The Heat Shock Protein 60 and Pap1 Participate in the Sporothrix schenckii-Host Interaction

Abstract

Sporothrixschenckii is one of the etiological agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. Its cell wall contains a glycoconjugate composed of rhamnose, mannose, glucuronic acid, and proteins, named peptidorhamnomannan, which harbors important Sporothrix-specific immunogenic epitopes. Although the peptidorhamnomannan carbohydrate moiety has been extensively studied, thus far, little is known about the protein core. Here, using LC-MS/MS, we analyzed the S.schenckii peptidorhamnomannan peptide fraction and generated mass signals of 325 proteins, most of them likely to be moonlighting proteins. Among the identified proteins, chaperonin GroEL/Hsp60 and the uncharacterized protein Pap1 were selected for further analysis. Both proteins were heterologously expressed in bacteria, and they showed adhesive properties to the extracellular matrix proteins laminin, elastin, fibrinogen, and fibronectin, although Pap1 also was bound to type-I and type-II collagen. The inoculation of concentrations higher than 40 μg of these proteins, separately, increased immune effectors in the hemolymph of Galleriamellonella larvae and protected animals from an S.schenckii lethal challenge. These observations were confirmed when yeast-like cells, pre-incubated with anti-rHsp60 or anti-rPap1 antibodies were used to inoculate larvae. The animals inoculated with pretreated cells showed increased survival rates when compared to the control groups. In conclusion, we report that Hsp60 and Pap1 are part of the cell wall peptidorhamnomannan, can bind extracellular matrix components, and contribute to the S.schenckii virulence. To our knowledge, this is the first report about moonlighting protein in the S.schenckii cell wall with an important role during the pathogen–host interaction.

Current Models to Study the Sporothrix-Host Interaction

“…In general terms, in an experimental model of systemic sporotrichosis, mice can be inoculated intraperitoneally or intravenously either in the tail vein or in the retro-orbital plexus (Tachibana et al, 1998;Fernandes et al, 1999). For a disseminated Tachibana et al, 1998;Fernandes et al, 1999;Nascimento and Almeida, 2005;Kong et al, 2006;Brito et al, 2007;Arrillaga-Moncrieff et al, 2009;Ruiz-Baca et al, 2009;Teixeira et al, 2009Teixeira et al, , 2010Almeida-Paes et al, 2012Castro et al, 2013;Clavijo-Giraldo et al, 2016;Martinez-Alvarez et al, 2019;García-Carnero et al, 2021;Song et al, 2021 Host innate response Shiraishi et al, 1992;Sassá et al, 2009;Guzman-Beltran et al, 2012;Guzman Beltrán et al, 2021;Romo-Lozano et al, 2012Sandig and Bulfone-Paus, 2012;Negrini Tde et al, 2013;de Almeida et al, 2017de Almeida et al, , 2018Martinez-Alvarez et al, 2017Lozoya-Perez et al, 2018Lozoya-Pérez et al, 2020;Lozoya-Perez et al, 2021;Rossato et al, 2019;Huang et al, 2021;Tamez-Castrellón et al, 202...…”

“…Clavijo-Giraldo et al, 2016;Jellmayer et al, 2017;Lozoya-Perez et al, 2018Lozoya-Pérez et al, 2020;Lozoya-Perez et al, 2021;Manente et al, 2018;Rossato et al, 2019;García-Carnero et al, 2021;Tamez-Castrellón et al, 2021 Subcutaneous infection X XTachibana et al, 1999;Kong et al, 2006;Brito et al, 2007;Arrillaga-Moncrieff et al, 2009;Castro et al, 2013;Fernandes et al, 2013;Della Terra et al, 2017;de Almeida et al, 2018 …”

Virulence Factors of Sporothrix schenckii

“…Another element of the S. schenckii cell wall is the peptidorhamnomannan (PRM), a glycoconjugate composed of mannose (57%), rhamnose (33.5%), proteins (14.2%), and galactose (1%) [ 35 ]. Although the PRM has been identified as one of the main antigenic components of the cell wall and, despite its carbohydrate moiety, has been widely studied [ 35 , 36 , 37 , 38 , 39 ], the proteins that compose it were only recently identified [ 40 ]. By mass spectrometry, it was found that this complex is composed of 325 proteins, most of which lack signal peptide and, therefore, could be moonlight proteins transported to the cell surface by non-canonical secretory pathways [ 40 , 41 ].…”

“…Although the PRM has been identified as one of the main antigenic components of the cell wall and, despite its carbohydrate moiety, has been widely studied [ 35 , 36 , 37 , 38 , 39 ], the proteins that compose it were only recently identified [ 40 ]. By mass spectrometry, it was found that this complex is composed of 325 proteins, most of which lack signal peptide and, therefore, could be moonlight proteins transported to the cell surface by non-canonical secretory pathways [ 40 , 41 ]. Two of these PRM proteins, Hsp60 and Pap1, were identified as adhesins that bind to different ECM proteins in the host cells [ 40 ].…”

“…By mass spectrometry, it was found that this complex is composed of 325 proteins, most of which lack signal peptide and, therefore, could be moonlight proteins transported to the cell surface by non-canonical secretory pathways [ 40 , 41 ]. Two of these PRM proteins, Hsp60 and Pap1, were identified as adhesins that bind to different ECM proteins in the host cells [ 40 ]. Hsp60 is a chaperone highly conserved in all living organisms and upregulated in response to stress conditions, playing an important role in the cell housekeeping functions [ 42 ].…”