The oral cavity is host to many microorganisms comprising bacteria, fungi and viruses. Both acquired and innate host immunity mechanisms prevent and control manifestations of local infection under most physiological conditions. Recent studies have emphasized the importance of oral infections since their effects may not be limited to the integrity of oral hard and soft tissues alone but also represent risks with regard to a variety of systemic diseases. Histatins are basic, small molecular weight, proteins secreted by both the parotid and submandibular/sublingual glands exhibiting direct and indirect antimicrobial activities. The broad and long term objective of this proposal is to understand mechanisms of the salivary host defense system at the molecular level providing opportunities to exploit such information for prevention and therapeutics. A major focus of investigation is the antifungal effect of histatins on the oral pathogen Candida albicans, causing the most frequently occurring oral fungal infection. While a number of molecular and cellular observations have been described the precise mechanism of histatin induced cell death has been elusive. The proposed studies represent a comprehensive and systematic approach for elucidating this mechanism using both quantitative global proteomics and genetics studies which include comparisons of single gene deletions and gene overexpression. These studies promise to be of translational importance considering the paucity of available antifungal agents and the alarming emergence of fungal resistance to available antimycotics.
Aim 1 focuses on proteomic changes in C. albicans cells which are associated with histatin susceptibility and resistance. For this purpose protein preparations of subcellular fractions of C. albicans which had been exposed to histatin 5 will be compared to controls over time. For quantitative comparisons three stable isotope methods will be employed. These include isotope coded affinity tag (ICAT), multiplexed isobaric isotope reagent (iTRAQ) and metabolic labeling. Data obtained by mass spectrometry will be subjected to database searches, bioinformatics and statistical analyses to reveal changes in protein expression levels in response to histatin 5 treatment.
In Aim 2 the genetic basis for histatin susceptibility will be investigated in a S. cerevisiae model. Histatin susceptibility will be assessed in comprehensive panels of yeast deletion and overexpression mutants using growth inhibition and cell killing assays. In addition, homologues of the functionally important genes in C. albicans will be disrupted by targeted deletion. Integration of the results from both proteomic and genetics approaches will allow us to identify the critical pathways of the histatin killing mechanism. Knowledge of such pathways represents the basis for the development of new second generation antimycotics.

Public Health Relevance

This project focuses on specific salivary proteins which are capable of killing yeasts causing important oral infections which can have consequences for systemic diseases. There are few antifungal agents available to clinicians and there is an alarming increase of new fungal strains which are resistant to available medications. The relevance of the proposed work is its ultimate goal of developing novel, effective and better tolerated antifungal drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE007652-24
Application #
8204952
Study Section
Oral, Dental and Craniofacial Sciences Study Section (ODCS)
Program Officer
Rodriguez-Chavez, Isaac R
Project Start
1986-04-01
Project End
2014-11-30
Budget Start
2011-12-01
Budget End
2014-11-30
Support Year
24
Fiscal Year
2012
Total Cost
$378,258
Indirect Cost
$145,484
Name
Boston University
Department
Dentistry
Type
Schools of Dentistry
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Iavarone, Federica; D'Alessandro, Alfredo; Tian, Na et al. (2014) High-resolution high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry characterization of a new isoform of human salivary acidic proline-rich proteins named Roma-Boston Ser?? (Phos) ? Phe varian J Sep Sci 37:1896-902
Trindade, Fábio; Oppenheim, Frank G; Helmerhorst, Eva J et al. (2014) Uncovering the molecular networks in periodontitis. Proteomics Clin Appl 8:748-61
Vukosavljevic, D; Hutter, J L; Helmerhorst, E J et al. (2014) Nanoscale adhesion forces between enamel pellicle proteins and hydroxyapatite. J Dent Res 93:514-9
Thomadaki, K; Bosch, Ja; Oppenheim, Fg et al. (2013) The diagnostic potential of salivary protease activities in periodontal health and disease. Oral Dis 19:781-8
Fernandez-Feo, M; Wei, G; Blumenkranz, G et al. (2013) The cultivable human oral gluten-degrading microbiome and its potential implications in coeliac disease and gluten sensitivity. Clin Microbiol Infect 19:E386-94
Carneiro, L G; Venuleo, C; Oppenheim, F G et al. (2012) Proteome data set of human gingival crevicular fluid from healthy periodontium sites by multidimensional protein separation and mass spectrometry. J Periodontal Res 47:248-62
Zamakhchari, Maram; Wei, Guoxian; Dewhirst, Floyd et al. (2011) Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract. PLoS One 6:e24455
Thomadaki, K; Helmerhorst, E J; Tian, N et al. (2011) Whole-saliva proteolysis and its impact on salivary diagnostics. J Dent Res 90:1325-30
Komatsu, Tomoko; Salih, Erdjan; Helmerhorst, Eva J et al. (2011) Influence of histatin 5 on Candida albicans mitochondrial protein expression assessed by quantitative mass spectrometry. J Proteome Res 10:646-55
Hanasab, Homan; Jammal, Daniel; Oppenheim, Frank G et al. (2011) The antifungal activity of human parotid secretion is species-specific. Med Mycol 49:218-21

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