30Paracoccidioides brasiliensis and P. lutzii, etiological agents of 31 paracoccidioidomycosis (PCM), develop as mycelia at 25-30 °C and as yeast at 35-37 °C. Only 32 a few Paracoccidioides spp. proteins are well characterized. Thus, we studied paracoccin 33 (PCN) from P. brasiliensis, its role in the fungus biology, and its relationship with the host 34 innate immune cells. Cloning and heterologous expression analysis revealed its lectin, 35 enzymatic, and immunomodulatory properties. Recently, we employed a system based on 36 Agrobacterium tumefaciens-mediated transformation to manipulate P. brasiliensis yeast genes 37 to obtain clones knocked-down for PCN, which after all, are unable to transit from yeast to 38 mycelium forms, causing a mild pulmonary disease. Herein, we generate P. brasiliensis 39 overexpressing PCN (ov-PCN). To date, it was not explored the overexpressing of endogenous 40 components in Paracoccidioides spp. Therefore, we investigate the role of PCN in fungal 41 biology and pathogenesis. Augmented levels of PCN mRNA and protein, and N-42 acetylglucosaminidase activity confirmed PCN overexpression in ov-PCN of P. brasiliensis 43 yeasts. Interestingly, PCN overexpression did not affect the yeasts' growth or viability and 44 favored cell separation. The ov-PCN clones transitioned faster to the mycelium form than the 45 wt-PCN yeasts. Concerning infection, while most of mice infected with the wt-yeasts (90%) 46 survive at least until the 70 th day, all mice infected with ov-PCN yeasts were already died at the 47 35 th day post-infection. In vitro assays showed that ov-PCN were more susceptible to 48 phagocytosis by macrophages. Finally, it was verified that the chitin particles isolated from the 49 ov-PCN cells were smaller than those obtained from the wt-PCN yeasts. Macrophages 50 stimulated with the chitin isolated from ov-PCN produce IL-10, whereas the particles with a 51 wider size range harvested from wt-PCN yeasts induced TNF-α and IL-1β secretion. The anti-52 inflammatory microenvironment from macrophage stimulation with small chitin particles 53 hampers the development of a protective immune response against the fungus. We postulated 3 54 that the high grade of chitin cleavage, as the results of augmented PCN expression, favors 55 pathogenesis following P. brasiliensis infection. Thus, PCN is a relevant virulence fungal 56 factor. 57 58 AUTHOR SUMMARY 59 Paracoccidioides spp. are pathogenic fungi that cause paracoccidioidomycosis (PCM) 60 in humans, the main deep mycosis of Latin America. Recently, by knocking down the 61 paracoccin gene, our group showed that this lectin is necessary for the morphological transition 62 from yeast to hyphae, and that this decrease results in low P. brasiliensis virulence. Here, after 63 overexpress PCN, we revealed the importance of the yeast chitin hydrolysis to the host 64 response. Infection of mice with ov-PCN yeasts causes severe lung disease compared to 65 moderate disease caused by wt-PCN yeasts. The release of smaller chitin particles ...