# The Heat Shock Protein 60 and Pap1 Participate in the _Sporothrix schenckii_-Host Interaction
## 3 Results
### Hsp60 and Pap1 Participate in the Sportthrix schenckii Adhesion to Extracellular Matrix Proteins  
Recombinant proteins and polyclonal anti-rHsp60 and anti-rPap1 antibodies were used in ELISA-based experiments to assess binding to immobilized laminin, fibrinogen, fibronectin, and type-II collagen. We selected these proteins because it was previously demonstrated that _S. schenckii_ yeast-like cells can adhere to them [59]. As a negative control, we included thrombospondin-1, an ECM component that is not bound by these cells [59], and elastin and type-I collagen, two ECM components previously uncharacterized in  
Figure 2: Expression of recombinant Hsp60 and Pap1 expressed in _Escherichia coli_. In (**A**), bacteria were transformed with pCold-_HSP60_ (rHsp60 lanes), pCold-_PAP1_ (rPap1 lanes), or the empty pCold I vector (EV) and grown under inducing condition (0.1 M isopropyl-ß-D-1-thiogalactopyranoside and 20 h at 15 \({}^{\circ}\)C). Protein samples were prepared from cultured cells and separated by denaturing SDS-PAGE in 12% (\(w/v\)) gels. Two independent clones for production of each recombinant protein were analyzed. The recombinant proteins were then subjected to purification as described in the main text and were either Commassie Blue-stained ((**B,C**) correspond to purified rHsp60 and rPap1, respectively) or silver-stained (**D**). MPM, molecular protein marker. terms of the _S. schenckii_ ability to adhere to them. The _S. schenckii_ yeast-like cells showed adhesion to all the ECM components tested with exception of thrombospondin-1, as reported [59] (Figure 3). Cells showed the highest adhesion to laminin and fibronectin, followed by type-I and type-II collagen, elastin, and fibrinogen (Figure 3). When cells were pre-incubated with anti-rHsp60 antibodies, and then used in similar adhesion experiments, there was a significant reduction in the cell ability to adhere to laminin, elastin, fibrinogen, and fibronectin (Figure 3). For the case of type-I and type-II collagen, the blocking of Hsp60 with the polyclonal antibodies did not affect the cells' ability to adhere to these two ECM components (Figure 3). When similar experiments were performed with cells pre-incubated with anti-rPap1 antibodies, a significant reduction was observed in the cells' ability to adhere to all the ECM proteins tested, including type-I and type-II collagen (Figure 3). This reduction was similar to that observed for cells pre-incubated with anti-rHsp60 antibodies, but was significantly different when compared to the case of adhesion to fibronectin (Figure 3). When cells were pre-incubated with antibodies against both recombinant proteins, the adhesion to laminin, elastin, and fibronectin was significantly reduced when compared to cells pre-incubated with only one antibody, suggesting both proteins contributed to the adhesion of these ECM components (Figure 3). For the case of fibrinogen, the reduction was not significantly different from the systems with cells pre-incubated with only one antibody (\(p\) = 0.14, and 0.11 when compared to cells pre-incubated with anti-rHsp60 or anti-Pap1, respectively). For the case of collagens, adhesion was not significantly affected, confirming the lack of participation of Hsp60 in the adhesion of these ECM components (Figure 3). Control experiments with pre-immune sera gave adhesion profiles similar to those with yeast-like cells without incubation with antibodies (Figure 3), and controls without secondary antibodies gave no detectable signals (data not shown). Collectively, these data suggest that both Hsp60 and Pap1 contribute to _S. schenckii_ adhesion to ECM, with differential adhesion properties. Figure 3: Adhesion of _Sporothrix schenckii_ yeast-like cells to immobilized extracellular matrix proteins. The extracellular matrix proteins were immobilized in the 96-well plates and used in ELISA-based analyses.