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

Department of Oral Biology, University at Buffalo, the State University of New York, Buffalo, NY 14214, USA.
FEMS Immunology & Medical Microbiology (Impact Factor: 3.08). 09/2008; 54(1):80-91. DOI: 10.1111/j.1574-695X.2008.00450.x
Source: PubMed


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.

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    • "Many of such protein complexes bind to bacteria and cause their agglutination, facilitating their clearance from the oral cavity [5]. For example, MUC7 contains a histatin-like binding domain against bacteria at its non-glycosylated N-terminus [6], which is known to work as an antifungal and antibacterial peptide [7]. "
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    ABSTRACT: Two types of mucins, MUC7 and MUC5B constitute the major salivary glycoproteins, however their metabolic turnover has not been elucidated in detail to date. This study was conducted to examine turnover of MUC7 and MUC5B in saliva, by focusing on the relationship between their deglycosylation and proteolysis. Whole saliva samples were collected from healthy individuals and incubated at 37°C in the presence of various protease inhibitors, sialidase, or a sialidase inhibitor. General degradation patterns of salivary proteins and glycoproteins were examined by SDS-polyacrylamide-gel-electrophoresis. Furthermore, changes of molecular sizes of MUC7 and MUC5B were examined by Western blot analysis. A protein band was identified as MUC7 by Western blot analysis using an antibody recognizing an N-terminal epitope. The MUC7 signal disappeared rapidly after 20-minutes of incubation. In contrast, the band of MUC7 stained for its carbohydrate components remained visible near its original position for a longer time indicating that the rapid loss of Western blot signal was due to the specific removal of the N-termimal epitope. Pretreatment of saliva with sialidase facilitated MUC7 protein degradation when compared with samples without treatment. Furthermore, addition of sialidase inhibitor to saliva prevented proteolysis of N-terminus of MUC7, suggesting that the desialylation is a prerequisite for the degradation of the N-terminal region of MUC7. The protein band corresponding to MUC5B detected in both Western blotting and glycoprotein staining showed little sign of significant degradation upon incubation in saliva up to 9 hours. MUC7 was highly susceptible to specific proteolysis in saliva, though major part of MUC5B was more resistant to degradation. The N-terminal region of MUC7, particularly sensitive to proteolytic degradation, has also been proposed to have distinct biological function such as antibacterial activities. Quick removal of this region may have biologically important implication.
    Preview · Article · Jul 2013 · PLoS ONE
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    • "The authors speculated that the impaired mitochondrial respiration leads to a decrease of the endogenous ROS level and as a result, to higher drug resistance. Comparable changes in the primary metabolism were also observed in a C. albicans mutant resistance to the human antimicrobial peptide MUC7 12-mer (Lis and Bobek, 2008). The application of fluconazole combined with the alkaloid berberine exerted a synergistic action against clinical, fluconazole-resistant C. albicans strains (Xu et al., 2009). "
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    ABSTRACT: Both fungi Candida albicans and Aspergillus fumigatus can cause a number of life-threatening systemic infections in humans. The commensal yeast C. albicans is one of the main causes of nosocomial fungal infectious diseases, whereas the filamentous fungus A. fumigatus has become one of the most prevalent airborne fungal pathogens. Early diagnosis of these fungal infections is challenging, only a limited number of antifungals for treatment are available, and the molecular details of pathogenicity are hardly understood. The completion of both the A. fumigatus and C. albicans genome sequence provides the opportunity to improve diagnosis, to define new drug targets, to understand the functions of many uncharacterised proteins, and to study protein regulation on a global scale. With the application of proteomic tools, particularly two-dimensional gel electrophoresis and LC/MS-based methods, a comprehensive overview about the proteins of A. fumigatus and C. albicans present or induced during environmental changes and stress conditions has been obtained in the past 5 years. However, for the discovery of further putative virulence determinants, more sensitive and targeted proteomic methods have to be applied. Here, we review the recent proteome data generated for A. fumigatus and C. albicans that are related to factors required for pathogenicity.
    Preview · Article · Jun 2011 · International journal of medical microbiology: IJMM
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    • "Our earlier results have shown that MUC7 12-mer binds to the surface of C. albicans cells where it accumulates and then, in a concentration-dependent, all-or-none manner breaks membrane integrity leading to an influx of peptide molecules and immediate cell death (Lis and Bobek 2008). One can envision a scenario where the peptide interacting with the surface of plasma membrane imposes a stress perceived by the cell as similar to other environmental cues triggering calcium response. "
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    ABSTRACT: 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.
    Full-text · Article · Aug 2010 · FEMS Yeast Research
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