5

Histatins are a group of antimicrobial peptides, found in the saliva of man and some higher primates, which possess antifungal properties. Histatins bind to a receptor on the fungal cell membrane and enter the cytoplasm where they target the mitochondrion. They induce the non-lytic loss of ATP from actively respiring cells, which can induce cell death. In addition, they have been shown to disrupt the cell cycle and lead to the generation of reactive oxygen species. Their mode of action is distinct from those exhibited by the conventional azole and polyene drugs, hence histatins may have applications in controlling drug-resistant fungal infections. The possibility of utilising histatins for the control of fungal infections of the oral cavity is being actively pursued with the antifungal properties of topical histatin preparations and histatin-impregnated denture acrylic being evaluated. Initial clinical studies are encouraging, having demonstrated the safety and efficacy of histatin preparations in blocking the adherence of the yeast Candida albicans to denture acrylic, retarding plaque formation and reducing the severity of gingivitis. Histatins may represent a new generation of antimicrobial compounds for the treatment of oral fungal infections and have the advantage, compared with conventional antifungal agents, of being a normal component of human saliva with no apparent adverse effects on host tissues and having a mode of action distinct to azole and polyene antifungals. Histatins — salivary antimicrobial peptides Histatins are small histidine-rich cationic peptides ranging in size from 7 to 38 amino-acid residues in length. They are secreted by the parotid and sub-mandibular salivary glands in man and some higher primates and are present in saliva at concentrations in the range 50–425 (Helmerhorst et al 1997). Histatins manifest an in vivo IC50 (the concentration which inhibits growth by 50%) value of approximately 1.4 M (Helmerhorst et al 1997). Histatins 1, 3 and 5 contain 38, 32 and 24 amino-acid residues, respectively, and the sequence of the first 22 amino acids of each histatin is identical. Histatins 1 and 3 are products of different genes while histatin 5 is a proteolytic cleavage product of histatin 3 (Oppenheim et al 1988). Histatins represent a group of antimicrobial peptides with some antibacterial properties and significant antifungal properties (Oppenheim et al 1988). The oral cavity is susceptible to a range of bacterial and fungal infections and histatins may have evolved to control infection in this region. Higher levels of histatins are present in the saliva of patients with recurrent oral candidosis than in un-infected controls, indicating their potential role in curtailing disease in cases of persistent infection (Bercier et al 1999). The elevated levels present in cases of recurrent infections may assist in curtailing more serious infection or the dissemination of the infection to other parts of the body. In contrast, however, the production of histatins decreases with age, as the incidence of oral fungal infection rises (Johnson et al 2000). Histatins are active against a range of pathogenic yeast and filamentous fungi resistant to conventional azole and polyene antifungal drugs (Helmerhorst et al 1999a). Histatin 5 is the most active histatin against the pathogenic yeast Candida albicans, which is a normal component of the body flora but is capable of causing lesions in the mouths of immuno-compromised patients. Histatin 5 retards the transition of C. albicans from the blastospore to the hyphal stage of growth — a process that may assist in arresting tissue penetration by the fungus (Helmerhorst et al 1997). Histatins are also capable of killing the yeast Cryptococcus neoformans (Tsai 1998) and Candida dubliniensis (a close relative of C. albicans) is susceptible to the effects of histatin 3 (Fitzgerald et al 2003). Although not normally associated with disease of the oral cavity, conidia of the pulmonary pathogen Aspergillus fumigatus demonstrate greater susceptibility to histatin 5 than amphotericin B (Helmerhorst et al 1999a). The efficacy of histatins in controlling fungal infection may be seen in patients who experience xerostomia (dry mouth), where there is an increased incidence of oral fungal infections in the absence of saliva containing these antimicrobial peptides. Their presence in saliva in normal patients helps to curtail (rather than eliminate entirely) the numbers of C. albicans in the oral cavity and thus prevent disease. Structure of histatin 5 Histatin 5 may be distinguished from the other histatins by the ability to form -helices in aqueous trifluoroethanol. This ability was initially thought to be important for the mode of action of histatin 5 but Situ et al (2000) demonstrated that a histatin variant (called 3P), with reduced ability to form a helix, that was formed by replacing three residues with proline, had an antifungal ability comparable with that of histatin 5. The fungicidal activity of histatin 5 resides in a region of 11–24 residues at the C terminal referred to as the functional domain (Driscoll et al 1995) or dh-5 (Helmerhorst et al 1997). Mutations in histatin 5 were created by replacing single residues and producing the altered protein in the bacterium Escherichia coli. Variant M21 was created by replacing Lys-13 with Thr, while M71 had Lys-13 replaced by Glu. Both variants demonstrated reduced fungicidal activity, indicating that the Lys-13 region was required for antifungal activity (Tsai et al 1996). A similar strategy was adopted in generating variants with multi-site substitutions in the dh-5 region. Two of the synthetic peptides (dhvar1 and dhvar 2) demonstrated enhanced anti-Candida properties having an IC50 (the concentration which inhibited growth by 50%) values of 0.6 and 0.8 , respectively, compared with an IC50 value of 1.4 for histatin 5 (Table 1). Peptides hvar1 and dhvar 2 demonstrated the ability to inhibit the growth ofC. albicans in an agar assay, in contrast to histatin 5, and manifested antibacterial activity against Prevotela intermedia, Streptococcus mutans and methicillinresistant Staphylococcus aureus (Helmerhorst et al 1997). The potential therapeutic application of dhvar 1 and dhvar 2 was abandoned when it was discovered that both displayed haemolytic activity in-vivo (Helmerhorst et al 1997). Mode of action of histatins Histatins demonstrate significant antifungal activity and represent an important element of the local immune response in the oral cavity. Understanding their mode of action may allow an enhancement (or fine-tuning) of their activity and open the possibility of employing them as antifungals, either as free agents or incorporated into materials that would normally be located in the oral cavity (e.g. denture acrylic, prosthetic implants). While histatins can manifest significant anti-Candida properties, their mode of action remains to be fully characterised. Histatin 5 was demonstrated to be capable of killing C. albicans pre-loaded with calcein without inducing significant release of the fluorescent dye, indicating that lysis of the cell membrane was not a significant effect of the peptide (Baev et al 2002). Histatins are also capable of killing spheroplasts (osmotically fragile cells retaining fragments of cell wall) of C. albicans, demonstrating that the fungal cell wall was not a target for their action (Driscoll et al 1996). Histatins appear to target a specific binding site in the fungal cell. Using cells fractionated with glass beads and separated by SDS-PAGE it was possible to demonstrate binding of I-histatin 5 to a protein of approximately 67 kDa, which may reside in the plasma membrane of the intact cell (Edgerton et al 1998). Recent work has demonstrated that histatin 5 binds heat shock protein 70 (Ssa1/2) — a protein located in the fungal cell envelope. Fungal mutants deleted in the genes for Ssa1/2 synthesis show a low level of susceptibility to histatin 5 compared with the unaltered parental isolate (Li et al 2003). It is unclear how histatin gains access to this binding site but it may cross the cell wall of C. albicans using an existing transporter and then target the binding site in the plasma membrane. One of the first indications that histatin 5 targets the mitochondrion came from the observation that nonrespiring cells were resistant to its activity (Helmerhorst et al 1999b). Fungal cells incapable of respiration (respiratory-deficient or petite mutants) are resistant to the action of histatins and C. albicans cells treated with the respiration inhibitor sodium azide are resistant to histatin 5 (Gyurko et al 2000). FITC-labelled histatin 5 accumulated in respiring C. albicans cells but not in those cells treated with azide, indicating the requirement for mitochondrial Table 1 Amino-acid sequences of histatin 5 and derivatives. Peptide Sequence IC50 Antifungal on agar Histatin 5 DSHAKRHHGYKRKFHEKHHSHRGY 1.4 M No Dhvar 1 KRLFKELKFSLRKY 0.6 M Yes Dhvar 2 KRLFKELLFSLRKY 0.8 M Yes Adapted from Helmerhorst et al (1997). 2 Kevin Kavanagh and Susan Dowd