Ação da fosfolipase B extracelular de Paracoccidioides brasiliensis na interação ex vivo com macrófagos alveolares
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Data
2010-03-26
Autores
SOARES, Deyze Alencar
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Editor
Universidade Federal de Goiás
Resumo
Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the most
prevalent systemic mycosis in Latin America, paracoccidioidomycosis. The phospholipase B
(PLB) enzyme is considered an important virulence factor in this dimorphic fungus, involved
in the immune response of the host-pathogen interaction. Our objective was to determine
whether a P. brasiliensis (Pb18) PLB is involved in adhesion / internalization of yeast and
evasion of host immune responses. The effect of PLB was analysed using specific inhibition
of PLB (alexidine dihydrochloride) and pulmonary surfactant in an ex vivo model (Pb18) of
alveolar macrophage (MHS cells) infection. PLB enzyme assays and real time RT-PCR (qRTPCR)
analysis of genes differentially expressed in the process of evasion: plb1 (phospholipase
B1), icl1 (isocitrate lyase) and sod3 (Cu, Zn dismutase) and immune responses: clec2 (C-type
lectin domain 2), cd14 (cluster of differentiation 14), tlr2 (toll-like receptor 2), nfkb (nuclear
factor kappa B), nkrf (NF-kappaB repressing factor), il1β (inteleukin-1β) and tnfα (tumor
necrosis factor alpha) were carried out using selective inhibition of PLB activity and
pulmonary surfactant. The levels of cytokines inteleukin 10 (IL-10), IL-12 and TNF-α) were
also determined by ELISA. PLB activity under adhesion conditions of P. brasiliensis (Pb18)
to alveolar macrophage cells was found at high levels up to 6 hours post-infection. In the
conditions of exposure to pulmonary surfactant and alexidine dihydrochloride, PLB activity
and the level of transcripts of genes related to phagocytosis and inflammatory response were
measured. We found that PLB activity had an influence on the phagocytic activity of alveolar
macrophages. Alexidine dihydrochloride (0,25 μM) selectively inhibited PLB activity by 66%
and decreased significantly the adhesion and internalization of yeast on MHS cells. Genes
involved in phagocytosis (trl2 and cd14) and inflammatory response (nrkf, tnfα and il1β) were
down-regulated in the presence of the PLB inhibitor. In contrast, the PLB activity and
internalization of fungal yeast cells increased significantly in the presence of pulmonary
surfactant (100 μg/mL) and genes such as clec2, important for effective phagocytosis by MHS
cells, and the pro-inflammatory inhibitor (nkrf) were up-regulated. Also, the pulmonary
surfactant did not alter cytokine production, while alexidine dihydrochloride decreased the
levels of IL-10 and increased the levels of IL-12 and TNF-α. In addition, through
simultaneous analyses of gene expression for the pathogen, P. brasiliensis, we found upregulation
of the genes sod3, icl1 and plb1, required for the evasion of alveolar macrophages.
P. brasiliensis PLB is important for the binding and internalization of yeast at macrophage
surfaces. The specific effect of inhibiting PLB enzyme activity indicates that adhesion may be
facilitated indirectly via fatty acid release from phospholipids of the membrane of host cells.
This is the first study to show that PLB activity may modulate immune responses to P.
brasiliensis infection.
Descrição
Palavras-chave
1. Interação patógeno-hospedeiro , 2. Surfactante
pulmonar , 2. Alexidine dihydrochloride , 4. Paracoccidioides brasiliensis , 1. Host-pathogen interaction , 2. Pulmonary surfactant , 3. Alexidine dihydrochloride , 4. Paracoccidioides brasiliensis , 1. Paracoccidioides brasiliensis;
2. Surfactante pulmonar;
3. Alexidine Dihydrochloride
Citação
SOARES, Deyze Alencar. Action of extracellular phospholipase B of Paracoccidioides brasiliensis interaction with alveolar macrophage ex vivo. 2010. 89 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2010.