The production of natural antibiotic peptides has emerged as an important mechanism of innate immunity in plants and animals. Defensins are diverse members of a large family of antimicrobial peptides, contributing to the antimicrobial action of granulocytes, mucosal host defence in the small intestine and epithelial host defence in the skin and elsewhere. This review, inspired by a spate of recent studies of defensins in human diseases and animal models, focuses on the biological function of defensins.

Defensins: antimicrobial peptides of innate immunity.

Mucins — large extracellular proteins that are heavily glycosylated with complex oligosaccharides — establish a selective molecular barrier at the epithelial surface and engage in morphogenetic signal transduction. Alterations in mucin expression or glycosylation accompany the development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion and immune surveillance. Mucins are used as diagnostic markers in cancer, and are under investigation as therapeutic targets for cancer.

Mucins in cancer: Protection and control of the cell surface

In the past decade, the frequency of diagnosed fungal infections has risen sharply due to several factors, including the increase in the number of immunosuppressed patients resulting from the AIDS epidemic and treatments during and after organ and bone marrow transplants. Linked with the increase in fungal infections is a recent increase in the frequency with which these infections are recalcitrant to standard antifungal therapy. This review summarizes the factors that contribute to antifungal drug resistance on three levels: (i) clinical factors that result in the inability to successfully treat refractory disease; (ii) cellular factors associated with a resistant fungal strain; and (iii) molecular factors that are ultimately responsible for the resistance phenotype in the cell. Many of the clinical factors that contribute to resistance are associated with the immune status of the patient, with the pharmacology of the drugs, or with the degree or type of fungal infection present. At a cellular level, antifungal drug resistance can be the result of replacement of a susceptible strain with a more resistant strain or species or the alteration of an endogenous strain (by mutation or gene expression) to a resistant phenotype. The molecular mechanisms of resistance that have been identified to date in Candida albicans include overexpression of two types of efflux pumps, overexpression or mutation of the target enzyme, and alteration of other enzymes in the same biosynthetic pathway as the target enzyme. Since the study of antifungal drug resistance is relatively new, other factors that may also contribute to resistance are discussed.

Clinical, cellular, and molecular factors that contribute to antifungal drug resistance.

Diversity of antimicrobial peptides and their mechanisms of action.

This review focuses on the role and regulation of mucin glycoproteins (mucins) in airway health and disease. Mucins are highly glycosylated macromolecules (≥50% carbohydrate, wt/wt). MUC protein ba...

Respiratory Tract Mucin Genes and Mucin Glycoproteins in Health and Disease

Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7).

Objectives:ToinvestigatethesusceptibilityofselectedbacteriaaswellasStreptococcusmutansbiofilmto MUC7 peptides and compare the activities with those of other known antimicrobial peptides. Methods:MICandMBCofpeptidesforS.mutans,Escherichiacoli,Streptococcusgordonii,Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Pseudomonas aeruginosa were determined using the microdilution method. For S. mutans, the effects of the peptides on the kinetics of growth inhibition, time-killing, and on biofilm formation and reduction were also examined. For biofilm studies, polystyrene microtitre plates, Calgary Biofilm Device (CBD) and hydroxylapatite (HA) discs, along with Crystal Violet and Alamar Blue dyes, and/or EM observations, were employed. Results: S. mutans was the most susceptible to all peptides tested (MICs of 9.4–25.0 mM), compared with the other species (MICs of 3.1–>100 mM). MUC7 peptides (except MUC7-12-mer-L4) exerted 2-fold higher activity against S. mutans than Hsn5-12-mer and magainin-II, and faster killing of S. mutans than Hsn512-mer. The MUC7 peptides also had an effect on S. mutans biofilm. On the polystyrene plates, they suppressed the biofilm formation, with MBIC50 of 6.25–12.5 mM, and reduced the 1 day developed biofilm inabatchculture,withMBRC50of25–50mM.OntheCBDpegs,theviabilitiesofthebiofilmweresuppressed by >95% in the presence of MUC7 peptides at 4· MIC (50 mM). One day developed biofilm viabilities were inhibited by 49–75%. On HA, the formation of biofilm (as observed by EM) was also considerably reduced. Conclusions:MUC7peptidespresentsomewhatpreferentialantimicrobialactivityagainstS.mutans.They also have an effect on in vitro formation and reduction of the preformed S. mutans biofilm.

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Effect of MUC7 peptides on the growth of bacteria and on Streptococcus mutans biofilm.

The cystatin superfamily of proteins, derived from a common ancestor, is comprised of a diverse group of potent cysteine proteinase inhibitors and antibacterial/viral agents grouped into several families. This review concentrates on family 2 cystatins, namely, the human salivary cystatins and cystatin C. Emphasis is given to their physicochemical and functional properties at both the protein and the molecular level. The role of cystatins in disease processes, including those in the oral cavity, is also discussed. Finally, future directions for cystatin research in oral biology are presented.

Cystatins--inhibitors of cysteine proteinases

Histatins constitute agroup ofsmall, cationic multifunctional proteins present inthesaliva ofhumanandsomenon- humanprimates. Themostsignificant function ofhistatins maybetheir anti-fungal activity against Candida albicans andCryptococcus neoformans. Histatins havebeenextensively studied atboththeprotein andgenelevels. Thestructure-functi on relationship ofhis- tatins withrespect totheir candidacidal activity hasalso beenstudied bymeansofrecombinant histatin variants, aswell asby chemically synthesized histatin fragments. Themechanism ofhistatins' action onCandida albicans isnotclear, butitappears tobe different fromthat ofazole-based anti-fungal drugs which interrupt ergosterol synthesis. During thepast20years, fungal infec- tions havebecome moreprevalent asaresult oftheemergence ofAIDS, aswell as,paradoxically, modern medical advances. The toxicity ofcurrent anti-fungal medicine, theemergence ofdrug-resistant strains, andtheavailability ofonly afewtypes ofanti- fungal agents arethemajordisadvantages ofcurrent anti-fungal therapy. Therefore, theimportance ofthesearch fornew,broad- spectrum anti-fungals with little ornotoxicity cannot beoveremphasized. Thefollowing properties makehistatins promising anti- fungal therapeutic agents: (1)Theyhavelittle ornotoxicity; (2)they possess high cidal activities against azole-resistant fungal species andmostofthefungal species tested; and(3)their candidacidal activity issimilar tothat ofazole-based antifungals. Current research efforts focus onthedevelopment ofimproved histatins with enhanced cidal activity andstability, andofsuitable andeffective histatin delivery systems. These andother approaches mayhelp tooutpace thegrowing list ofdrug-resistant and opportunistic fungi causing life-threatening , disseminating diseases. Thehistatins withimproved protective properties mayalso beusedascomponents ofartificial saliva forpatients with salivary dysfunction.

Human salivary histatins: promising anti-fungal therapeutic agents.

Human salivary histatin-5 (Hsn-5) is a 24-residue peptide that possesses potent antifungal activity in vitro. The MUC7 gene encodes human salivary low-molecular-weight mucin (MG2). The candidacidal activity of MUC7 domain 1 (MUC7 D1, the N-terminal 51 amino acid residues of MUC7) in vitro has also been demonstrated. In this study, we have investigated the antifungal therapeutic potential of Hsn-5, its two variants, R12I/K17N and R12I/H21L, and MUC7 D1. First, these peptides were tested for activities against different clinically important fungi. We found them to possess broad-spectrum antifungal activities; specifically, most exhibited excellent in vitro activity against eight clinically important fungal strains tested, including Candida albicans and Candida glabrata and their azole-resistant counterparts and Cryptococcus neoformans and its amphotericin B-resistant counterpart. These findings also suggest that the mechanism of action of both Hsn-5 and MUC7 D1 for these fungi is different from that of amphotericin B or azole antifungal agents. Second, we examined the stability of these peptides in whole human saliva and human serum. In saliva, the Hsn-5 variants R12I/K17N and R12I/H21L and MUC7 D1 degraded at a lower rate than Hsn-5. In human serum, MUC7 D1 was also more stable than Hsn-5; both peptides were more stable in serum than in saliva. Third, we examined the cytotoxicity of these peptides using human erythrocytes and two human cell lines (KB and HSG). No (or very low) hemolytic activity was observed with any of the four peptides, even at the highest protein concentration tested (200 μM), while amphotericin B caused 100% hemolysis at only 12.5 μM. The toxic effects of Hsn-5 and MUC7 D1 toward KB and HSG cells were also much lower than that of amphotericin B as measured by trypan blue exclusion. Together, these findings indicate that the investigated peptides possess high antifungal therapeutic potential, in particular for the treatment of drug-resistant fungal strains associated with immunocompromised (particularly human immunodeficiency virus-infected) patients. The same peptides could also be used as components of artificial saliva for patients with salivary dysfunction.

Libuse A. Bobek

Papers

Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7).

Effect of MUC7 peptides on the growth of bacteria and on Streptococcus mutans biofilm.

Cystatins--inhibitors of cysteine proteinases

Human salivary histatins: promising anti-fungal therapeutic agents.

In Vitro Assessment of Antifungal Therapeutic Potential of Salivary Histatin-5, Two Variants of Histatin-5, and Salivary Mucin (MUC7) Domain 1