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Digital archive of theses discussed at the University of Pisa


Thesis etd-06202016-113033

Thesis type
Tesi di dottorato di ricerca
Thesis title
Identification and functional characterisation of a Candida orthopsilosis adhesin
Academic discipline
Course of study
tutor Prof.ssa Tavanti, Arianna
  • adhesion
  • adhesion
  • adhesion
  • ALS genes
  • ALS genes
  • ALS genes
  • Candida orthopsilosis
  • Candida orthopsilosis
  • Candida orthopsilosis
Graduation session start date
Candida parapsilosis is recognised as an important nosocomial pathogen currently representing the second/third most common Candida species isolated from bloodstream infections. The intra specific genetic variability observed among clinical isolates has eventually led to the definition of three different species, named C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis. Retrospective DNA-based studies indicate that C. metapsilosis and C. orthopsilosis each represent 1–10% of the infections attributed to C. parapsilosis estimated from conventional biochemical tests. Although little is known with regard to the pathogenic potential of C. orthopsilosis and C. metapsilosis, there is increasing evidence that the former has a comparable virulence to C. parapsilosis, while C. metapsilosis is the least virulent member of the complex. The adhesion ability to host surfaces is a pivotal step in the early phases of infection, and in Candida spp Als (agglutinin-like sequence) cell-wall proteins play a key role in this process. The recent sequencing of C. orthopsilosis genome allowed the annotation of three ALS genes, named CORT0C04210, CORT0C04220, and CORT0B00800, thus setting the scene for a deeper evaluation of the molecular mechanisms underlying the adhesion ability of this species.
The present PhD research project was aimed at dissecting the contribution of Als proteins to the adhesion ability of C. orthopsilosis, by using both generation of null mutants and biophysical techniques. The results obtained confirmed that all three ALS genes are expressed in C. orthopsilosis. The presence of allelic variability was detected for each gene among clinical isolates, most likely due to differences in the length of the repetitive central region, as reported for C. albicans ALS genes. Although many authors suggested that longer ALS alleles may result in a more pronounced adhesive phenotype in this species, this association was not observed for C. orthopsilosis. The potential impact of amino acidic substitutions mapping in the N terminal domain of these proteins, which is involved in ligand binding, has also been addressed. The gene CORT0B00800 was selected as a first target for site directed mutagenesis, which was accomplished by using the SAT1-flipper cassette strategy, thus generating heterozygous and null mutant strains. Analysis of the phenotypic traits of the mutant family did not reveal any difference in the growing ability compared to the wild type strain, both in the absence and in the presence of cell-wall perturbing agents. Moreover, the transition to the filamentous morphology was unaffected by the lack of Als800p. The wild type and null mutant strains also adhered to a similar extent to human epithelial cells, although with minor differences due to the specific type of cells used in the assay (primary buccal cells or epithelial cell line). Nevertheless, the adhesive properties of the N terminal domain of Als800p (NTAls800) were demonstrated by using biophysical assays. Indeed, a soluble version of NTAls800 was expressed in Escherichia coli and purified, providing a protein suitable for Surface Plasmon Resonance (SPR), Fluorescence Polarisation (FP), and Nuclear Magnetic Spectroscopy (NMR) experiments used to evaluate its binding activity. NMR analysis confirmed the correct folding of NTAls800 and its ability to bind a hepta-Threonine peptide, which is a known binder for C. albicans Als3p. Moreover, FP based assays (direct and competitive binding experiments) demonstrated that synthetic ligands can enter the protein cleft with different affinity.
Collectively, these results indicate that CORT0B00800 deletion neither impair C. orthopsilosis growth and pseudohyphal formation, nor affect its ability to adhere to epithelial cells. However, biophysical techniques confirmed that Als800p displays binding activity, thus pointing out its potential role in C. orthopsilosis adhesion in different experimental settings involving other cell types. An FP-based screening of a library of potential biological ligands is currently underway, which will provide information on Als800p binding specificity.