Paracoccin distribution supports its role in Paracoccidioides brasiliensis growth and dimorphic transformation

Abstract

Paracoccidioides brasiliensis yeast was reported to express paracoccin, a GlcNAc-binding protein that displays N-acetyl-β-d-glucosaminidase (NAGase) activity. Highly specific anti-paracoccin antibodies have been previously used to examine the localization of paracoccin in yeast and inhibit its growth in vitro. In the present study, anti-paracoccin antibodies were used to characterize, by scanning confocal microscopy, the distribution of paracoccin in P. brasiliensis hyphae, transition forms from hyphae to yeast, and mature yeast. In the mycelial phase, paracoccin was detected mainly in the hyphae tips, where it demonstrated a punctate distribution, and was associated with the cell wall. During the first 48 hours after a temperature shift from 26°C to 37°C, paracoccin expression in the differentiating hyphae was mainly detected in the budding regions, i.e. lateral protrusions, and inside the new daughter cells. There was an increased number of chlamydoconidia that expressed a high concentration of paracoccin on their surfaces and/or in their interiors 72–96 hours after the temperature shift. After 120 hours, yeast cells were the predominant form and their cytoplasm stained extensively for paracoccin, whereas Wheat Germ Agglutinin (WGA) staining was predominant on their exterior walls. After 10 days at 37°C, the interior of both mother and daughter yeast cells, as well as the budding regions, stained intensely for paracoccin. The comparison of mRNA-expression in the different fungal forms showed that PCN transcripts, although detected in all evaluated morphological forms, were higher in hypha and yeast-to-hypha transition forms. In conclusion, the pattern of paracoccin distribution in all P. brasiliensis morphotypes supports prevalent beliefs that it plays important roles in fungal growth and dimorphic transformation.

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

“…These results show a faster ov-PCN yeast transition compared to wt-PCN or EV. Our data supports the critical effect of PCN in the morphological transition from yeast to mycelium and cell wall biogenesis of P. brasiliensis [21, 22, 32].…”

“…In a recent study, we compared the PCN transcripts in different morphotypes of P. brasiliensis. Although detected in all fungal forms, PCN mRNA expression was high in hypha and yeast-to-hypha transition forms [32]. Herein, we investigated PCN expression in yeasts for 24 h during THP-1 human macrophages infection.…”

“…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]. The relevant biological activities and peculiar localization of PCN motivated us to investigate the role the endogenous PCN could play in fungal biology, as well as its effect on the host immune response.…”

Influência de galectina-3 no curso da infecção por Cryptococcus neoformans

“…C. neoformans yeast cells (H99 strain) were cultured at 37°C for 24 h. The cells were incubated with Gal-3 (40 μg/ml) at 37°C for 1 h, followed by washing with PBS and fixation with PBS-buffered 3.7% formaldehyde at 25°C for 1 h. After fixation, cells were washed with PBS and embedded in OCT-Tissue Tek (Electron Microscopy Sciences, Hatfield, PA, USA). The samples were frozen and processed as described previously 49 . Slides were rinsed three times with PBS before and after treatment with glycine (0.1 M, 15 min), and blocked with BSA (1% in PBS) for 1 h. Samples were incubated overnight at 4°C with murine monoclonal antibody 18B7 (IgG1, 1 μg/ml), which binds cryptococcal glucuronoxylomannan (GXM) from all serotypes 50 .…”

Paracoccina: uma quitinase importante para a patobiologia e virulência de Paracoccidioides brasiliensis

“…Oliveira, Fernandes et al 2017), o que sugeriu seu envolvimento em processos de transição morfológica em P. brasiliensis.Paracoccina recombinante (rPCN), expressa em Escherichia coli, teve sua qualidade demonstrada pela reprodução das atividades biológicas conhecidas da proteína nativa. A disponibilidade da proteína recombinante permitiu avaliar o efeito da administração profilática ou terapêutica de paracoccina em camundongos (Alegre-Maller, Mendonca et al 2014; Alegre, Oliveira et al 2014).…”