# The Heat Shock Protein 60 and Pap1 Participate in the _Sporothrix schenckii_-Host Interaction
## 3 Results
### Hsp60 and Pap1 Contribute to Sporodrix schenckii Virulence  
The role of both Hsp60 and Pap1 in _S. schenckii_ virulence was next assessed in _G. mellonella_ larvae. We decided to use this system because it replicated the fungal virulence observed in the murine model and allowed us to include more animals per group than the mammalian system, strengthening the statistical power [7; 68; 69]. We first analyzed the inoculation effect of recombinant proteins on insect immune effectors. After the  
Figure 4: Adhesion of recombinant Hsp60 and Pap1 to immobilized extracellular matrix proteins. The extracellular matrix proteins were immobilized in 96-well plates and used in ELISA-based analyses. Cell homogenates from nontransformed bacteria (_Escherichia coli_) and bacteria transformed with the empty vector (EV) expressing rHsp60 (rHsp60) or rPap1 (rPap1) were incubated with the immobilized extracellular matrix proteins, and the protein–protein adhesion detected with the specific polyclonal anti-rHsp60 or anti-rPap1 and peroxidase-conjugated anti-rabbit IgG. Results are means \(\pm\) SD of three independent experiments performed by duplicate. \({}^{\star}\)\(p<0.05\) when compared to the other immobilized proteins. \({}^{\dagger}\)\(p<0.05\) when compared to cell homogenates containing rHsp60. inoculation of different doses of both recombinant proteins, no animal died in the 2-week observation period (data not shown). Hemocyte concentration, melanin production, and phenoloxidase activity, all measured in the insect hemolymph, are immunological parameters that were previously demonstrated to change in response to fungal or bacteria stimuli [7; 56; 61; 62; 70]. Thus, we measured these immunological parameters in animals inoculated with recombinant proteins 5 days after the inoculation. Doses of 10 and 20 ug rHsp60 did not change these three parameters, but inoculation of 40 ug rHsp60 positively affected hemocyte count, melanin production, and phenoloxidase activity (Table 2). The levels of these three parameters were significantly increased when animals were inoculated with 80 ug rHsp60, and these figures did not increase upon inoculation of 160 ug rHsp60 (Table 2). When experiments were performed with rPap1, a similar result was obtained with 10 ug recombinant protein, i.e., no significant changes in the three parameters analyzed (Table 2). However, with doses ranging from 20 to 80 ug rPap1, a dose-dependent increment in the levels of hemocytes, melanin, and phenoloxidase were observed in the _G. mellonella_ hemolymph (Table 2). Animals inoculated with 80 or 160 ug recombinant protein showed similar hemocyte, melanin, and phenol oxidase levels (Table 2). Next, we assessed the effect of the recombinant protein inoculation before a lethal challenge of _S. schenckii_ yeast-like cells. We inoculated the animal groups with the different doses of recombinant proteins, and 5 days later, applied an inoculation of \(1\times 10^{5}\) yeast-like cells, a dose that kills most of the animal population in the 2-week observation period [60]. Cells inoculated only with PBS, then challenged with the fungal cells, showed a typical kill curve, with a median survival of 6.0 \(\pm\) 1.0 days and 83.3 \(\pm\) 3.0% population death during the observation period, which was similar to data reported previously for this fungal strain [7; 30; 56; 60; 68; 69]. The inoculation of 10 or 20 ug rHsp60 did not significantly modify the kill curve, but inoculation of 40 ug rHsp60 significantly changed the kill curve with a median survival of more than 15 days and only 53.0 \(\pm\) 6.0% animal death at the end of the observation period (Figure 5A). Inoculation of 80 ug or 160 ug rHsp60 before the challenge with the fungal cells significantly affected _S. schenckii_ cells' ability to kill the animal population, with only three and one death observed, respectively (Figure 5A). When compared, both curves were not significantly different (\(p\) = 0.53). Following the same experimental setting, the inoculation of 10 ug rPap1 and the subsequent challenge with _S.