Bottom-Up Proteomics Analysis Pseudomonas aeruginosa Under the Effects of Antimicrobial Peptides
Disciplines
Bacteriology | Biochemistry
Abstract (300 words maximum)
Pseudomonas aeruginosa (P.aeruginosa) is identified by the World Health Organization as a critical multidrug-resistant pathogen. Its pathogenicity is due to Quorum Sensing (QS), a regulatory system that enables communication and coordination in response to population density. QS governs behaviors such as motility, virulence, and biofilm formation, the latter making P. aeruginosa infections especially difficult to treat. Key genes to this system are lasR, rhlR, and pqsE and their protein expression. The aim of this research is to investigate how antimicrobial peptides, including Temporin L and its analogs, influence protein production in P. aeruginosa, with a focus on QS gene expression. Peptide-based antibiotics offer advantages such as broad antimicrobial activity, lower toxicity, and reduced risk of resistance development, making them promising candidates for combating multidrug-resistant infections. The methods of this experiment start with antimicrobial activity assessed by determining the MIC (minimum inhibitory concentration) and NIC (lowest concentration slowing growth) of peptides against P. aeruginosa. 3mL of culture is grown until an OD of 0.5, representing actively dividing, resistant gene-expressing cells. Peptides at varying concentrations (150 µL) were introduced, and OD measurements were recorded every 30 minutes until cultures reached an OD of 1.0. Growth inhibition patterns established MIC and NIC values. The cultures were retained for LC-MS analysis. Samples were centrifuged to collect pellets, washed and purified to isolate proteins. Protein samples were prepared for bottom-up proteomic analysis. Preliminary results of Temporin L showed OD (cell growth) values decreasing as peptide concentration increased. Temporin L and TLP-1 both showed significant bacterial growth reduction where TLP-3 showed no inhibition. Proteomics analysis revealed 52 proteins in control samples versus 83 in TL treated samples, suggesting stress-induced protein upregulation. This experiment will be repeated with a variety of other peptides to identify which best target P. aeruginosa antimicrobial resistance.
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Academic department under which the project should be listed
CSM – Chemistry and Biochemistry
Primary Investigator (PI) Name
Mohammad Halim
Bottom-Up Proteomics Analysis Pseudomonas aeruginosa Under the Effects of Antimicrobial Peptides
Pseudomonas aeruginosa (P.aeruginosa) is identified by the World Health Organization as a critical multidrug-resistant pathogen. Its pathogenicity is due to Quorum Sensing (QS), a regulatory system that enables communication and coordination in response to population density. QS governs behaviors such as motility, virulence, and biofilm formation, the latter making P. aeruginosa infections especially difficult to treat. Key genes to this system are lasR, rhlR, and pqsE and their protein expression. The aim of this research is to investigate how antimicrobial peptides, including Temporin L and its analogs, influence protein production in P. aeruginosa, with a focus on QS gene expression. Peptide-based antibiotics offer advantages such as broad antimicrobial activity, lower toxicity, and reduced risk of resistance development, making them promising candidates for combating multidrug-resistant infections. The methods of this experiment start with antimicrobial activity assessed by determining the MIC (minimum inhibitory concentration) and NIC (lowest concentration slowing growth) of peptides against P. aeruginosa. 3mL of culture is grown until an OD of 0.5, representing actively dividing, resistant gene-expressing cells. Peptides at varying concentrations (150 µL) were introduced, and OD measurements were recorded every 30 minutes until cultures reached an OD of 1.0. Growth inhibition patterns established MIC and NIC values. The cultures were retained for LC-MS analysis. Samples were centrifuged to collect pellets, washed and purified to isolate proteins. Protein samples were prepared for bottom-up proteomic analysis. Preliminary results of Temporin L showed OD (cell growth) values decreasing as peptide concentration increased. Temporin L and TLP-1 both showed significant bacterial growth reduction where TLP-3 showed no inhibition. Proteomics analysis revealed 52 proteins in control samples versus 83 in TL treated samples, suggesting stress-induced protein upregulation. This experiment will be repeated with a variety of other peptides to identify which best target P. aeruginosa antimicrobial resistance.