# The Heat Shock Protein 60 and Pap1 Participate in the _Sporothrix schenckii_-Host Interaction
## 3 Results
### Hsp60 and Pap1 Contribute to Sporodrix schenckii Virulence
schenckii_ inoculum of 1 \(\times\) 10\({}^{5}\) yeast-like cells was injected into larvae, which were kept at 37 \({}^{\circ}\)C, and mortality was recorded daily. PBS, control group inoculated and challenged with PBS. _S. schenckii_, animal group inoculated with PBS and challenged with 1 \(\times\) 10\({}^{5}\) yeast-like cells. Moreover, cell-free LDH activity was measured as an indicator of cellular cytotoxicity, as previously reported [7; 64]. Animal groups pre-incubated with 10 or 20 \(\mathrm{\SIUnitSymbolMicro g}\) Hsp60 showed similar cytotoxicity levels to the control group pre-inoculated with PBS and challenged with yeast-like cells (Figure 6), but pre-inoculation of 40 \(\mathrm{\SIUnitSymbolMicro g}\) Hsp60 significantly reduced cytotoxicity levels, and these were even more diminished with pre-inoculation of either 80 or 160 \(\mathrm{\SIUnitSymbolMicro g}\) Hsp60 (Figure 6). When rPap1 was used in the pre-inoculation stage, 10 \(\mathrm{\SIUnitSymbolMicro g}\) did not affect cytotoxicity levels, but 20 \(\mathrm{\SIUnitSymbolMicro g}\) significantly reduced the values of free LDH, and those were even more reduced with pre-inoculation of either 40 or 80 \(\mathrm{\SIUnitSymbolMicro g}\) rPap1 (Figure 6). The cytotoxicity associated with the pre-incubation of 160 \(\mathrm{\SIUnitSymbolMicro g}\) rPap1 was similar to that obtained with 80 \(\mathrm{\SIUnitSymbolMicro g}\) Pap1 (Figure 6). Next, we assessed the potential of the polyclonal anti-rHsp60 and anti-rPap1 to protect against experimental systemic sporotrichosis. The pre-immune sera or the polyclonal antibodies did not affect the animal viability or immunological parameter in the 2-week observation period (data not shown). When fungal cells were pre-incubated with polyclonal anti-Hsp60 and these cells were used to challenge \(G\). _mellonella_ larvae, most of the animal population survived to the observation period with median survivals of more than 15 days (Figure 7). Similarly, cells pre-incubated with anti-rPap1 antibodies showed attenuation in the ability to kill larvae, and this was not significantly different when compared with anti-rHsp60-treated cells (Figure 7). Animal groups inoculated with yeast-like cells or fungal cells pre-incubated with control sera showed no significant differences (Figure 7). Additionally, no animal death was observed in the control group inoculated with PBS (Figure 7). The fungal burden showed a correlation with the effect of polyclonal antibodies on the ability of \(S\). _schenckii_ cells to kill animals. Similar fungal burdens were observed in animals inoculated with yeast-like cells or fungal cells pre-incubated with pre-immune sera (Table 4), and CFUs were significantly reduced in the animal groups challenged with yeast-like cells pre-incubated with either anti-rHsp60 or anti-rPap1 (Table 4). Both cellular and humoral immune effectors, named hemocyte levels and phenoloxidase and melanin production, respectively, showed a similar trend in the animal group analyzed. These three immune effectors were high and similar in animal groups inoculated with \(S\). _schenckii_ yeast-like cells and fungal cells pre-incubated with pre-immune sera against rHsp60 or rPap1 (Table 4). However, animal groups inoculated with yeast-like cells pre-incubated with either anti-rHps60 or anti-rPap1 showed a significant reduction in the three immunological parameters (Table 4). When cytotoxicity was analyzed in these animal groups, this was significantly lower and similar in larvae inoculated with fungal