Presentation Title

Protocol development to test the activity of the antimicrobial peptide human defensin 2 against Mycobacterium smegmatis

Faculty Mentor

Edith Porter

Start Date

18-11-2017 2:15 PM

End Date

18-11-2017 3:15 PM

Location

BSC-Ursa Minor 72

Session

Poster 3

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Background: Mycobacterium tuberculosis (Mtb), a pathogenic acid-fast bacterium causes ten million infections and two million deaths per year. Multidrug resistance of Mtb has been in part attributed to its lipid rich cell wall and tendency to aggregate. Mucosal surfaces in the respiratory tract secrete antimicrobial peptides including human beta-defensins (HBD) which typically exert their effects at low μM concentrations. HBD2 has been thought to primarily act as chemoattractant. However, considering its association with lipid membranes direct activity against Mtb is conceivable. Objective: Using Mycobacterium smegmatis (Ms) as a model organism for Mtb, this study aimed to establish a protocol to homogenize Ms and test the effectiveness of HBD-2 against Ms. Methods: Ms was grown for 48 h in 7H9 medium, adjusted to McF 0.5, sonicated for 3, 5, and 9 rounds of five second durations, and diluted 1:50 in 7H9 media. Aliquots in five replicates were further incubated in a microtiter plate for 48 h and metabolic activity was measured with a fluorescence assay employing resazurin. The non-fluorescent resazurin is reduced by bacterial metabolites to fluorescent resorufin. HBD-2 was 1:2 serially diluted and added as 10 x fold stocks to yield final concentrations of 0.0195 µM - 5 µM. Ms boiled for 10 min served as killing control and 7H9 media only as growth control. Results: Nine sonication rounds maximized a homogenous mixture of Ms without killing the bacteria. As little as 0.039 µM HBD2 effected growth inhibition of Ms. Conclusion: HBD-2 is active against Ms at nanomolar concentrations and could be exploited for novel prophylactic and therapeutic treatment against Mtb. Acknowledgements: Dr. Wuyuan Lu for HBD2 peptide synthesis and the Louis Stokes Alliance for Minority Participation (LSAMP) for registration funds and poster assistance.

Keywords: Tuberculosis, defensins, antimicrobial peptides, epithelial cells

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Nov 18th, 2:15 PM Nov 18th, 3:15 PM

Protocol development to test the activity of the antimicrobial peptide human defensin 2 against Mycobacterium smegmatis

BSC-Ursa Minor 72

Background: Mycobacterium tuberculosis (Mtb), a pathogenic acid-fast bacterium causes ten million infections and two million deaths per year. Multidrug resistance of Mtb has been in part attributed to its lipid rich cell wall and tendency to aggregate. Mucosal surfaces in the respiratory tract secrete antimicrobial peptides including human beta-defensins (HBD) which typically exert their effects at low μM concentrations. HBD2 has been thought to primarily act as chemoattractant. However, considering its association with lipid membranes direct activity against Mtb is conceivable. Objective: Using Mycobacterium smegmatis (Ms) as a model organism for Mtb, this study aimed to establish a protocol to homogenize Ms and test the effectiveness of HBD-2 against Ms. Methods: Ms was grown for 48 h in 7H9 medium, adjusted to McF 0.5, sonicated for 3, 5, and 9 rounds of five second durations, and diluted 1:50 in 7H9 media. Aliquots in five replicates were further incubated in a microtiter plate for 48 h and metabolic activity was measured with a fluorescence assay employing resazurin. The non-fluorescent resazurin is reduced by bacterial metabolites to fluorescent resorufin. HBD-2 was 1:2 serially diluted and added as 10 x fold stocks to yield final concentrations of 0.0195 µM - 5 µM. Ms boiled for 10 min served as killing control and 7H9 media only as growth control. Results: Nine sonication rounds maximized a homogenous mixture of Ms without killing the bacteria. As little as 0.039 µM HBD2 effected growth inhibition of Ms. Conclusion: HBD-2 is active against Ms at nanomolar concentrations and could be exploited for novel prophylactic and therapeutic treatment against Mtb. Acknowledgements: Dr. Wuyuan Lu for HBD2 peptide synthesis and the Louis Stokes Alliance for Minority Participation (LSAMP) for registration funds and poster assistance.

Keywords: Tuberculosis, defensins, antimicrobial peptides, epithelial cells