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Secreted aspartyl proteinase (PbSap) contributes to the virulence of Paracoccidioides brasiliensis infection.
Castilho DG
,
Chaves AFA
,
Navarro MV
,
Conceição PM
,
Ferreira KS
,
da Silva LS
,
Xander P
,
Batista WL
.
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Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and is caused by fungi from the Paracoccidioides genus. Virulence factors are important fungal characteristics that support the development of disease. Aspartyl proteases (Saps) are virulence factors in many human fungal pathogens that play an important role in the host invasion process. We report here that immunization with recombinant Sap from Paracoccidioides brasiliensis (rPbSap) imparted a protective effect in an experimental PCM model. The rPbSap-immunized mice had decreased fungal loads, and their lung parenchyma were notably preserved. An aspartyl protease inhibitor (pepstatin A) significantly decreased pulmonary injury and reduced fungal loads in the lung. Additionally, we observed that pepstatin A enhanced the fungicidal and phagocytic profile of macrophages against P. brasiliensis. Furthermore, PbSAP expression was highly altered by environmental conditions, including thermal stress, dimorphism switching and low pH. Hence, our data suggest that PbSap is an important virulence regulator in P. brasiliensis.
Fig 1. Reverse transcriptase qPCR of PbSAP from P. brasiliensis under multiple conditions and schematic representation of the PbSAP 5’ UTR region.PbSAP (PADG_00634) transcript levels were measured during the mycelium to yeast (M-Y) (A) or yeast to mycelium (Y-M) transition (B). Schematic representation showing the putative non-conventional heat shock elements (ncHSE) motifs in the promoter region of PbSAP (C). Quantitative real time RT-PCR of PbSAP from Pb18 yeast cells after heat shock at 42°C for 30 or 60 min (D), treatment with 2 mM H2O2 at 2 or 6 h (E) and different NaCl concentrations (F), as indicated. The change in transcriptional levels was calculated via the 2-ΔΔCt method, with two housekeeping genes (α-TUB and 18S). All of the data shown in this figure were analyzed using Student’s t-test. Error bars correspond to the standard deviation of measurements performed in triplicate, and asterisks indicate statistically significant differences in expression (*p < 0.05, **p<0.01 and ***p<0.001).
Fig 2. SDS-PAGE and western blot analysis of the recombinant PbSap.Total bacterial extracts from recombinant bacteria expressing rPbSap (A) and the respective protein eluted from Ni-NTA columns with pH 4.5 buffer (B). Western blot analysis of mouse immune sera showing the production of anti-rPbSap (C) and analysis using the total protein extract (25 μg) of P. brasiliensis yeast (D). Immunoblot reactivity of PCM patient’s sera with rPbSap. Controls were serum from a healthy individual (NHS) and human sera were tested at a 1:200 dilution (E).
Fig 3. SDS-PAGE, western blot and RT-PCR analysis of PbSap in P. brasiliensis yeast cells cultured in low pH.Profile of the Coomassie brilliant blue stained gel (12% SDS-PAGE) using the total protein extract (25 μg) of P. brasiliensis yeast cultured in pH 6.5 or pH 4.0 with or without BSA. (A). Immunobloting analysis of intracellular and secreted proteins in low pH (B). Relative densitometric values of bands were normalized by dry mass and the results are shown in the bar graphs. the arrowhead indicates the non-glycosylated form of PbSap (44 kDa).
Fig 4. Immunofluorescence assay of PbSap in P. brasiliensis yeast cells cultured in different pH.Confocal microscopy observation of calcofluor white and FITC-antibody double-stained yeast cells. White bars correspond to 10 μm.
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