Talks
Presentation Type
Oral Presentation
Discipline Track
Biomedical Science
Abstract Type
Research/Clinical
Abstract
Background: Clostridioides difficile is a gram-positive, spore-forming anaerobic bacterium that is the leading cause of nosocomial infections. Due to its detergent-resistant spores and antibiotic resistance, C. difficile infection is an urgent threat since it is difficult to eliminate, allowing it to persist in healthcare institutions. Inadequate treatments available for this infection lead to the problem that C. difficile infection rates may rise in coming years due to an increase in hospital-acquired infections following the COVID-19 pandemic, and therefore the discovery of new antimicrobial compounds is of utmost importance. Antimicrobial peptides (AMPs) have become a promising alternative due to their unique mechanism, resulting in less antimicrobial resistance. Protein synthesis is a metabolic process that is a validated target for antibiotics. Initiation factor 1 (IF1) is a key component of protein synthesis and plays a key role in forming the pre-initiation complex with the 30S ribosome. The solution structure of Cd-IF1 has been determined and it consists of 5 β strands and 1 α helix forming a β-barrel arranged in a β1-β2-β3-α1-β4-β5 topology.
Methods: Protein expression and purification of 15N-labeled Cd-IF1 were isolated using Fast Protein Liquid Chromatography (FPLC). NMR Titration studies of Cd-IF1 and the 30S ribosomal subunit were carried out at 298 K on a Bruker Ultrashield Plus 600 NMR spectrometer. Minimum Inhibitory Concentration (MIC) Assays were conducted for antimicrobial susceptibility testing. Cytotoxicity assays were conducted using Human Embryonic Kidney-293 (HEK-293) cells to determine the toxicity of the Cd-IF1 AMP.
Results: The MIC for C. difficile was 0.13 mg/ml (66 μM) and for gram-positive species, the range of MIC was between 0.14 mg/ml and 0.37 mg/ml. The range MIC values for gram-negative species were between 1.0 – 1.5 mg/ml. The cytotoxicity assay determined that Cd-IF1 AMP showed no toxicity against HEK-293 cells.
Conclusion: NMR titration studies of the 30S ribosomal subunit and Cd-IF1 reveal key interactions between the α -helical strand and the 30S ribosome. A peptide was derived from the α helical strand and tested against C. difficile and various bacteria for antimicrobial abilities. From these studies, the C. difficile antimicrobial peptide (AMP) displayed broad-spectrum inhibition against a wide range of bacteria but not HEK-293 cells and may be a candidate for antibiotic development.
Academic/Professional Position
Faculty
Recommended Citation
Alanis, Elvira; Alanis, Miguel; Dean, Frank B.; and Zhang, Yonghong, "Rational Design of an Antimicrobial Peptide" (2024). Research Symposium. 4.
https://scholarworks.utrgv.edu/somrs/2023/talks/4
Included in
Rational Design of an Antimicrobial Peptide
Background: Clostridioides difficile is a gram-positive, spore-forming anaerobic bacterium that is the leading cause of nosocomial infections. Due to its detergent-resistant spores and antibiotic resistance, C. difficile infection is an urgent threat since it is difficult to eliminate, allowing it to persist in healthcare institutions. Inadequate treatments available for this infection lead to the problem that C. difficile infection rates may rise in coming years due to an increase in hospital-acquired infections following the COVID-19 pandemic, and therefore the discovery of new antimicrobial compounds is of utmost importance. Antimicrobial peptides (AMPs) have become a promising alternative due to their unique mechanism, resulting in less antimicrobial resistance. Protein synthesis is a metabolic process that is a validated target for antibiotics. Initiation factor 1 (IF1) is a key component of protein synthesis and plays a key role in forming the pre-initiation complex with the 30S ribosome. The solution structure of Cd-IF1 has been determined and it consists of 5 β strands and 1 α helix forming a β-barrel arranged in a β1-β2-β3-α1-β4-β5 topology.
Methods: Protein expression and purification of 15N-labeled Cd-IF1 were isolated using Fast Protein Liquid Chromatography (FPLC). NMR Titration studies of Cd-IF1 and the 30S ribosomal subunit were carried out at 298 K on a Bruker Ultrashield Plus 600 NMR spectrometer. Minimum Inhibitory Concentration (MIC) Assays were conducted for antimicrobial susceptibility testing. Cytotoxicity assays were conducted using Human Embryonic Kidney-293 (HEK-293) cells to determine the toxicity of the Cd-IF1 AMP.
Results: The MIC for C. difficile was 0.13 mg/ml (66 μM) and for gram-positive species, the range of MIC was between 0.14 mg/ml and 0.37 mg/ml. The range MIC values for gram-negative species were between 1.0 – 1.5 mg/ml. The cytotoxicity assay determined that Cd-IF1 AMP showed no toxicity against HEK-293 cells.
Conclusion: NMR titration studies of the 30S ribosomal subunit and Cd-IF1 reveal key interactions between the α -helical strand and the 30S ribosome. A peptide was derived from the α helical strand and tested against C. difficile and various bacteria for antimicrobial abilities. From these studies, the C. difficile antimicrobial peptide (AMP) displayed broad-spectrum inhibition against a wide range of bacteria but not HEK-293 cells and may be a candidate for antibiotic development.