The Effect Of L-DOPA On Melanin Production Of Penicillium Marneffei, Drug Susceptibility And Preparation Of Antibody Against The Melanin

Objective The research is to investigate the effect of L-DOPA onmelanin production of Penicillium marneffei (PM), drug susceptibility ofantifungal agents against PM and the antigenicity of PM melanin.Methods PM strain of human clinical isolates GXMU121011wascultured on Sabouraud (SDA) at37℃for7days, the effects of differentconcentrations of L-DOPA (0.1mM,1mM,3mM,6mM,10mM) anddifferent PM inoculation density (105CFU/ml,106CFU/ml,107CFU/ml)on PM melanin synthesis were observed. The inhibition zones ofitraconazole(ITC), fluconazole(FLU) and amphotericin B(AMB) againsteight PM strains with L-DOPA culture or without L-DOPA culture weremeasured test in vitro. The monoclonal antibody against PM melanin wasprepared by immunized BALB/c mice through intraperitoneal injectionfollowed with the spleen cells fusion with the myeloma cells by PEGmethods. The antibodies against PM melanin were detected byagglutination test, ELISA method and immunofluorescence method. Inaddition, the serum antibodies from the patients of penicilosis were detected by agglutination test.Results (1)①When PM strain culture on SDA plates supplementedwith different concentration of L-DOPA at37℃for7days, black pigmentproduction was observed in the colonies or around colonies in varyingdegrees;②Black pigment increased when L-DOPA concentrationincreased from0.1mM to1mM, the color of colonies showed most darklyat the L-DOPA concentration of1mM or3mM, but the pigment ofcolonies decreased with the concentration of L-DOPA increased. Thecolonies were shrinkage at the L-DOPA concentration of10mM.(2) Thecolonies of PM showed pigment production in varies inoculation densitywith L-DOPA culture on SDA plate at37℃for7days. The dark pigmentproduction of colonies increased parallel to inoculation density.(3) Theinhibition zones of eight PM strains were measured after48h to96hincubation on SDA with L-DOPA or without L-DOPA. The averageinhibition zones of ITC, AMB and FLU against PM were as follows: ITC,26.88±1.81mm (control group,34.13±1.81mm)；AMB,17.13±2.59mm(control group,22.25±4.80mm); FLU,19.50±6.46mm (control group,25.87±7.34mm). There were significant differences between L-DOPAculture and no L-DOPA culture (P<0.05).(4)①The mice suffered fromdifferent degrees of ascites and hair loss after four weeks immunizationwith PM melanin by intraperitoneal injection;②The ascites, mice serumand supernatant of myeloma fusion cells had agglutination reaction withPM melanin granules and synthetic DOPA melanin. ELISA showed TheOD values of antibodies from ascites, mice serum and supernatant ofmyeloma fusion cells were1.635±0.085,1.820±0.249and0.671±0.142respectively. Observation under fluorescence microscope, the cell wall of PM yeast with L-DOPA culture appeared green fluorescence when theyeast cells added with serum of immunized mice. The serum frompatients of PM infection also had agglutination reaction with PMmelanin.Conclusions (1) The L-DOPA concentration and inoculation densityof colonies have effects on PM melanin synthesis.(2) The PM yeast cellscultured with L-DOPA decrease susceptibilities to ITC, FLU and AMB.(3) The PM melanin particles can stimulate immune system to produceantibodies.