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Tesi etd-01252018-173907


Thesis type
Tesi di laurea magistrale
Author
FIORENTINI, FABRIZIO
URN
etd-01252018-173907
Title
Targeted gene disruption demonstrates a role of CPAR2_404790 in Candida parapsilosis virulence and pathogenicity
Struttura
BIOLOGIA
Corso di studi
BIOTECNOLOGIE MOLECOLARI
Commissione
relatore Prof.ssa Tavanti, Arianna
Parole chiave
  • host-pathogen interaction
  • murine vaginal candidiasis
  • Candida parapsilosis
  • adhesion
  • ALS genes
  • virulence factors
Data inizio appello
12/02/2018;
Consultabilità
parziale
Data di rilascio
12/02/2021
Riassunto analitico
Candida parapsilosis is an important nosocomial opportunistic pathogen, second in frequency only to Candida albicans and commonly associated with mucosal and systemic infection. The adhesion ability to host surface is a pivotal step in early phases of infection. C. parapsilosis diploid genome encompasses 5 genes encoding agglutinin-like sequence proteins (Als): CPAR2_404800, CPAR2_404770, CPAR2_404780, CPAR2_404790, CPAR2_500660. The aim of this study was to evaluate the role of CPAR2_404790 (further indicated as CpALS6), an orthologue of C. albicans adhesion encoding gene ALS6, in the adhesion ability of C. parapsilosis. C. parapsilosis heterozygous and null mutant strains, lacking one or both copies of CpALS6 gene, respectively, were generated. The deletion of CpALS6 was performed using the SAT1-flipper cassette strategy. C. parapsilosis reference strain ATCC 22019 was used as parental strain for the creation of heterozygous (CpALS6 H) and null mutant (CpALS6 KO) strains. After the first transformation round, leading to the creation of a heterozygous strain, SAT1-flipper cassette was excised from the genome by growing the strain in Yeast Nitrogene Base (YNB) medium containing maltose, a transcript inducer of flippase recombinase. This step allowed the cassette to be recycled for second round of transformation and generation of null mutants. Analysis of the phenotypic traits of the mutant family did not reveal any difference compared to wild type, in terms of growing ability, both in the absence and in the presence of cell-wall perturbing agents (congo red, calcofluor white, fluconalzole, caffeine). Furthermore, the ability to produce filamentous forms (pseudhyphae) under inducing condition did not differ between mutant strains and wild type. These results indicate that deletion of CpALS6 does not alter replication ability and morphogenesis of C. parapsilosis heterozygous and null mutants. Mutant strains were then characterized for their ability to adhere to human buccal cells (HBECs). Each strain was co-incubated with HBECs for 45 minutes at 37°C, in 1000:1 ratio. The results obtained indicate that the deletion of both alleles of CpALS6 gene results in a decreased ability of C. parapsilosis to adhere on HBECs, compared to wild type strain, as demonstrate by a ≥60% reduction in adhesion index (P<0.001). Quantitative expression of CpALS genes was determined in C. parapsilosis mutant and wild type strains following 45 minutes co-incubation with HBECs. As expected, no CpALS6 transcript was detected in the null mutant strain, confirming the deletion of the gene of interest, while no significant changes of other CpALS transcriptional levels were observed. Furthermore, in order to investigate the contribution of CpALS6 in C. parapsilosis pathogenicity, the mutant collection was tested in a murine model of vaginal candidiasis. Preliminary data indicates that the deletion of CpALS6 affects the ability of C. parapsilosis to colonize and persist in the vaginal mucosa. The overall results obtained in this study provide the first evidence for a direct role of the CpAls6 protein in C. parapsilosis adhesion to host surfaces as well as in the pathogenic potential of this fungal species.
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