Paracoccin Overexpression in Paracoccidioides brasiliensis Enhances Fungal Virulence by Remodeling Chitin Properties of the Cell Wall

Abstract

Background The thermo-dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis. Although poorly studied, paracoccin (PCN) from P. brasiliensis has been shown to harbor lectinic, enzymatic, and immunomodulatory properties that impact disease development. Methods Mutants of P. brasiliensis overexpressing PCN (ov-PCN) were constructed by Agrobacterium tumefaciens-mediated transformation. Ov-PCN strains were analyzed and inoculated intranasally or intravenously to mice. Fungal burden, lung pathology, and survival were monitored to evaluate virulence. Electron microscopy was used to evaluate the size of chito-oligomer particles released by ov-PCN or wild-type strains to growth media. Results ov-PCN strains revealed no differences in cell growth and viability, although PCN overexpression favored cell separation, chitin processing that results in the release of smaller chito-oligomer particles, and enhanced virulence. Our data show that PCN triggers a critical effect in the cell wall biogenesis through the chitinase activity resulting from overexpression of PCN. As such, PCN overexpression aggravates the disease caused by P. brasiliensis. Conclusions Our data is consistent with a model in which PCN modulates the cell wall architecture via its chitinase activity. These findings highlight the potential for exploiting PCN function in future therapeutic approaches.

Virulence Vs. Immunomodulation: Roles of the Paracoccin Chitinase and Carbohydrate-Binding Sites in Paracoccidioides brasiliensis Infection

“…The contrasting disease patterns produced by kd-PCN and ov-PCN yeast are consistent with the verified in vitro features of the transformed yeast, in the sense that kd-PCN yeast are susceptible (Fernandes et al, 2017) and ov-PCN yeasts are resistant to the fungicidal activity of phagocytes (Gonçales et al, 2021).…”

“…These observations lead us to consider PCN as just an immunomodulatory agent that favors host resistance to P. brasiliensis infection (Ricci-Azevedo et al, 2017). Therefore, the results obtained with mice infected with kd-PCN and ov-PCN yeast (Fernandes et al, 2017;Gonçales et al, 2021), seemed to conflict with our assumptions. We expected that ov-PCN yeast would cause an auto-resolving illness due to their high content of an agent able to drive host immunity toward a Th1 response.…”

“…The difficulties known to be inherent to P. brasiliensis molecular manipulation were overcome with the indispensable collaboration of Dr. Fernando Rodrigues (School of Health Sciences, University of Minho, Braga, Portugal) using the technique based on Agrobacterium tumefaciens mediated transformation (ATMT). We successfully modified the gene PADG_03,347 (Gene ID: 22582669) and generated PCN knocked-down (kd-PCN) ( Fernandes et al, 2017 ) and PCN overexpressing (ov-PCN) ( Gonçales et al, 2021 ) yeast. Levels of PCN mRNA expression were reduced 60% in kd-PCN, and augmented 7-fold in ov-PCN compared to wt-yeast.…”

“…The particles isolated from wt-PCN varied widely in size and ranged up to 240 nm 2 , while those from ov-PCN were smaller and had a narrower size distribution ranging up to 60 nm 2 . PCN overexpression, associated with increased chitinase-activity, leads to greater hydrolysis of the yeast's cell wall chitin into smaller chito-oligomers, a reduced β-1,4-GlcNAc content, and diminished cell wall thickness ( Gonçales et al, 2021 ). These alterations culminate in altering the cell wall architecture.…”

“…Soluble recombinant PCN, administered to P. brasiliensis infected hosts, acts as an immunomodulatory agent that favors protection against the fungus ( Alegre-Maller et al, 2014 ; Alegre et al, 2014 ). In contrast, endogenous PCN, expressed by the infecting fungus itself, acts as a P. brasiliensis virulence factor ( Fernandes et al, 2017 ; Gonçales et al, 2021 ) that aggravates the infection in a murine model. The first P. brasiliensis protein explored concerning this functional duality in the host-pathogen relationship was a surface glycoprotein of 43 kDa, known as gp43, which acts as a P. brasiliensis adhesin that binds laminin and fibronectin ( Vicentini et al, 1994 ).…”