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Antifungal Activity and Physicochemical Properties of a Novel Antimicrobial Protein AMP-17 from Musca domestica

LONG-BING YANG
LONG-BING YANG
, 
GUO GUO
GUO GUO
, 
XIN-YU ZHAO
XIN-YU ZHAO
, 
PEI-PEI SU
PEI-PEI SU
, 
PING FU
PING FU
, 
JIAN PENG
JIAN PENG
, 
JIANG-FAN XIU
JIANG-FAN XIU
 y 
BO-YAN LI
BO-YAN LI
   | 03 sept 2019
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Article Category: original-paper
Publicado en línea: 03 sept 2019
Páginas: 383 - 390
Recibido: 15 jun 2019
Aceptado: 04 ago 2019
DOI: https://doi.org/10.33073/pjm-2019-041
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© 2019 Anton Pyskun et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Time- kill curves of the AMP-17 against the fungal species tested. The growth of five fungi was monitored in the presence of 100 μg/ml AMP-17. Growth of C. albicans (A), C. tropicalis (B), C. krusei (C), C. parapsilosis (D), and C. neoformans with AMP-17 were monitored by reading the optical density (OD562) of the cultures. 100-μg/ml fluconazole was used as a positive control for fungi. Sabouraud’s dextrose broth (0 μg/ml AMP-17) was used as a negative control. The OD562 values of the remaining viable cells were monitored in 96-well plates at different times.
Time- kill curves of the AMP-17 against the fungal species tested. The growth of five fungi was monitored in the presence of 100 μg/ml AMP-17. Growth of C. albicans (A), C. tropicalis (B), C. krusei (C), C. parapsilosis (D), and C. neoformans with AMP-17 were monitored by reading the optical density (OD562) of the cultures. 100-μg/ml fluconazole was used as a positive control for fungi. Sabouraud’s dextrose broth (0 μg/ml AMP-17) was used as a negative control. The OD562 values of the remaining viable cells were monitored in 96-well plates at different times.

Fig. 2.

Influences of various NaCl or MgCl2 concentrations on the antifungal activity of AMP-17. The MIC value of AMP-17 against C. albicans was determined under the influence of different concentrations of NaCl or MgCl2 according to the conditions recommended by CLSI (2008).
Influences of various NaCl or MgCl2 concentrations on the antifungal activity of AMP-17. The MIC value of AMP-17 against C. albicans was determined under the influence of different concentrations of NaCl or MgCl2 according to the conditions recommended by CLSI (2008).

Fig. 3.

Influences of trypsin/pepsin/proteinase K on AMP-17 when used against C. albicans. Fifty μl of the proteases (at a concentration of 1 mg/ml) and 50 μl AMP-17 (at a concentration of 37.5 μg/ml) were mixed in a 96-well plate and placed in a 37°C water bath for 2, 20, 40, 60, 80, 100, and 120 min. Then, it was subjected to CFU enumeration after being treated with 100 μl of the fungal suspension for 18 hours. PBS was used as a negative control. Compared with PBS control, *P < 0.05; **P < 0.01.
Influences of trypsin/pepsin/proteinase K on AMP-17 when used against C. albicans. Fifty μl of the proteases (at a concentration of 1 mg/ml) and 50 μl AMP-17 (at a concentration of 37.5 μg/ml) were mixed in a 96-well plate and placed in a 37°C water bath for 2, 20, 40, 60, 80, 100, and 120 min. Then, it was subjected to CFU enumeration after being treated with 100 μl of the fungal suspension for 18 hours. PBS was used as a negative control. Compared with PBS control, *P < 0.05; **P < 0.01.

Fig. 4.

Hemolytic activities of AMP-17 in vitro. The diluted human red blood cells (100 μl) were incubated with AMP-17 at 37°C for 1 h and spun at 225×g for 10 minutes. The supernatants (100 μl) were transferred to a 96-well plate for measurement of the absorbance of the supernatants at 450 nm by the ultra-microplate spectrophotometer. 0.1% Triton-X 100 and 0.01M PBS were used as a positive control and a negative control, respectively. Fig. 4A is based on Fig. 4B calculated by the hemolysis rate formula. Fig. 4B was the original experimental diagram.
Hemolytic activities of AMP-17 in vitro. The diluted human red blood cells (100 μl) were incubated with AMP-17 at 37°C for 1 h and spun at 225×g for 10 minutes. The supernatants (100 μl) were transferred to a 96-well plate for measurement of the absorbance of the supernatants at 450 nm by the ultra-microplate spectrophotometer. 0.1% Triton-X 100 and 0.01M PBS were used as a positive control and a negative control, respectively. Fig. 4A is based on Fig. 4B calculated by the hemolysis rate formula. Fig. 4B was the original experimental diagram.

MIC values of AMP-17 against the fungi species tested.

PeptideMIC (μg/ml)
C. albicansATCC10231C. kruseiIFM56881C. tropicalisIFM57016C. parapsilosisATCC22019C. neoformansIFM51426
AMP-1718.7518.7518.7518.759.375

MFC values of AMP-17 against the fungi species tested.

PeptideMIC (μg/ml)
C. albicansATCC10231C. kruseiIFM56881C. tropicalisIFM57016C. parapsilosisATCC22019C. neoformansIFM51426
AMP-1737.537.537.537.518.75

Influences of temperature and the number of freeze-thaw times on the antifungal activity of AMP-17.

Temperature (98°C)(min)Colony count(x¯ ± SD)Temperature (−80°C)(Times)Colony count(x¯ ± SD)
569.50 ± 4.95**1(240.50 ± 3.54**) × 102
206.00 ± 2.83**2(273.50±3.54**) × 102
304.50 ± 2.12**4(342.00 ± 8.49**) × 102
606.50 ± 0.71**6(357.00 ± 1.41**) × 102
909.50 ± 3.54**8(351.50 ± 6.36**) × 102
1203.50 ± 0.71**10(370.50 ± 3.54**) × 102
Negative control (ddH2O)565900.00 ± 17112.00Negative control (ddH2O)(5507.00 ± 147.08) × 102
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