Cell Wall Proteome Profiling of a Candida albicans Fluconazole-Resistant Strain from a Lebanese Hospital Patient Using Tandem Mass Spectrometry-A Pilot Study.

Candida albicans is an opportunistic pathogenic fungus responsible for high mortality rates in immunocompromised individuals. Azole drugs such as fluconazole are the first line of therapy in fungal infection treatment. However, resistance to azole treatment is on the rise. Here, we employ a tandem mass spectrometry approach coupled with a bioinformatics approach to identify cell wall proteins present in a fluconazole-resistant hospital isolate upon drug exposure. The isolate was previously shown to have an increase in cell membrane ergosterol and cell wall chitin, alongside an increase in adhesion, but slightly attenuated in virulence. We identified 50 cell wall proteins involved in ergosterol biosynthesis such as Erg11, and Erg6, efflux pumps such as Mdr1 and Cdr1, adhesion proteins such as Als1, and Pga60, chitin deposition such as Cht4, and Crh11, and virulence related genes including Sap5 and Lip9. Candidial proteins identified in this study go a long way in explaining the observed phenotypes. Our pilot study opens the way for a future large-scale analysis to identify novel proteins involved in drug-resistance mechanisms.

Phenotypic and Cell Wall Proteomic Characterization of a DDR48 Mutant Candida albicans Strain.

Candida albicans is an opportunistic fungus possessing multiple virulence factors controlling pathogenicity. Cell wall proteins are the most important among these factors, being the first elements contacting the host. Ddr48 is a cell wall protein consisting of 212 amino acids. A DDR48 haploinsufficient mutant strain was previously found necessary for proper oxidative stress response and drug resistance. In this study, we aimed to further elucidate the role of Ddr48 by performing additional phenotypic characterization assays. A combinatory proteomic and bioinformatics approach was also undertaken to determine differentially expressed cell wall proteins. Results showed that the mutant strain exhibited a 10% decrease in adhesion mirrored by a 20% decrease in biofilm formation, and slight sensitivity to menadione, diamide, and SDS. Both strains showed similar hyphae formation, virulence, temperature tolerance, and calcofluor white and Congo red sensitivities. Furthermore, a total of 8 and 10 proteins were identified exclusively in the wild-type strain grown under filamentous and nonfilamentous conditions respectively. Results included proteins responsible for superoxide stress resistance (Sod4 and Sod6), adhesion (Als3, Hyr4, Pmt1, and Utr2), biofilm formation (Hsp90, Ece1, Rim9, Ipp1, and Pra1) and cell wall integrity (Utr2 and Pga4). The lack of detection of these proteins in the mutant strain correlates with the observed phenotypes.

Proteomic analysis of a Candida albicans pga1 Null Strain.

We previously characterized Pga1, a Candida albicans (C. albicans) cell wall protein necessary for proper virulence, adhesion, and resistance to oxidative stress. By utilizing tandem mass spectrometry coupled with bioinformatics to investigate cell wall proteome expression in a pga1 null fourteen and 36 proteins were identified in the wild type grown under filamentous and non-filamentous conditions respectively, but were not detected in the mutant, including members of the PGA GPI anchored family. Virulence and adhesion proteins such as Hsp 90, Sap10, Cdc11, Int 3 and members of the lipase family were also identified exclusively in the wild type.

Tandem Mass Spectrometric Cell Wall Proteome Profiling of a Candida albicans hwp2 Mutant Strain.

BACKGROUND: Candida albicans is present as part of the normal gut flora and detected in the oral cavities and GI tracts of around fifty percent of adults. Benign colonization can turn pathogenic causing a variety of mild to severe infections. In a pathogen, the cell wall and cell surface proteins are major antigenic determinants and drug targets as they are the primary structures that contact the host. Cell surface proteins perform a variety of functions necessary for virulence such as adhesion, host degradation, resistance to oxidative stress, and drug resistance. We have previously characterized Hwp2, a C. albicans cell wall adhesin shown to play a major role in the cell wall architecture and function as hwp2 mutants were deficient in chitin deposition, filamentation, adhesion and invasive growth, virulence, and resistance to oxidative stress. OBJECTIVE/METHOD: Here, we utilized tandem mass spectrometry coupled with a bioinformatics approach to differentially profile the cell wall proteome of a wild-type strain compared to an hwp2 null mutant to determine key differentially expressed proteins. RESULT: Many proteins identified exclusively in the wild-type go a long way in explaining the abovementioned phenotypes. These include virulence factors such as members of the SAP family including Sap4, Sap5, and Sap10, as well as several lipases involved in host degradation. We also identified members of the PGA family of proteins Pga28, Pga32, Pga41 and Pga50, which function in adhesion, Cht2 a chitinase involved in chitin remodeling, and several proteins that function in promoting filamentation such as Phr1, Mts1, and Rbr1.

Proteomic analysis of a Candida albicans pir32 null strain reveals proteins involved in adhesion, filamentation and virulence.

We have previously characterized Pir32, a Candia albicans cell wall protein that we found to be involved in filamentation, virulence, chitin deposition, and resistance to oxidative stress. Other than defining the cell shape, the cell wall is critical for the interaction with the surrounding environment and the point of contact and interaction with the host surface. In this study, we applied tandem mass spectrometry combined with bioinformatics to investigate cell wall proteome changes in a pir32 null strain. A total of 16 and 25 proteins were identified exclusively in the null mutant strains grown under non-filamentous and filamentous conditions. These proteins included members of the PGA family with various functions, lipase and the protease involved in virulence, superoxide dismutases required for resisting oxidative stress, alongside proteins required for cell wall remodeling and synthesis such as Ssr1, Xog1, Dfg5 and Dcw1. In addition proteins needed for filamentation like Cdc42, Ssu81 and Ucf1, and other virulence proteins such as Als3, Rbt5, and Csa2 were also detected. The detection of these proteins in the mutant and their lack of detection in the wild type can explain the differential phenotypes previously observed.