# 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_ cells generated a kill curve similar to that observed with animals inoculated with PBS then challenged with yeast-like cells (Figure 5B), with a mean survival of 6.0 \(\pm\) 0.5 days and 79.3 \(\pm\) 6.0% animal population death at the end of the observation period. However, \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Inoculated with** & **Hemocytes (\(\times 10^{6}\)) mL\({}^{-1}\)a** & **Phenoloxidase b** & **Melanin c** \\ \hline None & 3.3 \(\pm\) 0.4 & 0.5 \(\pm\) 0.3 & 0.7 \(\pm\) 0.4 \\ PBS d  & 3.1 \(\pm\) 0.7 & 0.8 \(\pm\) 0.5 & 1.1 \(\pm\) 0.2 \\
10 ug rHsp60 & 2.9 \(\pm\) 0.6 & 0.7 \(\pm\) 0.4 & 1.3 \(\pm\) 0.5 \\
20 ug rHsp60 & 3.2 \(\pm\) 0.5 & 1.0 \(\pm\) 0.3 & 1.0 \(\pm\) 0.3 \\
40 ug rHsp60 & 6.9 \(\pm\) 0.8 *  & 1.9 \(\pm\) 0.6 *  & 2.8 \(\pm\) 0.4 *  \\
80 ug rHsp60 & 9.8 \(\pm\) 0.4 *  & 2.7 \(\pm\) 0.6 *  & 4.2 \(\pm\) 0.8 *  \\
160 ug rHsp60 & 10.1 \(\pm\) 0.7 *  & 3.1 \(\pm\) 0.7 *  & 3.8 \(\pm\) 0.4 *  \\
10 ug rPap1 & 3.3 \(\pm\) 0.8 & 0.9 \(\pm\) 0.2 & 1.1 \(\pm\) 0.2 \\
20 ug rPap1 & 5.3 \(\pm\) 0.6 *  & 1.6 \(\pm\) 0.4 *  & 2.1 \(\pm\) 0.4 *  \\
40 ug rPap1 & 8.2 \(\pm\) 0.5 *  & 2.4 \(\pm\) 0.4 *  & 3.2 \(\pm\) 0.4 *  \\
80 ug rPap1 & 10.2 \(\pm\) 0.6 *  & 3.4 \(\pm\) 0.3 *  & 4.3 \(\pm\) 0.6 *  \\
160 ug rPap1 & 9.8 \(\pm\) 0.6 *  & 3.3 \(\pm\) 0.5 *  & 4.0 \(\pm\) 0.5 *  \\ \hline \hline \end{tabular}  
* Larvae were injected with the indicated inoculum and monitored for 15 days, then hemolymph was collected and used to calculate hemocyte concentration. b defined as the \(\Delta_{\mathrm{Som}}\) per min per ug protein. c defined as the absorbance at 405 nm of the cell-free hemolymph. d Animal control group inoculated with PBS.
* \(p\) < 0.05 when compared with the values obtained with animals inoculated with PBS. f \(p\) < 0.05 when compared with values obtained with different concentrations of the same recombinant protein.  
\end{table} 
Table 2: Hemocyte count, phenoloxidase, and melanin production in _Galleria mellonella_ larvae inoculated with either recombinant Hsp60 or Pap1. inoculation of 20 ug rPap1 significantly changed the kill curve (\(p<0.05\)), with a median survival of 10.0 \(\pm\) 1.5 days and 60.0 \(\pm\) 2.0% dead animals (Figure 5B). The inoculation of 40 ug rPap1 decreased animal mortality even further, with a median survival of more than 15 days and a mortality of 24.0 \(\pm\) 7.0% (Figure 5B). The _S. schenckii_ cells were incapable of killing larvae when either 80 or 160 ug rPap1 were previously inoculated in the animal groups (Figure 5B). When colony-forming units were calculated in the animal groups pre-inoculated with rHsp60 and challenged with yeast-like cells, we did not observe changes in the fungal burden when 10 or 20 ug rHsp60 were inoculated or when compared to animals inoculated only with PBS, but the inoculation of 40 ug rHps60 5 days before the fungal challenge significantly reduced the CFUs, and this was even more evident when 80 or 160 ug recombinant protein were pre-inoculated (Table 3). The immunological parameters analyzed, named hemocytes concentration, phenoloxidase activity, and melanin, were positively affected only with the pre-inoculation of 80 or 160 ug rHsp60 (Table 3). For the case of the pre-inoculation of rPap1, 10 ug did not modify the parameters under analysis (Table 3), but a dose-dependent reduction in the CFUs was observed with pre-inoculation of rPap1 in the range of 20 to 80 ug; although figures calculated with a dose of 160 ug were similar to those obtained with 80 ug (Table 3). The immunological parameters analyzed were significantly incremented when animal groups were pre-inoculated with 40, 80, or 160 ug rPap1, but in this case, a dose-dependent behavior was not observed (Table 3). Figure 5: Effect of recombinant Hsp60 and Pap1 on the _Galleria mellonella_ killed by _Sporothrix schonckii_. _G. mellonella_ larvae were inoculated with either phosphate-buffered saline (PBS) or the indicated amount of rHsp60 (**A**) or rPap1 (**B**) and incubated for 5 days at 37 \({}^{\circ}\)C. Then, a lethal _S.