Periathamby Antony Raj

Candidacidal Activity of Salivary Histatins IDENTIFICATION OF A HISTATIN 5-BINDING PROTEIN ON Candida albicans

Candida albicans is the predominant species of yeast isolated from patients with oral candidiasis, which is frequently a symptom of human immunodeficiency virus infection and is a criterion for staging and progression of AIDS. Salivary histatins (Hsts) are potent in vitro antifungal agents and have great promise as therapeutic agents in humans with oral candidiasis. The molecular mechanisms by which Hsts kill yeast cells are not known. We report here, that unlike other antimicrobial proteins, Hsts do not display lytic activities to lipid membranes, measured by release and dequenching of the fluorescent dye calcein. Analysis of the magnitude and time course of Hst-induced calcein release from C. albicans cells further showed that loss of cell integrity was a secondary effect following cell death, rather than the result of primary disruption of the yeast cell membrane.125I-Hst 5 binding studies indicated that C. albicans expressed a class of saturable binding sites (K D = 1 μm), numbering 8.6 × 105 sites/cell. Both Hst 3 and Hst 4 competed for these binding sites with similar affinities, which is consistent with the micromolar concentration of Hsts required for candidacidal activity. Specific 125I-Hst 5 binding was not detected to C. albicans spheroplasts, which were 14-fold less susceptible to Hst 5 killing, compared with intact cells in candidacidal assays. In overlay experiments, 125I-Hst 5 bound to a 67-kDa protein detected in C. albicans whole cell lysates and crude membrane fractions, but not in the yeast cell wall fraction. Consistent with the overlay data, cross-linking of 125I-Hst 5 toC. albicans resulted in the appearance of a specific 73-kDa125I-Hst 5-containing complex that was not detected in the cell wall. 125I-Hst 5-binding protein of similar size was also observed in susceptible S. cerevisiae strain TI#20. This is the first description of Hst 5 binding sites on C. albicans which mediate cell killing and identification of a 67-kDa yeast Hst 5-binding protein. The binding characteristics of Hst 5 are in agreement with the observed potency of its biological effect and provide crucial information to the use of Hst 5 as a therapeutic agent. The presence of a specific C. albicans Hst 5-binding protein provides further insight into the potential mechanism of yeast killing and suggests a basis for differential activity between yeast killing and the nontoxic nature of Hsts to humans.

Functional domain and poly-L-proline II conformation for candidacidal activity of bactenecin 5.

The functional domain for candidacidal activity of bactenecin 5 has been determined by synthesizing bactenecin 5 and its fragments [1–22 (BN22), 7–22 (BN16) and 20–43 (BC24)]. The N‐terminal sequence BN16 retained the candidacidal potency of the parent molecule and this region appears to be the candidacidal domain. The circular dichroism spectra of these peptides indicate the presence of largely poly‐l‐proline II conformations in aqueous solutions and in lipid vesicles. The coupling constant (JNH‐CαH) values, and a set of medium‐ and short‐range nuclear Overhauser effects observed for the N‐terminal peptide (BN16) in the two‐dimensional nuclear magnetic resonance suggest that poly‐l‐proline II helix could be the biologically active conformation.

Denture polymers with antimicrobial properties: a review of the development and current status of anionic poly(methyl methacrylate) polymers

The denture base polymer poly(methyl methacrylate) (PMMA) is highly susceptible for microbial colonization resulting in denture-associated infections. Over the years research has focused on ways to modify the PMMA properties via surface and chemical modification. These studies led to the development of new denture polymers that include anionic PMMA polymers. The new anionic polymers presented the possibility of compromising the physical and mechanical properties required for denture fabrication. These obstacles were overcome by generating anionic PMMA polymers with physical and mechanical properties suitable for denture fabrication. A large body of literature is available on the anionic PMMA polymers, their antimicrobial properties and their potential for the commercial and clinical application as dental biomaterials. This article describes a review and evaluation of the anionic PMMA polymers for their suitability to serve as denture base polymers, their antimicrobial properties, their efficacy to prevent denture-induced infection and their safety in the oral environment.

New phosphated poly(methyl methacrylate) polymers for the prevention of denture-induced microbial infection: an in vitro study

Purpose Poly(methyl methacrylate) (PMMA) has been widely used as a denture-base acrylic resin due to its excellent physical and mechanical properties. However, the material is highly prone to microbial fouling that often leads to Candida-associated denture stomatitis. Incorporation of phosphate groups into PMMA could facilitate adsorption of salivary antimicrobials and inhibit microbial adherence on the polymer surface. An in vitro study evaluated PMMA polymers containing varying amounts of phosphate group for their efficacy to inhibit Candida albicans adhesion, adsorb salivary histatin 5, and exhibit candidacidal activity. Methods Six PMMA polymers containing 0%, 5%, 15%, 10%, 20%, and 25% of phosphate group were synthesized by bead (suspension) polymerization technique using mixtures of methyl methacrylate and methallyl phosphate as monomers. The efficacy of the polymers to inhibit the adherence of C. albicans was examined by using human saliva-coated polymer beads and radio-labeled C. albicans cells, as compared with that of PMMA. The potency of the phosphated PMMA polymers to adsorb histatin 5 was determined by measuring the radioactivity of the adsorbed labeled-peptide on the polymer surface. The candidacidal activity of the histatin 5-adsorbed polymers was assessed by using the fluorescence technique. The percent release of the fluorescent probe calcein from the C. albicans membrane caused by the disruption of the cell membrane was determined. The data were analyzed statistically by one-way ANOVA followed by Scheffé’s test (α = 0.05 and n = 6). Results The presence of ≥15% phosphate content in PMMA significantly reduced the saliva-mediated adhesion of C. albicans. Phosphated PMMA polymers showed significantly enhanced adsorption of histatin 5 in a phosphate density-dependent manner. The candidacidal activity of the histatin 5-bound polymers increased significantly with the increase in the phosphate content of the polymer. Conclusion Phosphated PMMA polymers have the potential to serve as novel denture-base resins, which may reduce C. albicans colonization and prevent denture stomatitis.

Novel molecules for intra-oral delivery of antimicrobials to prevent and treat oral infectious diseases

New molecules were designed for efficient intra-oral delivery of antimicrobials to prevent and treat oral infection. The salivary statherin fragment, which has high affinity for the tooth enamel, was used as a carrier peptide. This was linked through the side chain of the N-terminal residue to the C-terminus of a defensin-like 12-residue peptide to generate two bifunctional hybrid molecules, one with an ester linkage and the other with an anhydride bond between the carrier and the antimicrobial components. They were examined for their affinity to a HAP (hydroxyapatite) surface. The extent of the antimicrobial release in human whole saliva was determined using 13C-NMR spectroscopy. The candidacidal activity of the molecules was determined as a function of the antimicrobial release from the carrier peptide in human saliva. The hybrid-adsorbed HAP surface was examined against Candida albicans and Aggregatibacter actinomycetemcomitans using the fluorescence technique. The bifunctional molecules were tested on human erythrocytes, GECs (gingival epithelial cells) and GFCs (gingival fibroblast cells) for cytotoxicity. They were found to possess high affinity for the HAP mineral. In human whole saliva, a sustained antimicrobial release over a period of more than 40-60 h, and candidacidal activity consistent with the extent of hybrid dissociation were observed. Moreover, the bifunctional peptide-bound HAP surface was found to exhibit antimicrobial activity when suspended in clarified human saliva. The hybrid peptides did not show any toxic influence on human erythrocytes, GECs and GFCs. These novel hybrids could be safely used to deliver therapeutic agents intra-orally for the treatment and prevention of oral infectious diseases.

MiniReviewCurrent status of defensins and their role in innate and adaptive immunity

Naturally occurring antimicrobial cationic polypeptides play a major role in innate and adaptive immunity. These polypeptides are found to be either linear and unstructured or structured through disulfide bonds. Among the structured antimicrobial polypeptides, defensins comprise a family of cysteine-rich cationic polypeptides that contribute significantly to host defense against the invasion of microorganisms in animals, humans, insects and plants. Their wide-spread occurrence in various tissues of these diverse organisms, and their importance in innate and adaptive immunity have led to their identification, isolation and characterization. A large volume of literature is available on defensins’ occurrence, structural characterization, gene expression and regulation under normal and pathological conditions. Much has also been published regarding their antimicrobial, antiviral and chemoattractive properties, and their molecular and cellular interactions. In this review, we describe the current status of our knowledge of defensins with respect to their molecular, cellular and structural biology, their role in host defense, future research paradigms and the possibility of their utilization as a new class of non-toxic antimicrobial agents and immuno-modulators.