# The Heat Shock Protein 60 and Pap1 Participate in the _Sporothrix schenckii_-Host Interaction
## 4 Discussion  
The fungal cell wall is an attractive place to find new targets for antifungal drugs because the molecular nature of its compounds is different from the proteins, polysaccharides, and oligosaccharides found in the mammalian host and because some of these are ubiquitous molecules found in the fungal cell wall, such as glucans and chitin, providing the advantage of having wide-range targets. The _S. schenckii_ cell wall has a unique component that confers the fungus immunogenicity and specific epitopes, the PRM [20]. Although PRM is a thoroughly studied _S. schenckii_ cell wall component, the identities of the proteins composing this glyoconjugate remain unknown. Our mass spectrometry results showed that PRM is composed of at least 325 proteins, and the bioinformatics analysis predicted that only one out of 26 proteins listed in Table 1, named GPI-anchored cell wall beta-1,3-endoglucanase Eglc, has a signal peptide and is predicted to be secreted and localized in the cell wall. It has to be acknowledged that the protocol for PRM isolation does not preclude the contamination of cell walls with intracellular proteins. However, the harsh conditions used here and the PRM enrichment by chromatographies make it unlikely that any contaminant protein could co-purify along with PRM. Moreover, the detection of proteins listed in Table 1 was constant in all the experimental replicates analyzed by mass spectrometry, and we found biological activities at least for Hsp60 and Pap1 linked to the cell wall, named adhesion properties, and an effect on host mortality upon pre-incubation of yeast-like cells with polyclonal anti-rHsp60 or anti-rPaP1 antibodies, making it unlikely these proteins were intracellular contaminants. Based on this, we suggest that most of the PRM proteins might be moonlighting proteins that are transported to the cell wall via noncanonical secretory pathways [71]. A moonlighting protein is defined as a multifunctional protein that participates in more than one independent biological process, usually in different cell compartments [72], and since most of the identified proteins found in the PRM have putative housekeeping intracellular functions or participate in metabolic pathways, the PRM proteins may have a second function in the _S. schenckii_ cell wall, probably participating in host-fungus interaction. Moonlighting proteins in the cell wall of other pathogenic fungi participate in cell adhesion to different host components. In _Candida albicans_, Ssa1 and Ssa2, members of the Hsp70 family, bind to the host-cell cadherins [73], enolase binds to plasminogen [74], glyceraldehyde-3-phosphate dehydrogenase 1 and 2 bind to fibronectin, laminin, and complement regulators [32; 75], and phosphate-glycerate mutase bind to plasminogen and complement regulators [76; 77], among others [35]. In _H. capsulatus_, Hsp60 is the major ligand to the complement receptor 3 (CR3; CD11b/CD18) in alveolar macrophages [37]; and in _Cryptococcus neoformans_, phosphoglycer kinase and fructose biphosphate aldolase were found to bind to plasminogen [78]. The presence of moonlighting proteins in the _S. schenckii_ cell wall was previously suggested. The cell wall proteomic analysis of cells responding to meandione, an oxidative agent, found 13 proteins that could be involved in the fungal oxidative stress response, some of which were already reported as proteins with dual localization and functions in _Candida_ spp. [79]. Another study suggested that the proteins Hsp60, chaperonin GroEL, elongation factor 1\(\beta\), and mitochondrial peroxiredoxin participate in oxidative stress, playing a role in _S. schenckii_ survival within the host [80]. However, these studies did not confirm the proteins' second function nor localization within the _S. schenckii_ cell. Out of the PRM proteins found, two stood out due to their high coverage and Andromeda scores, Hsp60 and the previously uncharacterized Pap1.