Semester

Fall

Date of Graduation

2010

Document Type

Thesis

Degree Type

MS

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Donald H Beezhold

Abstract

Fungi are ubiquitous microorganisms that commonly cause diseases with high mortality rates in immunocompromised hosts. In order to successfully treat the infection and subsequently decrease mortality rates, it is necessary to rapidly identify the causal organism(s) and promptly begin the proper treatment. Common identification methods, such as viable culture characterization, are often time consuming or not species-specific, as is the case with many molecular or immunodiagnostic assays. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to discriminate between many clinically relevant microorganisms. Recently, MALDI-TOF MS has been used to discriminate between monilaceous fungal species, however, darkly pigmented fungi yield poor mass spectra with only a few peaks of low relative abundance. Darkly pigmented fungi are of interest because they are common indoor contaminants, and frequently a cause of disease in immunocompromised patients. In this study, the effect of dark fungal pigments on the ionization of standard peptide and protein solutions during MALDI-TOF MS is investigated. Solutions of human serum albumin and angiotensin II were spiked with varying concentrations of both synthetic melanin and fungal pigments extracted from Aspergillus niger, A. fumigatus, and A. terreus. Serum albumin and angiotensin II [M+H]+ signals were suppressed in a concentration dependent manner. Examination of the MALDI sample deposit under magnification showed significant heterogeneity, with regions of highly concentrated pigment appearing black. MALDI-TOF mass spectra acquired from darkly pigmented regions of the sample deposit yielded poor or no [M+H]+ ion signal from the standard. In contrast, nonpigmented regions within the sample deposit and liquid hyphal negative control extracts of A. niger, A. fumigatus, and A. terreus produced no such inhibition. Gas chromatography analysis of each fungal pigment extract revealed the presence of putative cell wall components including aliphatic chains and fatty acids. Field emission scanning electron microscopy (FESEM) showed A. niger and A. fumigatus extracts retain original spore morphology, while the A. terreus extract was homogeneous and morphologically similar to synthetic melanin. This study demonstrates that synthetic and fungal pigmentation derived from darkly pigmented fungi inhibits the desorption/ionization process during MALDI-TOF MS, however these fungi may be successfully analyzed by MALDI-TOF MS when culture methods that suppress pigment expression are employed.

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