Chemical genomic screening of a Saccharomyces cerevisiae genomewide mutant collection reveals genes required for defense against four antimicrobial peptides derived from proteins found in human saliva.

To compare the effects of four antimicrobial peptides (MUC7 12-mer, histatin 12-mer, cathelicidin KR20, and a peptide containing lactoferricin amino acids 1 to 11) on the yeast Saccharomyces cerevisiae, we employed a genomewide fitness screen of combined collections of mutants with homozygous deletions of nonessential genes and heterozygous deletions of essential genes. When an arbitrary fitness score cutoffs of 1 (indicating a fitness defect, or hypersensitivity) and -1 (indicating a fitness gain, or resistance) was used, 425 of the 5,902 mutants tested exhibited altered fitness when treated with at least one peptide. Functional analysis of the 425 strains revealed enrichment among the identified deletions in gene groups associated with the Gene Ontology (GO) terms "ribosomal subunit," "ribosome biogenesis," "protein glycosylation," "vacuolar transport," "Golgi vesicle transport," "negative regulation of transcription," and others. Fitness profiles of all four tested peptides were highly similar, particularly among mutant strains exhibiting the greatest fitness defects. The latter group included deletions in several genes involved in induction of the RIM101 signaling pathway, including several components of the ESCRT sorting machinery. The RIM101 signaling regulates response of yeasts to alkaline and neutral pH and high salts, and our data indicate that this pathway also plays a prominent role in regulating protective measures against all four tested peptides. In summary, the results of the chemical genomic screens of S. cerevisiae mutant collection suggest that the four antimicrobial peptides, despite their differences in structure and physical properties, share many interactions with S. cerevisiae cells and consequently a high degree of similarity between their modes of action.

Humanized mouse model used to monitor MUC gene expression in nasal polyps and to preclinically evaluate the efficacy of montelukast in reducing mucus production.

OBJECTIVES: To determine whether MUC gene expression could be down-regulated in nasal polyps by the leukotriene receptor antagonist montelukast, we developed a system in which nondisrupted human nasal polyps could be successfully implanted into severely immunocompromised mice, and in which the histopathology of the original nasal polyp tissue could be preserved for long periods. In addition, the histopathologic changes in the human nasal polyps were carefully examined to determine the origin of the submucosal glands (SMGs) that develop in true nasal polyps found in the anterior third of the nose. METHODS: Small, nondisrupted pieces of human nasal polyp tissues were subcutaneously implanted into NOD-scid IL-2rgamma(null) mice. Xenograft-bearing mice were treated with either montelukast or saline solution. Xenografts at 8 to 12 weeks after implantation were examined histologically, and expression of MUC genes 4, 5AC, and 7 was studied in the polyps before implantation and in the 8-week xenograft. Alzet pumps were inserted into the mice, and montelukast (Singulair) was continuously delivered to determine its effect on goblet cell hyperplasia, mucus production, and the enlargement of nasal polyps over an 8-week period. RESULTS: The xenografts were maintained in a viable and functional state for up to 3 months and retained a histopathology similar to that of the original tissue, but with a noticeable increase in goblet cell hyperplasia and marked mucus accumulation in the SMGs. MUC4 and MUC5AC were significantly increased in the xenograft 8 weeks after implantation, but MUC7 was significantly decreased compared to the preimplantation polyps. Inasmuch as MUC7 is found exclusively in serous glands, the findings suggest that serous glands are not found in polyps in the anterior third of the nose. The histopathologic findings confirm the original findings of Tos et al suggesting that the SMGs are derived from pinching-off of the epithelium of the enlarging polyp following inflammatory changes. These SMGs have the same epithelium as surface epithelium and consist of multiple goblet cells that secrete periodic acid Schiff stain-positive mucin into the interior of the SMGs. A progressive increase in the volume of the xenografts was observed, with little or no evidence of mouse cell infiltration into the human leukocyte antigen-positive human tissue. An average twofold increase in polyp volume was found 2 months after engraftment. Montelukast did not decrease the growth of the xenograft in the 8-week NOD-scid mice, nor did it affect MUC gene expression. CONCLUSIONS: The use of innate and adaptive immunodeficient NOD-scid mice homozygous for targeted mutations in the IL-2 gamma-chain locus NOD-scid IL-2r gamma(null) for establishing engraftment of nondisrupted pieces of human nasal polyp tissues represents a significant advancement in studying chronic inflammation over a long period of time. In the present study, we utilized this humanized mouse model to confirm our prediction that MUC genes 4 and 5AC are highly expressed and significantly increased over those of preimplanted polyps. The overexpression of these 2 MUC genes correlates with both the goblet cell hyperplasia and the excessive mucus production that are found in nasal polyp xenografts. MUC7, which is primarily associated with the submucosa, as opposed to MUC4 and MUC5AC, which are primarily expressed in the epithelium, was significantly decreased in the nasal polyp xenografts. Montelukast had no significant effect on MUC gene expression in the xenografts. In addition to the MUC gene expression patterns, the histology of the xenografts supports the concept that mucinous glands that are characteristic of true nasal polyps are significantly different from those in the mucosa found in the lateral wall of the nose in patients with chronic sinusitis without nasal polyps. The mucinous glands seen in nasal polyps (which appear to be derived from an invagination of hyperplastic epithelial mucosa containing large numbers of goblet cells) are histologically distinct from the seromucinous glands found in the submucosa of hyperplastic middle turbinates. The data presented here establish a humanized mouse model as a viable approach to study nasal polyp growth, to assess the therapeutic efficacy of various drugs in this chronic inflammatory disease, and to contribute to our understanding of the pathogenesis of this disease.

Regulation of Human MUC7 Mucin Gene Expression by Cigarette Smoke Extract or Cigarette Smoke and Pseudomonas aeruginosa Lipopolysaccharide in Human Airway Epithelial Cells and in MUC7 Transgenic Mice.

OBJECTIVE: The human MUC7 gene encodes a low-molecular-weight mucin glycoprotein that functions in lubrication/protection of epithelial surfaces of the oral cavity and respiratory tract. This study was designed to evaluate the effect of cigarette smoke extract (CSE), cigarette smoke (CS), and Pseudomonas aeruginosa lipopolysaccharide (LPS), either alone or in the combination, on MUC7 expression in vitro and in vivo. MATERIALS AND METHODS: qRT-PCR was used to determine the levels of mucin gene transcription in the human lung carcinoma cell line NCI-H292 (in vitro) and MUC7 transgenic mouse tissues (in vivo). ELISA was used to assess mucin glycoprotein levels in the cell line, and immunohistochemistry to assess mucins in lung and trachea sections. RESULTS: In vitro treatment of cells with LPS (10 (microg/ml) or CSE (0.5, 1, 2.5 and 5%) alone, resulted in a statistically significant increase of MUC7 transcripts only with 1%CSE (3.2-fold). The combined CSE/LPS treatment resulted in a synergistic increase of MUC7 with 0.5%CSE/LPS (4.4 fold). MUC7 glycoprotein levels increased only minimally, the highest increase was seen with the 0.5%CSE/LPS combination treatment (1.3-fold). In vivo exposure of MUC7 transgenic mice to CS, LPS or CS/LPS combination resulted in significant increase in MUC7 transcripts only with LPS treatment (in both trachea and lung). Immunohistochemistry indicated variable increase in MUC7 glycoprotein with CS and LPS treatment, both in the trachea and lungs, but CS/LPS exposure appeared to yield the highest increase. CONCLUSION: In vitro, CSE and a combination of CSE/LPS treatment upregulated MUC7 gene transcription. In vivo, LPS upregulated MUC7 transcription, and a combination of CS/LPS appeared to increase MUC7 glycoprotein.

Antimicrobial peptide MUC7 12-mer activates the calcium/calcineurin pathway in Candida albicans.

MUC7 12-mer is a cationic antimicrobial peptide derived from the N-terminal region of human low-molecular-weight salivary mucin. In order to gain new insights into the modes of action of the 12-mer against opportunistic fungal pathogen Candida albicans, we examined changes in the gene expression profile of C. albicans upon exposure to this peptide. Cells at an early logarithmic phase were exposed to 6 muM peptide and grown until an OD(600 nm) of approximately 0.4 was reached. Changes in gene expression were determined by microarray analysis and showed that 19 out of the total of 531 genes, whose expression was elevated in response to the peptide, are regulated by the calcium/calcineurin signalling pathway. Inactivation of this pathway by deletions, or by FK506, caused hypersensitivity to the peptide, demonstrating the importance of this pathway to the defense of C. albicans against the MUC7 peptide. Other differentially expressed genes that were detected include those encoding subunits of proteasome, and genes involved in cell stress, iron metabolism, cell wall maintenance and small-molecule transport. The presented results suggest that the calcium/calcineurin signalling pathway plays a role in the adaptation of C. albicans to the MUC7 antimicrobial peptide.

Exploring the mode of action of antimicrobial peptide MUC7 12-mer by fitness profiling of Saccharomyces cerevisiae genomewide mutant collection.

The MUC7 12-mer (RKSYKCLHKRCR) is a cationic antimicrobial peptide derived from the human salivary mucin MUC7. To study its effect/mechanism of action on fungi, we performed a fitness screen of a tagged, diploid, homozygous gene deletion mutant pool of the yeast Saccharomyces cerevisiae grown in the presence of the MUC7 peptide. Forty-five strains exhibiting reduced fitness and 13 strains exhibiting increased fitness (sensitivity or resistance, respectively) were identified by hybridization intensities to tag arrays. The strongest fitness defects were observed with deletions in genes encoding elements of the RIM101 signaling pathway (regulating response to alkaline and neutral pH and other environmental conditions) and of the endosomal sorting complex required for transport (ESCRT; functioning mainly in protein sorting for degradation, but also required for activation of the RIM101 pathway). Other deletions identified as conferring fitness defect or gain are in genes associated with a variety of functions, including transcription regulation, protein trafficking, transport, metabolism, and others. The results of the pool fitness screen were validated by a set of mutant strains tested individually in the presence of the MUC7 12-mer. All tested RIM101-related deletion strains showing fitness defects confirmed their sensitivities. Taken together, the results led us to conclude that deletions of genes associated with the RIM101 pathway confer sensitivity to the peptide by preventing activation of this pathway and that this stress response plays a major role in the protection of S. cerevisiae against damage inflicted by the MUC7 12-mer peptide.

Proteomic and metabolic characterization of a Candida albicans mutant resistant to the antimicrobial peptide MUC7 12-mer.

MUC7 12-mer is a cationic peptide derived from the N-terminal portion of human mucin MUC7, exhibiting potent antibacterial and antifungal properties. To advance our knowledge regarding the mechanisms of action of MUC7 peptide against an opportunistic fungal pathogen Candida albicans, we sought to develop and characterize mutant(s) resistant to this peptide. One of the selected mutants, designated #37, was much less susceptible to the MUC7 12-mer in a killing assay than the parental strain (ED(50)>40 vs. c. 6 microM, respectively). Difference gel electrophoresis (DIGE) analysis of the mutant revealed elevation of several glycolytic enzymes. The mutant also exhibited lowered ATP contents along with a relatively lower rate of oxygen consumption, as well as inability to grow on nonfermentable carbon sources. These properties are likely to be associated with changes in metabolic regulation, rather than lack of functional mitochondria, as determined by rhodamine 123 staining. Analysis of interaction between fluorescently labeled peptide and cells of both strains revealed that resistance of the mutant #37 is associated with changes in the process of transition between surface-bound state of the peptide to its internalization marking cell death.

A comparative analysis of bone formation induced by human demineralized freeze-dried bone and enamel matrix derivative in rat calvaria critical-size bone defects.

BACKGROUND: Human demineralized freeze-dried bone allograft (DFDBA) and enamel matrix derivative (EMD) have been used with varying success for the treatment of bone and periodontal defects. The purpose of this study was to compare qualitatively and quantitatively the bone formation induced by DFDBA and EMD to that of a positive control, recombinant human bone morphogenetic protein 2 (rhBMP-2), in the 8-mm rat calvaria critical-size bone defect. METHODS: Five groups of five rats each were used. The two test groups were DFDBA and EMD. A negative control consisted of a defect without any biomaterial implanted, a positive control consisted of a defect filled with collagen carrying rhBMP-2, and a non-surgical control consisted of the intact rat calvaria. Eight weeks after implantation of the biomaterials, histologic analysis was used for qualitative assessments and microcomputed tomography was used for quantitative assessments of bone formation. Statistical evaluation was performed by analysis of variance followed by the Fisher's least significant difference multiple-comparison test. RESULTS: In the negative control and EMD groups, the histologic analysis showed no bone formation within the center of the defect and limited bone repair at its margins. In the DFDBA group, granules of DFDBA were still present 8 weeks after implantation, and a limited degree of osteoinduction was seen at the center of the defect. The microcomputed tomography quantitative analysis showed a limited capacity of DFDBA and EMD to induce bone formation, and no statistically significant difference was detected among DFDBA, EMD, and the negative control. On the contrary, the positive control (rhBMP-2) consistently showed regeneration of bone throughout the critical-size defects. CONCLUSION: Unlike rhBMP-2, DFDBA and EMD had limited ability to induce bone formation in the rat calvaria critical-size bone defect; therefore, they may not be effective as bone-regenerative therapy for critical-size defects.

Factors affecting antimicrobial activity of MUC7 12-mer, a human salivary mucin-derived peptide.

BACKGROUND: MUC7 12-mer (RKSYKCLHKRCR), a cationic antimicrobial peptide derived from the human low-molecular-weight salivary mucin MUC7, possesses potent antimicrobial activity in vitro. In order to evaluate the potential therapeutic application of the MUC7 12-mer, we examined the effects of mono- and divalent cations, EDTA, pH, and temperature on its antimicrobial activity. METHODS: Minimal Inhibitory Concentrations (MICs) were determined using a liquid growth inhibition assay in 96-well microtiter plates. MUC7 12-mer was added at concentrations of 1.56-50 microM. MICs were determined at three endpoints: MIC-0, MIC-1, and MIC-2 (the lowest drug concentration showing 10%, 25% and 50% of growth, respectively). To examine the effect of salts or EDTA, a checkerboard microdilution technique was used. Fractional inhibitory concentration index (FICi) was calculated on the basis of MIC-0. The viability of microbial cells treated with MUC7 12-mer in the presence of sodium or potassium was also determined by killing assay or flow cytometry. RESULTS: The MICs of MUC7 12-mer against organisms tested ranged from 6.25-50 microM. For C. albicans, antagonism (FICi 4.5) was observed for the combination of MUC7 12-mer and calcium; however, there was synergism (FICi 0.22) between MUC7 12-mer and EDTA, and the synergism was retained in the presence of calcium at its physiological concentration (1-2 mM). No antagonism but additivity or indifference (FICi 0.55-2.5) was observed for the combination of MUC7 12-mer and each K+, Na+, Mg2+, or Zn2+. MUC7 12-mer peptide (at 25 microM) also exerted killing activity in the presence of NaCl, (up to 25 mM for C. albicans and up to 150 mM for E. coli, a physiological concentration of sodium in the oral cavity and serum, respectively) and retained candidacidal activity in the presence of KCl (up to 40 mM). The peptide exhibited higher inhibitory activity against C. albicans at pH 7, 8, and 9 than at pH 5 and 6, and temperature up to 60 degrees C did not affect the activity. CONCLUSION: MUC7 12-mer peptide is effective anticandidal agent at physiological concentrations of variety of ions in the oral cavity. These results suggest that, especially in combination with EDTA, it could potentially be applied as an alternative therapeutic agent for the treatment of human oral candidiasis.

Calcium sulfate and platelet-rich plasma make a novel osteoinductive biomaterial for bone regeneration.

BACKGROUND: With the present study we introduce a novel and simple biomaterial able to induce regeneration of bone. We theorized that nourishing a bone defect with calcium and with a large amount of activated platelets may initiate a series of biological processes that culminate in bone regeneration. Thus, we engineered CS-Platelet, a biomaterial based on the combination of Calcium Sulfate and Platelet-Rich Plasma in which Calcium Sulfate also acts as an activator of the platelets, therefore avoiding the need to activate the platelets with an agonist. METHODS: First, we tested CS-Platelet in heterotopic (muscle) and orthotopic (bone) bone regeneration bioassays. We then utilized CS-Platelet in a variety of dental and craniofacial clinical cases, where regeneration of bone was needed. RESULTS: The heterotopic bioassay showed formation of bone within the muscular tissue at the site of the implantation of CS-Platelet. Results of a quantitative orthotopic bioassay based on the rat calvaria critical size defect showed that only CS-Platelet and recombinant human BMP2 were able to induce a significant regeneration of bone. A non-human primate orthotopic bioassay also showed that CS-Platelet is completely resorbable. In all human clinical cases where CS-Platelet was used, a complete bone repair was achieved. CONCLUSION: This study showed that CS-Platelet is a novel biomaterial able to induce formation of bone in heterotopic and orthotopic sites, in orthotopic critical size bone defects, and in various clinical situations. The discovery of CS-Platelet may represent a cost-effective breakthrough in bone regenerative therapy and an alternative or an adjuvant to the current treatments.

Functional analysis of human MUC7 mucin gene 5'-flanking region in lung epithelial cells.

The human MUC7 gene encodes a low-molecular-mass mucin glycoprotein that functions in modulation of microbial flora in the oral cavity and respiratory tracts. MUC7 gene expression is tissue- and cell-specific, with dominant expression in salivary gland acinar cells. To begin to understand the molecular mechanisms responsible for controlling MUC7 gene expression, we analyzed the promoter activity of MUC7 5'-flanking region in a human lung epithelial cell line A549. We demonstrated that MUC7 gene is expressed constitutively in this cell line and is upregulated by TNF-alpha stimulation. The promoter activities of a 2,762-bp fragment of the human genomic DNA (-2,732/+30 bp) and its deletion series, subcloned into a luciferase reporter vector, were characterized at the basal level and under stimulation by TNF-alpha. The results indicated that the minimal functional MUC7 promoter is in the region of -138/+30 bp. This region also revealed the greatest increase in the promoter activity upon TNF-alpha stimulation. Two putative AP1-binding elements and one NF-kappaB-binding element were identified within the proximal promoter. Further analyses demonstrated that mutations of these elements dramatically reduced specific DNA-protein binding ability and reporter gene expression. AP1 elements played an essential role in the constitutive expression, while the NF-kappaB element was crucially important in the response to TNF-alpha stimulation, demonstrating that TNF-alpha activates MUC7 transcription via NF-kappaB signaling pathway.

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

OBJECTIVES: To investigate the susceptibility of selected bacteria as well as Streptococcus mutans biofilm to MUC7 peptides and compare the activities with those of other known antimicrobial peptides. METHODS: MIC and MBC of peptides for S. mutans, Escherichia coli, Streptococcus gordonii, 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 microM), compared with the other species (MICs of 3.1->100 microM). 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 Hsn5-12-mer. The MUC7 peptides also had an effect on S. mutans biofilm. On the polystyrene plates, they suppressed the biofilm formation, with MBIC(50) of 6.25-12.5 microM, and reduced the 1 day developed biofilm in a batch culture, with MBRC(50) of 25-50 microM. On the CBD pegs, the viabilities of the biofilm were suppressed by >95% in the presence of MUC7 peptides at 4x MIC (50 microM). 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: MUC7 peptides present somewhat preferential antimicrobial activity against S. mutans. They also have an effect on in vitro formation and reduction of the preformed S. mutans biofilm.

Modulation of MUC7 mucin expression by exogenous factors in airway cells in vitro and in vivo.

The human MUC7 gene encodes a low-molecular-mass mucin that participates in the maintenance of healthy epithelium in the oral cavity, and possibly in respiratory tracts, by promoting the clearance of various bacteria. We examined whether MUC7 gene is expressed in primary normal human tracheobronchial epithelial cells and whether the expression is modulated by exogenous factors. By assessing MUC7 transcripts, we found that the MUC7 gene was induced by culturing the normal human tracheobronchial epithelial cells at the air-liquid interface, in which the cells were well differentiated. When the cells were treated with a panel of cytokines (IL-1beta, IL-4, IL-13, and TNF-alpha), epidermal growth factor, or a bacterial product (Pseudomonas aeruginosa lipopolysaccharide [LPS]), MUC7 transcripts and glycoprotein products were increased 1.7- to 3.2-fold. The effect of LPS on MUC7 gene expression was also studied in the airway tissues of MUC7 gene transgenic mice. In the in vitro cultured trachea and lung explants, the LPS-treated tissues showed over 2-fold increased levels of MUC7 mRNA compared with the untreated specimens. These results were confirmed by in vivo studies using the lungs and tracheas harvested from the transgenic mice irritated by LPS through the tracheal instillation. By immunohistochemistry, MUC7 glycoprotein was localized in tracheal submucosa within the serous cells. Upon LPS stimulation, the overexpressed MUC7 remains confined to the serous glands. In the lungs, MUC7 seems to be expressed within the respiratory epithelium at the level of the bronchioles. Upon stimulation with LPS, it seems to be overexpressed within the same cells and within the stromal tissue.

Human salivary mucin MUC7 12-mer-L and 12-mer-D peptides: antifungal activity in saliva, enhancement of activity with protease inhibitor cocktail or EDTA, and cytotoxicity to human cells.

MUC7 12-mer-L exhibits potent in vitro antifungal activity in low-ionic-strength buffers. In this study, we investigated the anticandidal activity and stability of MUC7 12-mer-L and its all-D-amino-acid isomer, along with Hsn5 12-mer (P113) and magainin-II, in human clarified and unclarified saliva in the absence or presence of protease inhibitor cocktail (PIC, which includes EDTA) or EDTA alone. In the absence of PIC or EDTA in saliva, only MUC7 peptides showed significant candidacidal activity. At a 100 microM concentration in clarified saliva and unclarified saliva, MUC7 12-mer-D demonstrated 94 versus 64% killing, respectively; MUC7 12-mer-L showed 57 versus 32% killing; Hsn5 12-mer showed 16 versus 0% killing; and magainin-II showed no killing. Addition of PIC or EDTA to either saliva caused the enhancement of antifungal activities of all peptides, although to different degrees. Taken together, the results suggest that EDTA (a metal-dependent protease inhibitor and/or divalent cation chelator) enhanced the antifungal activity of all four peptides mainly by chelation of divalent cations present in saliva (known to inhibit peptide antifungal activity), and PIC enhanced the activity of the three L peptides above that achievable by EDTA alone through inhibition of all classes of proteases. Peptide stability in saliva monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no degradation of MUC7 12-mer-D and 23, 60, and 75% degradation of MUC7 12-mer-L, Hsn5 12-mer, and magainin-II, respectively. Cytotoxicity assays determined that, at 100 microM peptide concentrations, MUC7 12-mer-D and 12-mer-L caused 3.5 and 4.3% hemolysis in phosphate-buffered saline and no toxicity to the HOK-16B cell line (derived from normal human oral keratinocytes). In summary, MUC7 12-mer peptides appear to be excellent candidates for investigation of antifungal activity in in vivo models of oral candidiasis.

In vitro synergic antifungal effect of MUC7 12-mer with histatin-5 12-mer or miconazole.

OBJECTIVES: MUC7 12-mer (RKSYKCLHKRCR), a cationic peptide derived from human salivary MUC7 mucin, exhibits potent in vitro antifungal activity, as determined by killing assays in phosphate buffer. In this study we examined the MUC7 12-mer antifungal activity alone or in combination with other antifungal agents in LYM medium (modified RPMI 1640). METHODS: Antifungal activities of MUC7 12-mer and other compounds against several fungal strains were first measured by MIC and minimum fungicidal concentration (MFC) tests using broth microdilution assay. The viability of Candida albicans and Cryptococcus neoformans were also determined by killing assays and time kinetics of peptide-mediated killing. Antifungal activities of MUC7 12-mer in combination with other compounds [histatin-5 (Hsn5) 12-mer: AKRHHGYKRKFH, amphotericin B or miconazole] against C. albicans and C. neoformans were determined by chequerboard assays and confirmed by killing assays. Toxicities of individual compounds were determined by haemolytic assays. RESULTS: MICs and MFCs of MUC7 12-mer ranged from 3.13 to 6.25 mg/L for most of the strains tested, and were, in most cases, comparable to those of amphotericin B and miconazole (0.78-6.25 mg/L). ED(50) values of MUC7 12-mer and Hsn5 12-mer were 7.1 and 7.4 micro M (or 11.2 and 11.6 mg/L), respectively, for C. albicans; and 1.2 and 1.1 micro M (or 1.9 and 1.7 mg/L), respectively, for C. neoformans. The killing of C. albicans and C. neoformans was achieved after 30 and 10 min exposure to the peptides, respectively. Combinations of MUC7 12-mer and Hsn5 12-mer, and of MUC7 12-mer and miconazole have a synergic antifungal effect on C. neoformans, with a fractional inhibitory concentration index (FICI) of 0.37 and 0.25, respectively; and a slightly lower than synergic effect on C. albicans, with a FICI of 0.63 and 0.56, respectively. In addition, using human erythrocytes, the two salivary peptides showed low levels of haemolytic activity. CONCLUSIONS: This study suggests that MUC7 12-mer and Hsn5 12-mer peptides may be suitable candidates for use in combination antifungal therapy.

Efficacy of human salivary mucin MUC7-derived peptide and histatin 5 in a murine model of candidiasis.

MUC7 16-mer (residues 36-51 of human salivary mucin, MUC7) and histatin 5 possess potent in vitro antifungal activity. In the present study, we have evaluated the efficacy of these peptides in vivo using the experimental model of murine vulvo-vaginal candidiasis. The treatment groups included MUC7 16-mer, histatin 5, clotrimazole (all in pluronic F127 gel), and placebo (gel alone). Mice were treated intravaginally for 7 consecutive days. At the end of the treatment, anticandidal activities were assessed by colony counts and by histological examination. All groups except clotrimazole presented positive cultures; no statistically significant differences were found in fungal burden amongst placebo and any treatment group except clotrimazole. Histopathological findings confirmed the microbiological results; all groups with the exception of clotrimazole showed variable signs of infection.

Human salivary MUC7 mucin peptides: effect of size, charge and cysteine residues on antifungal activity.

We have previously shown that MUC7 (human salivary low-molecular-mass mucin) 20-mer: LAHQKPFIRKSYKCLHKRCR (residues 32-51 of the parent MUC7, with a net positive charge of 7) possesses a broad-spectrum antimicrobial activity [Bobek and Situ (2003) Antimicrob. Agents Chemother. 47, 645-652]. The aims of the present study were to determine the minimum peptide chain length and its location within the 20-mer region that retains potent antifungal activity against Candida albicans and Cryptococcus neoformans and to examine the effect of net charge of the peptide as well as the role of cysteine residues on the fungicidal activity. First, several C-terminal truncated MUC7 20-mer fragments (16-mer, 12-mer, 11-mer, 10-mer and 8-mer) and one N-terminal fragment (8-mer-N) were synthesized and tested. The results showed that MUC7 12-mer, located at the C-terminal region of MUC7 20-mer, having a net charge of +6 and exhibiting an amphipathic helical conformation, not only retained but exceeded the antifungal activity of that of 20-mer. Secondly, several variants of the 12-mer peptide containing a lower or no net positive charge, or no cysteine residues were synthesized and tested. A clear correlation between the net positive charge of the 12-mer, its potency and initial interaction of peptide with fungal cells was found by killing assays, fluorescence microscopy and fungal cell-membrane potential measurements. Furthermore, cysteine residues, which play a critical role in bacterial binding, were found to be not important for the fungicidal activity of these peptides. These results identified MUC7 12-mer as a potential candidate for development into a novel antifungal therapeutic agent.

MUC7 20-Mer: investigation of antimicrobial activity, secondary structure, and possible mechanism of antifungal action.

This study was aimed at examining the spectrum of antimicrobial activity of MUC7 20-mer (N-LAHQKPFIRKSYKCLHKRCR-C; residues 32 to 51 of MUC7, the low-molecular-weight human salivary mucin, comprised of 357 residues) and comparing its antifungal properties to those of salivary histatin 5 (Hsn-5). We also examined the secondary structure of the 20-mer and the possible mechanism of its antifungal action. Our results showed that MUC7 20-mer displays potent killing activity against a variety of fungi and both gram-positive and gram-negative bacteria at micromolar concentrations. Time-dependent killing of Candida albicans and Cryptococcus neoformans by MUC7 20-mer and Hsn-5 indicated differences in killing rates between MUC7 20-mer and Hsn-5. The secondary structure prediction showed that MUC7 20-mer adopts an amphiphilic helix with distinguishable hydrophilic and hydrophobic faces (a characteristic that is associated with antimicrobial activity). In comparison to that of Hsn-5, the fungicidal activity of MUC7 20-mer against C. albicans seems to be independent of fungal cellular metabolic activity, as evidenced by its killing potency at a low temperature (4 degrees C) and in the presence of inhibitors of oxidative phosphorylation in the mitochondrial system. Fluorescence microscopy showed the ability of MUC7 20-mer to cross the fungal cell membrane and to accumulate inside the cells. The internalization of MUC7 20-mer was inhibited by divalent cations. Confocal microscopy of cells doubly labeled with MUC7 20-mer and a mitochondrion-specific dye indicated that mitochondria are not the target of MUC7 20-mer for either C. albicans or C. neoformans.