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Two-dimensional polyacrylamide gel electrophoresis isolation and microsequencing of Pseudomonas aeruginosa proteins
M Michéa-Hamzehpour1, J C Sanchez, S F Epp
1Département de Génétique et Microbiologie, Centre Médical Universitaire, Genève, Suisse.
Abstract:
Outer membrane (OM) proteins of beta-lactam-susceptible and -resistant strains of Pseudomonas aeruginosa were analyzed by 2-D polyacrylamide gel electrophoresis. Carrier ampholytes, pH 4-8, and immobilized pH gradient (IPG), pH 3.5-10.0, procedures were used. An acidic-protein spot (pI = 5.2) detected in susceptible but not in an imipenem-resistant strain was sequenced and twenty-five N-terminal amino acids had total homology with the OM protein D, the imipenem-specific porin of P. aeruginosa. A basic-protein spot (pI = 9.0) detected in ceftazidime-resistant, but not in a susceptible strain was sequenced and fourteen N-terminal amino acids had homology with a beta-lactamase encoded by the ampC gene of P. aeruginosa. The IPG procedure allows identification of more than one hundred proteins of the OM fraction from a single gel. Detection of beta-lactamase in OM fractions might reflect a periplasmic contamination, but its anchorage within the OM cannot be ruled out.
Insights
Outer membrane proteins of Pseudomonas aeruginosa were analyzed, revealing key differences in susceptible and resistant strains. This study identified specific proteins linked to imipenem and ceftazidime resistance in this bacterium.
Area of Science:
- Microbiology
- Protein analysis
- Drug resistance mechanisms
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen known for its intrinsic and adaptive resistance to antibiotics.
- Outer membrane proteins play a crucial role in P. aeruginosa's defense against antimicrobial agents, including beta-lactams.
- Understanding the proteomic changes in the outer membrane is vital for deciphering resistance mechanisms.
Purpose of the Study:
- To analyze and compare the outer membrane proteomes of beta-lactam-susceptible and -resistant Pseudomonas aeruginosa strains.
- To identify specific outer membrane proteins associated with resistance to imipenem and ceftazidime.
- To evaluate the utility of 2-D polyacrylamide gel electrophoresis with different pH gradients for proteomic analysis.
Main Methods:
- Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was employed to separate outer membrane proteins.
- Both carrier ampholyte (pH 4-8) and immobilized pH gradient (IPG, pH 3.5-10.0) systems were utilized for protein separation.
- Protein spots of interest were sequenced to determine N-terminal amino acid homology with known proteins.
Main Results:
- An acidic protein (pI 5.2) homologous to outer membrane protein D (imipenem-specific porin) was found in susceptible strains but absent in an imipenem-resistant strain.
- A basic protein (pI 9.0) homologous to beta-lactamase (ampC gene) was detected in ceftazidime-resistant strains but not in susceptible strains.
- The IPG method enabled the identification of over one hundred outer membrane proteins from a single gel.
Conclusions:
- Outer membrane protein D may be involved in imipenem susceptibility in P. aeruginosa.
- The presence of beta-lactamase in outer membrane fractions of resistant strains suggests a role in ceftazidime resistance, though periplasmic contamination is a possibility.
- 2-D PAGE with IPG is a powerful tool for comprehensive outer membrane proteomic analysis in P. aeruginosa.