Paracoccin is an N-acetyl-glucosamine-binding lectin from Paracoccidioides brasiliensis, which can be obtained in small amounts either from culture supernatants or yeast cell extracts. In the present work, immunoelectron microscopy with mouse anti-paracoccin IgG localized the antigen to the cell wall of P. brasiliensis yeast forms. Paracoccin interacted with chitin, and colocalized with beta-1,4-homopolymer of GlcNAc to the budding sites of P. brasiliensis yeast cell. In order to evaluate the role of paracoccin on fungal growth, yeast cells were cultivated in the presence of anti-paracoccin antibodies. A significant reduction of both colony forming units and individual yeast cells was observed as well as morphological alterations such as smaller colonies and cells more loosely aggregated than in control cultures without the antibody. A role of paracoccin on the cell wall organization was reinforced by alterations in the labeling pattern of chitin when yeasts were treated with anti-paracoccin antibodies. Binding of specific antibodies to paracoccin may disrupt the paracoccin/chitin interactions, resulting in the inhibition of P. brasiliensis growth.
"Biotinylated anti-paracoccin was dialyzed against water in a Centricon system YM-50 (Millipore CentriconH), and stored at 220uC. The specificity of the anti-paracoccin antibody has previously been demonstrated by Ganiko et al, 2007. Briefly, anti-paracoccin antibodies were adsorbed in paracoccin (500 ng/dot) immobilized on nitrocellulose membrane. "
[Show abstract][Hide abstract] ABSTRACT: The fungus Paracoccidioides brasiliensis is a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The cell wall of P. brasiliensis is a network of glycoproteins and polysaccharides, such as chitin, that perform several functions. N-linked glycans are involved in glycoprotein folding, intracellular transport, secretion, and protection from proteolytic degradation. Here, we report the effects of tunicamycin (TM)-mediated inhibition of N-linked glycosylation on P. brasiliensis yeast cells. The underglycosylated yeasts were smaller than their fully glycosylated counterparts and exhibited a drastic reduction of cell budding, reflecting impairment of growth and morphogenesis by TM treatment. The intracellular distribution in TM-treated yeasts of the P. brasiliensis glycoprotein paracoccin was investigated using highly specific antibodies. Paracoccin was observed to accumulate at intracellular locations, far from the yeast wall. Paracoccin derived from TM-treated yeasts retained the ability to bind to laminin despite their underglycosylation. As paracoccin has N-acetyl-β-d-glucosaminidase (NAGase) activity and induces the production of TNF-α and nitric oxide (NO) by macrophages, we compared these properties between glycosylated and underglycosylated yeast proteins. Paracoccin demonstrated lower NAGase activity when underglycosylated, although no difference was detected between the pH and temperature optimums of the two forms. Murine macrophages stimulated with underglycosylated yeast proteins produced significantly lower levels of TNF-α and NO. Taken together, the impaired growth and morphogenesis of tunicamycin-treated yeasts and the decreased biological activities of underglycosylated fungal components suggest that N-glycans play important roles in P. brasiliensis yeast biology.
PLoS ONE 12/2011; 6(12):e29216. DOI:10.1371/journal.pone.0029216 · 3.23 Impact Factor
"Host cell exposure to recombinant enolase had enhanced fungal adhesion, possibly due to an increased exposure of N -acetylglucosamine, which might be recognized by paracoccin, a TNF-α stimulatory lectin (Coltri et al., 2006). Paracoccin is a cell wall chitin-binding protein with N acetyl-β-d-glucosaminidase activity that plays a role in fungal growth, supposedly by participating in cell wall remodeling and organization (Ganiko et al., 2007; dos Reis Almeida et al., 2010). Secreted gp43 is a major diagnostic P. brasiliensis antigen (Puccia and Travassos, 1991) that partly resides on the cell wall (Straus et al., 1996). "
[Show abstract][Hide abstract] ABSTRACT: The cell wall of pathogenic fungi plays import roles in the interaction with the host, so that its composition and structure may determine the course of infection. Here we present an overview of the current and past knowledge on the cell wall constituents of Paracoccidioides brasiliensis and P. lutzii. These are temperature-dependent dimorphic fungi that cause paracoccidioidomycosis, a systemic granulomatous, and debilitating disease. Focus is given on cell wall carbohydrate and protein contents, their immune-stimulatory features, adhesion properties, drug target characteristics, and morphological phase specificity. We offer a journey toward the future understanding of the dynamic nature of the cell wall and of the changes that may occur when the fungus infects the human host.
Frontiers in Microbiology 12/2011; 2:257. DOI:10.3389/fmicb.2011.00257 · 3.99 Impact Factor
"We have previously demonstrated that P.brasiliensis yeasts release and express a GlcNAc-binding lectin designated paracoccin on their surface , . Besides interacting in a sugar-recognition dependent manner with laminin, paracoccin stimulates macrophages to produce high levels of TNF-α and nitric oxide, which are involved in the fungicidal activity of these cells. "
[Show abstract][Hide abstract] ABSTRACT: This study aimed to investigate the immunological mechanisms involved in the gender distinct incidence of paracoccidioidomycosis (pcm), an endemic systemic mycosis in Latin America, which is at least 10 times more frequent in men than in women. Then, we compared the immune response of male and female mice to Paracoccidioides brasiliensis infection, as well as the influence in the gender differences exerted by paracoccin, a P. brasiliensis component with carbohydrate recognition property. High production of Th1 cytokines and T-bet expression have been detected in the paracoccin stimulated cultures of spleen cells from infected female mice. In contrast, in similar experimental conditions, cells from infected males produced higher levels of the Th2 cytokines and expressed GATA-3. Macrophages from male and female mice when stimulated with paracoccin displayed similar phagocytic capability, while fungicidal activity was two times more efficiently performed by macrophages from female mice, a fact that was associated with 50% higher levels of nitric oxide production. In order to evaluate the role of sexual hormones in the observed gender distinction, we have utilized mice that have been submitted to gonadectomy followed by inverse hormonal reconstitution. Spleen cells derived from castrated males reconstituted with estradiol have produced higher levels of IFN-gamma (1291+/-15 pg/mL) and lower levels of IL-10 (494+/-38 pg/mL), than normal male in response to paracoccin stimulus. In contrast, spleen cells from castrated female mice that had been treated with testosterone produced more IL-10 (1284+/-36 pg/mL) and less IFN-gamma (587+/-14 pg/mL) than cells from normal female. In conclusion, our results reveal that the sexual hormones had a profound effect on the biology of immune cells, and estradiol favours protective responses to P. brasiliensis infection. In addition, fungal components, such as paracoccin, may provide additional support to the gender dimorphic immunity that marks P. brasiliensis infection.
PLoS ONE 05/2010; 5(5):e10757. DOI:10.1371/journal.pone.0010757 · 3.23 Impact Factor
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