# The Heat Shock Protein 60 and Pap1 Participate in the _Sporothrix schenckii_-Host Interaction
## 3 Results
### Hsp60 and Pap1 Contribute to Sporodrix schenckii Virulence
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Inoculated with** & **Colony-Forming Units (\(\times\)10\({}^{5}\)) a** & **Hemocytes (\(\times\)10\({}^{6}\)) mL\({}^{-1}\) a** & **Phenoloxidase b** & **Melanin c** \\ \hline None & 0.0 \(\pm\) 0.0 & 3.6 \(\pm\) 0.2 & 0.4 \(\pm\) 0.4 & 0.5 \(\pm\) 0.3 \\ PBS d  & 0.0 \(\pm\) 0.0 & 3.3 \(\pm\) 0.3 & 0.9 \(\pm\) 0.3 & 1.2 \(\pm\) 0.4 \\ PBS \(+\)\(S\). _schenckii_ & 2.4 \(\pm\) 0.4 & 8.0 \(\pm\) 0.6 & 3.2 \(\pm\) 0.6 & 3.5 \(\pm\) 0.6 \\
10 \(\mathrm{\SIUnitSymbolMicro g}\) rHsp60 \(+\)\(S\). _schenckii_ & 2.7 \(\pm\) 0.3 & 7.6 \(\pm\) 0.5 & 3.7 \(\pm\) 0.3 & 3.3 \(\pm\) 0.3 \\
20 \(\mathrm{\SIUnitSymbolMicro g}\) rHsp60 \(+\)\(S\). _schenckii_ & 2.3 \(\pm\) 0.4 & 8.1 \(\pm\) 0.3 & 3.2 \(\pm\) 0.5 & 3.3 \(\pm\) 0.3 \\
40 \(\mathrm{\SIUnitSymbolMicro g}\) rHsp60 \(+\)\(S\). _schenckii_ & 1.3 \(\pm\) 0.2 \({}^{*}\) & 7.2 \(\pm\) 0.5 & 2.6 \(\pm\) 0.5 & 3.0 \(\pm\) 0.2 \\
80 \(\mathrm{\SIUnitSymbolMicro g}\) rHsp60 \(+\)\(S\). _schenckii_ & 0.4 \(\pm\) 0.2 \({}^{*}\) & 10.5 \(\pm\) 0.5 \({}^{*}\) & 4.4 \(\pm\) 0.4 \({}^{*}\) & 4.4 \(\pm\) 0.3 \({}^{*}\) \\
160 \(\mathrm{\SIUnitSymbolMicro g}\) rHsp6 \(+\)\(S\). _schenckii_ & 0.2 \(\pm\) 0.2 \({}^{*}\) & 10.3 \(\pm\) 0.3 \({}^{*}\) & 4.6 \(\pm\) 0.2 \({}^{*}\) & 4.6 \(\pm\) 0.5 \({}^{*}\) \\
10 \(\mathrm{\SIUnitSymbolMicro g}\) rPap1 \(+\)\(S\). _schenckii_ & 2.7 \(\pm\) 0.5 & 7.3 \(\pm\) 0.7 & 2.9 \(\pm\) 0.5 & 3.1 \(\pm\) 0.5 \\
20 \(\mathrm{\SIUnitSymbolMicro g}\) rPap1 \(+\)\(S\). _schenckii_ & 1.9 \(\pm\) 0.3 \({}^{*}\) & 7.7 \(\pm\) 0.6 & 3.3 \(\pm\) 0.3 & 3.2 \(\pm\) 0.5 \\
40 \(\mathrm{\SIUnitSymbolMicro g}\) rPap1 \(+\)\(S\). _schenckii_ & 1.1 \(\pm\) 0.2 \({}^{*}\) & 10.2 \(\pm\) 0.3 \({}^{*}\) & 4.4 \(\pm\) 0.3 \({}^{*}\) & 4.1 \(\pm\) 0.3 \({}^{*}\) \\
80 \(\mathrm{\SIUnitSymbolMicro g}\) rPap1 \(+\)\(S\). _schenckii_ & 0.3 \(\pm\) 0.2 \({}^{*}\) & 10.6 \(\pm\) 0.4 \({}^{*}\) & 4.6 \(\pm\) 0.5 \({}^{*}\) & 4.7 \(\pm\) 0.5 \({}^{*}\) \\
160 \(\mathrm{\SIUnitSymbolMicro g}\) rPap1 \(+\)\(S\). _schenckii_ & 0.2 \(\pm\) 0.1 \({}^{*}\) & 10.8 \(\pm\) 0.8 \({}^{*}\) & 4.5 \(\pm\) 0.3 \({}^{*}\) & 4.5 \(\pm\) 0.4 \({}^{*}\) \\ \hline \hline \end{tabular}  
* Larvae were injected with the indicated inoculum and monitored for 15 days, then hemolymph was collected and used to calculate colony-forming units and hemocyte concentration. b Defined as the \(\Delta_{\mathrm{Japan}}\) per min per \(\mathrm{\SIUnitSymbolMicro g}\) 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 pre-inoculated with PBS and challenged with \(S\). _schenckii_ yeast-like cells.  
\end{table} 
Table 3: Colony-forming units, hemocyte counts, phenoloxidase, and melanin production in _Galeria mellonella_ larvae pre-inoculated with either recombinant Hsp60 or Pap1 and challenged with _Sporothorax schenckii_ yeast-like cells. cells pre-incubated with anti-rHsp60 or anti-rPap1 antibodies, when compared to the free LDH activity quantified in the hemolymph of animals inoculated with yeast-like cells with no pre-incubation step or pre-incubated with the pre-immune sera (Figure 8). The control group inoculated only with PBS showed low and similar cytotoxicity levels to those of noninoculated animals (Figure 8). \begin{table}
\begin{tabular}{c c c c c} \hline
**Inoculated with** & **Colony-Forming Units (\(\times\)10\({}^{5}\)) 1 [FOOTNOTE:1]Footnote 1: \(\mathrm{L}\mathrm{L}\mathrm{L}\mathrm{L}\mathrm{L}\mathrm{L}\mathrm{L}\mathrm{L}  
Collectively, these data strongly suggest that Hsp60 and Pap1 are proteins that contribute to the _S. schenckii_'s ability to kill _G. mellonella_ larvae. Figure 8: Effect of anti-recombinant Hsp60 and Pap1 antibodies on the cytotoxicity associated with _Galeria mellonella_ killed by _Sporothrix schenckii. G. mellonella_ larvae were inoculated with phosphate-buffered saline (PBS), \(1\times 10^{5}\) yeast-like cells (_S.