Extracellular Paracoccidioides brasiliensis phospholipase B involvement in alveolar macrophage interaction

Abstract

Background: Phospholipase B (PLB) has been reported to be one of the virulence factors for human pathogenic fungi and has also been described as necessary for the early events in infection. Based on these data, we investigated the role of PLB in virulence and modulation of the alveolar pulmonary immune response during infection using an in-vitro model of host-pathogen interaction, i.e. Paracoccidioides brasiliensis yeast cells infecting alveolar macrophage (MH-S) cells.

Results: The effect of PLB was analyzed using the specific inhibitor alexidine dihydrochloride (0.25 μM), and pulmonary surfactant (100 μg mL-1), during 6 hours of co-cultivation of P. brasiliensis and MH-S cells. Alexidine dihydrochloride inhibited PLB activity by 66% and significantly decreased the adhesion and internalization of yeast cells by MH-S cells. Genes involved in phagocytosis (trl2, cd14) and the inflammatory response (nfkb, tnf-α, il-1β) were down-regulated in the presence of this PLB inhibitor. In contrast, PLB activity and internalization of yeast cells significantly increased in the presence of pulmonary surfactant; under this condition, genes such as clec2 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 interleukin-10 (IL-10) and increased the levels of IL-12 and tumor necrosis factor-α (TNF-α). In addition, gene expression analysis of plb1, sod3 and icl1 suggests that P. brasiliensis gene re-programming is effective in facilitating adaptation to this inhospitable environment, which mimics the lung-environment interaction.

Conclusion: P. brasiliensis PLB activity is involved in the process of adhesion and internalization of yeast cells at the MH-S cell surface and may enhance virulence and subsequent down-regulation of macrophage activation.

Figure 1

Paracoccidioides brasiliensis isolate Pb18 yeast cell viability and infection index after co-culture with alveolar macrophage (MH-S) cells. (A) CFU of P. brasiliensis isolate Pb18 yeast cells; (B) Infection index of in-vitro MH-S cells in the presence of alexidine dihydrochloride (0.25 μM) and pulmonary surfactant (100 μg.mL^-1). Percentage of MH-S cells infected with P. brasiliensis yeast cells - adhered (black bar) or internalized (white bar). In all experiments, MH-S cells and opsonized yeast cells were incubated at a yeast-to-macrophage ratio of 1:5, at 37°C in an atmosphere of 5% CO₂ as described in the Materials and Methods. Data shown are derived from two in-vitro independent experiments performed in triplicate (mean ± SEM, with *significance assumed in the range of P < 0:05); ns = non-significantly (P < 0.05); **Significantly different from the untreated control P < 0.001 by the paired 2-tailed Student's t-test.

Figure 2

Real-Time RT-PCR. Analysis of the transcript level of Paracoccidioides brasiliensis genes related to oxidative stress - superoxide dismutase (sod3); metabolism - isocitrate lyase (icl1) and hydrolytic enzyme phospholipase B (plb1). The assay was carried out in triplicate (mean ± SEM); Significantly different from controls: (*P < 0:05 and **P < 0:001) by the paired 2-tailed Student's t-test.

Figure 3

Real-Time RT-PCR. Analysis of the transcript level of macrophage genes related to phagocytosis (clec2, trl2, and cd14) and inflammation (nkrf, nfkb, tnf-α, and il-1β). The assay was carried out in triplicate (mean ± SEM, with *significance assumed in the range of P < 0:05); **Significantly different from controls: P < 0.001 by the paired 2-tailed Student's t-test.

Figure 4

Amount of cytokines and tumor necrosis factor-α released by alveolar macrophage (MH-S) cells infected with Paracoccidioides brasiliensis. The assay was carried out in triplicate (mean ± SEM); ns = non-significantly and *significantly different from controls: P < 0.05 by the paired 2-tailed Student's t-test.

Figure 5

Model of expression differential genes in presence of the surfactant and alexidine, respectively. The small arrows indicate induced (↑) and repressed (↓) genes. Paracoccidioides brasiliensis survival in macrophage phagosome and burst oxidative: plb1, icl1, and sod3. Macrophage genes: clec2, trl2, cd14, nfkb, nkrf, tnf-α, and il-1β.