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

Date Published: December 4, 2014

Publisher: Public Library of Science

Author(s): Ana Claudia Paiva Alegre-Maller, Flávia Costa Mendonça, Thiago Aparecido da Silva, Aline Ferreira Oliveira, Mateus Silveira Freitas, Ebert Seixas Hanna, Igor C. Almeida, Nicholas J. Gay, Maria Cristina Roque-Barreira, Joseph M. Vinetz.

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.

Partial Text: Paracoccidioidomycosis (PCM), first reported by Adolf Lutz in 1908 in Brazil, is an acute-chronic systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis. PCM is autochthonous to Latin America, and its incidence extends from southern Mexico to northern Argentina [1]. Infection is initiated by the inhalation of airborne propagules, derived from conidia, which transform into pathogenic yeast in the lung [2].

Dramatic increases in the incidence of human fungal diseases worldwide as well as the toxicity and limited efficacy of anti-fungal drugs, especially without the help of host immune reactivity, require the development of new strategies for confronting fungal infections. Immunomodulation strategies are thought to hold promise. Their design entails a deep understanding of fungal interaction with various innate immune receptors [13], a necessity that became more obvious by the finding that an anti-fungal agent (amphotericin B) needs TLRs for efficacy [25]. Some exogenous lectins exert immunomodulatory effects in mammals by interacting with host-cell glycans [26]–[28], a fact that opens new perspectives in the design of strategies to control infectious diseases. ArtinM is the most studied immunomodulatory exogenous substance acting through carbohydrate recognition on cells of innate [23], [29]–[40] and adaptive [41] cells. Its effects favor host resistance against diseases caused by several pathogens [31], [36], [42], [43], including P. brasiliensis[14], [34], [44]. Nonetheless, as a plant lectin, ArtinM itself has restricted application in the prophylaxis or therapy of human diseases. On the other hand, paracoccin, as a lectin constituent of P. brasiliensis, the causal agent of PCM, could be potentially used for therapy against this endemic fungal disease in Latin America. This perspective has motivated us to characterize PCN effects in the course of PCM and elucidate the mechanisms that account for its property of inducing host Th1 immunity. This was discovered by administering PCN prophylactically to mice prior to be infected with P. brasiliensis yeasts. Remarkably, they became resistant to the infection, a fact that was attributed to the augmented pulmonary levels of pro-inflammatory mediators [20]. PCN was shown herein to be efficient also as a therapy against the ongoing mycosis, and the observed Th1 immunity was attributed to PCN interaction with N-glycans of TLR2 and TLR4.



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