Therapeutic Administration of Recombinant Paracoccin Confers Protection against Paracoccidioides brasiliensis Infection: Involvement of TLRs

Abstract

BackgroundParacoccin (PCN) is an N-acetylglucosamine-binding lectin from the human pathogenic fungus Paracoccidioides brasiliensis. Recombinant PCN (rPCN) induces a T helper (Th) 1 immune response when prophylactically administered to BALB/c mice, protecting them against subsequent challenge with P. brasiliensis. In this study, we investigated the therapeutic effect of rPCN in experimental paracoccidioidomycosis (PCM) and the mechanism accounting for its beneficial action.Methodology/Principal FindingsFour distinct regimens of rPCN administration were assayed to identify which was the most protective, relative to vehicle administration. In all rPCN-treated mice, pulmonary granulomas were less numerous and more compact. Moreover, fewer colony-forming units were recovered from the lungs of rPCN-treated mice. Although all therapeutic regimens of rPCN were protective, maximal efficacy was obtained with two subcutaneous injections of 0.5 µg rPCN at 3 and 10 days after infection. The rPCN treatment was also associated with higher pulmonary levels of IL-12, IFN-γ, TNF-α, nitric oxide (NO), and IL-10, without IL-4 augmentation. Encouraged by the pulmonary cytokine profile of treated mice and by the fact that in vitro rPCN-stimulated macrophages released high levels of IL-12, we investigated the interaction of rPCN with Toll-like receptors (TLRs). Using a reporter assay in transfected HEK293T cells, we verified that rPCN activated TLR2 and TLR4. The activation occurred independently of TLR2 heterodimerization with TLR1 or TLR6 and did not require the presence of the CD14 or CD36 co-receptors. The interaction between rPCN and TLR2 depended on carbohydrate recognition because it was affected by mutation of the receptor's N-glycosylation sites. The fourth TLR2 N-glycan was especially critical for the rPCN-TLR2 interaction.Conclusions/SignificanceBased on our results, we propose that PCN acts as a TLR agonist. PCN binds to N-glycans on TLRs, triggers regulated Th1 immunity, and exerts a therapeutic effect against P. brasiliensis infection.

Impact of Paracoccin Gene Silencing on Paracoccidioides brasiliensis Virulence

“…Several studies have shown that P. brasiliensis genes may be associated with fungal virulence (21–25) and thereby influence the host immune response (18–20, 26–31). In addition to participating in fungal morphogenesis, an important role was recently attributed to PCN in P. brasiliensis infection (15, 16).…”

Paracoccin overexpression in Paracoccidioides brasiliensis reveals the influence of chitin hydrolysis on fungal virulence and host immune response

“…The gene (annotated as PADG_03347) codes for PCN, whose previous cloning and heterologous expression allowed the identification of this multidomain protein that exerts biological activities of lectin and binds to GlcNAc and chitinase [19], which hydrolyzes chitin [20]. It also acts as an immunomodulatory agent [44, 45]. Yeast transformation was mediated by ATMT [23, 46], a system reported to be successful when employed to obtain P. brasiliensis knocked-down clones for proteins playing relevant roles in the fungal virulence or pathogenesis, e.g., CDC42 [17], PbHAD32 [24], asSconC [16], PbGP43 [26], PbP27 [27], Pb14-3-3 [28], and PbSOD3 [29].…”

“…Our interest on the PCN gene manipulation comes from demonstrations that the subcutaneous administration of recombinant PCN (rPCN) to infected mice with P. brasiliensis promotes modulation of the host immune response and confers protection against the fungal disease [44, 45]. The response is triggered by the PCN lectin domain interaction with N-glycans of TLR2 and TLR4 [45, 47]. Both receptors are expressed on the surface of macrophages, which undergo M1 polarization followed by high production of pro-inflammatory mediators, such as the Th1 polarizing cytokine IL-12 [48].…”

“…Both receptors are expressed on the surface of macrophages, which undergo M1 polarization followed by high production of pro-inflammatory mediators, such as the Th1 polarizing cytokine IL-12 [48]. We demonstrated that the developed Th1 immune response accounts for the host resistance to the P. brasiliensis infection conferred by the PCN administration [45]. A subsequent study, focused on PCN distribution in P. brasiliensis , revealed its association with chitin and prominent localization in structures related to fungal growth, such as hyphae tips and budding regions of yeast cell wall [32].…”

Lectins from Synadenium carinatum (ScLL) and Artocarpus heterophyllus (ArtinM) Are Able to Induce Beneficial Immunomodulatory Effects in a Murine Model for Treatment of Toxoplasma gondii Infection

“…Thus, the original use of lectins in vaccination protocols has been expanded to other applications. Hence, besides its use as therapeutic agents in infectious and parasitic diseases, lectins have been applied as new potential antineoplastic agents targeting apoptosis, autophagy, and anti-angiogenesis in preclinical or clinical trials for cancer therapeutics, and in neurodegenerative disorders, as well (Alegre-Maller et al, 2014; Bogoeva et al, 2014; Aminou et al, 2016; Barbosa-Lorenzi et al, 2016; Dar et al, 2016; Freitas et al, 2016; Ricci-Azevedo et al, 2016). …”