Article Figures & Data
Figures
- FIG. 1.
Multiple-sequence alignment of sequences of HAD superfamily hydrolase proteins from diverse human fungal pathogens: Blastomyces dermatitidis (Bd), Histoplasma capsulatum (Hc), Coccidioides posadasii (Cp), Penicillium marneffei (Pm), Aspergillus fumigatus (Af), and Fusarium graminearum (Fg) revealed 84%, 82%, 77%, 67%, 66%, and 58% identities, respectively, compared with the sequence of P. brasiliensis (Pb).
- FIG. 2.
Generation of P. brasiliensis PbHAD32 aRNA strains. (A) T-DNA construct for aRNA silencing of PbHAD32 in P. brasiliensis via ATMT. PbHAD32 aRNA oligonucleotides AS1 (base pairs 1 to 112 of PbHAD32; exon 1), AS2 (base pairs 175 to 309 of PbHAD32; exon 2), and AS3 (base pairs 376 to 536 of PbHAD32; exon 2) were amplified, individually placed under the control of the calcium-binding protein (CBP1) promoter, and later on inserted into the T-DNA of the binary vector pUR5750 for ATMT of P. brasiliensis. (B) Gene expression levels of PbHAD32 in PbWt, PbWt transformed with the empty vector (PbWt + EV), and PbHAD32 aRNA yeast cells after subculture for 15, 45, and 90 days (gene expression levels obtained by RT-PCR were normalized to the level of expression of the internal reference, TUB2; *, P < 0.05 compared with PbWt and PbWt transformed with the empty vector). (C) Gene expression levels of PbHAD32 in PbWt and PbHAD32 aRNA yeast cells after the yeast-to-mycelium transition (Y-M), production of conidia (M-C), and transition into yeast cells (C-Y) (the complete process was Y-M-C-Y) (gene expression levels obtained by RT-PCR were normalized to the level of expression of the internal reference, TUB2; *, P < 0.05 compared with PbWt). (D) Western blot analysis of total protein extracts from PbHAD32 in PbWt and PbHAD32 aRNA yeast cells. Lane 1, PbWt; lane 2, PbHAD32 aRNA.
- FIG. 3.
Silencing of PbHAD32 leads to distinct P. brasiliensis yeast cell morphology without affecting cell vitality. (A) Microscopic evaluation of PbWt yeast cells and yeast cells from two different PbHAD32 aRNA strains generated with different aRNA oligonucleotides (PbHAD32 aRNA1, AS1; PbHAD32 aRNA2, AS2). Magnifications, ×40. (B) Vitality of PbWt, PbWt transformed with the empty vector (PbWt + EV), and PbHAD32 aRNA yeast cells. (C) Cell viability (represented on top of the bars) and gene expression levels of PbHAD32 in PbWt, PbWt transformed with the empty vector, and PbHAD32 aRNA yeast cells during batch culture growth (gene expression levels obtained by RT-PCR were normalized to the level of expression of the internal reference, TUB2; *, P < 0.05 compared with PbWt and PbWt transformed with the empty vector).
- FIG. 4.
PbHad32p plays an essential role during adherence to epithelial cells. (A) Adherence of PbWt and PbHAD32 aRNA yeast cells to A549 epithelial human lung cells at different times postinfection (1 and 3 h). The percent adherence was expressed as the number of CFU adhered to epithelial cells divided by the number of CFU from wells without epithelial cells (*, P < 0.05 compared with PbWt). (B) Gene expression levels of PbHAD32 during infection of epithelial cells with PbWt yeasts cells (*, P < 0.05 compared with PbHAD32 aRNA). (C) Gene expression levels of PbHAD32 in PbWt and PbHAD32 aRNA yeast cells growing in the absence of epithelial cells.
- FIG. 5.
Silencing of PbHAD32 decreases the virulence of P. brasiliensis in a murine model of infection. Representative survival curve of an experimental infection carried out in BALB/c mice via i.t. infection with 2 × 106 PbWt or PbHAD32 aRNA yeast cells (P < 0.0001).
