Investigation of the in vivo interaction between Ã-lactamase and its inhibitor protein
The affinity of Ã-lactamase inhibitory protein (BLIP) for TEM-1 Ã-lactamase has raised hopes in the challenge of protein-based inhibitor discovery for Ã-lactamase-mediated antibiotic resistance. Currently, the effect of the formation of the Ã-lactamase:BLIP complex in vivo in Ã-lactam resistant bacteria is an open question. The scarcity of information to the extent to which BLIP can impair Ã-lactamase activity inside cells has urged us to assess the in vivo efficacy of BLIP as a potent Ã-lactamase inhibitor. To this end, Ã-lactamase and BLIP were coexpressed in Escherichia coli. Simultaneous expression of Ã-lactamase and BLIP and the formation of the TEM-1 Ã-lactamase:BLIP complex in the periplasmic space of E. coli were verified by electrophoretic and Western blot techniques. Growth profiles of the cells expressing both Ã-lactamase and its protein inhibitor, complemented with Ã-lactamase activity measurements, suggested that BLIP synthesis retarded cell growth and reduced Ã-lactamase activity. Although co-expression of Ã-lactamase and its protein inhibitor did not completely impair cell growth, the specificity of BLIP enabled it to bind Ã-lactamase in the bacterial periplasm, regardless of the crowding components.
Investigation of the in vivo interaction between Ã-lactamase and its inhibitor protein
The affinity of Ã-lactamase inhibitory protein (BLIP) for TEM-1 Ã-lactamase has raised hopes in the challenge of protein-based inhibitor discovery for Ã-lactamase-mediated antibiotic resistance. Currently, the effect of the formation of the Ã-lactamase:BLIP complex in vivo in Ã-lactam resistant bacteria is an open question. The scarcity of information to the extent to which BLIP can impair Ã-lactamase activity inside cells has urged us to assess the in vivo efficacy of BLIP as a potent Ã-lactamase inhibitor. To this end, Ã-lactamase and BLIP were coexpressed in Escherichia coli. Simultaneous expression of Ã-lactamase and BLIP and the formation of the TEM-1 Ã-lactamase:BLIP complex in the periplasmic space of E. coli were verified by electrophoretic and Western blot techniques. Growth profiles of the cells expressing both Ã-lactamase and its protein inhibitor, complemented with Ã-lactamase activity measurements, suggested that BLIP synthesis retarded cell growth and reduced Ã-lactamase activity. Although co-expression of Ã-lactamase and its protein inhibitor did not completely impair cell growth, the specificity of BLIP enabled it to bind Ã-lactamase in the bacterial periplasm, regardless of the crowding components.
___
- Adelowo OO, Fagade OE (2012). Phylogenetic characterization, antimicrobial susceptibilities, and mechanisms of resistance in bacteria isolates from a poultry waste-polluted river, southwestern Nigeria. Turk J Biol 36: 37–45.
- Alaybeyoglu B, Sariyar Akbulut B, Ozkirimli E (2015). A novel chimeric peptide with antimicrobial activity. J Pept Sci, accepted for publication.
- Bebrone C, Moali C, Mahy F, Rival S, Docquier JD, Rossolini GM, Fastrez J, Pratt RF, Frere JM, Galleni M (2001). CENTA as a chromogenic substrate for studying beta-lactamases. Antimicrob Agents Ch 45: 1868–1871.
- Blakesley RW, Boezi JA (1977). A new staining technique for proteins in polyacrylamide gels using Coomassie Brilliant Blue G250. Anal Biochem 82: 580–582.
- Blazquez J, Baquero MR, Canton R, Alos I, Baquero F (1993). Characterization of a new TEM-type β-lactamase resistant to clavulanate, sulbactam, and tazobactam in a clinical isolate of Escherichia coli. Antimicrob Agents Ch 37: 2059–2063.
- Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
- Chanda S, Rakholiya K, Dholakia K, Baravalia Y (2013). Antimicrobial, antioxidant, and synergistic properties of two nutraceutical plants: Terminalia catappa L. and Colocasia esculenta L. Turk J Biol 37: 81–91.
- Doran JL, Leskiw BK, Aippersbach S, Jensen SE (1990). Isolation and characterization of a beta-lactamase-inhibitory protein from Streptomyces clavuligerus and clonning and analysis of the corresponding gene. J Bacteriol 172: 4909–4918.
- Essack SY (2001). The development of beta-lactam antibiotics in response to the evolution of beta-lactamases. Pharm Res 18: 1391–1399.
- Frère JM (1995). Beta-lactamases and bacterial resistance to antibiotics. Mol Microbiol 16: 385–395.
- Fryszczyn BG, Brown NG, Huang W, Balderas MA, Palzkill T (2011). Use of periplasmic target protein capture for phage display engineering of tight-binding protein-protein interactions. Protein Eng Des Sel 24: 819–828.
- Hanes MS, Ratcliff K, Marqusee S, Handel TM (2010). Protein- protein binding affinities by pulse proteolysis: application to TEM-1/BLIP protein complexes. Protein Sci 19: 1996–2000.
- Huang W, Petrosino J, Hirsch M, Shenkin PS, Palzkill T (1996). Amino acid sequence determinants of β-lactamase structure and activity. J Mol Biol 258: 688–703.
- Huang W, Beharry Z, Zhang Z, Palzkill T (2003). A broad-spectrum peptide inhibitor of beta-lactamase identified using phage display and peptide arrays. Protein Eng 16: 853–860.
- Jones RN, Wilson HW, Novick WJ, Barry AL, Thornsberry C (1982). In vitro evaluation of CENTA, a new beta-lactamase- susceptible chromogenic cephalosporin reagent. J Clin Microbiol 15: 954–958.
- Khait R, Schreiber G (2012). FRETex: a FRET-based, high- throughput technique to analyze protein-protein interactions. Protein Eng Des Sel 25: 681–687.
- Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680– 685.
- Majiduddin FK, Materon IC, Palzkill TG (2002). Molecular analysis of β-lactamase structure and function. Int J Med Microbiol 292: 127–137.
- Martinez JL, Cercenado E, Rodriguez-Creixems M, Vicente-Perez, Delgado-Iribarren A, Baquero F (1987). Resistance to beta- lactam/clavulanate. Lancet 2: 1473.
- Matagne A, Lamotte-Brasseur J, Frere JM (1998). Catalytic properties of class A β-lactamases: efficiency and diversity. Biochem J 330: 581–598.
- Nossal NG, Heppel LA (1966). The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 241: 3055–3062.
- Ozbalci C, Unsal C, Kazan D, Sariyar Akbulut B (2010). Proteomic response of Escherichia coli to the alkaloid extract of Papaver polychaetum. Ann Microbiol 60: 709–717.
- Phichith D, Bun S, Padiolleau-Lefevre S, Guellier A, Banh S, Galleni M, Frere JM, Thomas D, Friboulet A, Avalle B (2010). Novel peptide inhibiting both TEM-1 b-lactamase and penicillin- binding proteins. FEBS J 277: 4965–4972.
- Phillip Y, Kiss V, Schreiber G (2012a). Protein-binding dynamics imaged in a living cell. Proc Natl Acad Sci 109: 1461–1466.
- Phillip Y, Harel M, Khait R, Qin S, Zhou HX, Schreiber G (2012b). Contrasting factors on the kinetic path to protein complex formation diminish the effects of crowding agents. Biophys J 103: 1011–1019.
- Robert CH, Janin J (1998). A soft, mean-field potential derived from crystal contacts for predicting protein-protein interactions. J Mol Biol 283: 1037–1047.
- Rudgers GW, Huang W, Palzkill T (2001). Binding properties of a peptide derived from beta-lactamase inhibitory protein. Antimicrob Agents Ch 45: 3279–3286.
- Shevchenko A, Thomas H, Havlis J, Olsen JV, Mann M (2007). In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1: 2856–2860.
- Strynadka NC, Jensen SE, Johns K, Blanchard H, Page M, Matagne A, Frere JM, James MN (1994). Structural and kinetic characterization of a beta-lactamase-inhibitor protein. Nature 368: 657–660.
- Yuan J, Huang W, Chow DC, Palzkill T (2009). Fine mapping of the sequence requirements for binding of beta lactamase inhibitory protein (BLIP) to TEM-1 beta-lactamase using a genetic screen for BLIP function. J Mol Biol 389: 401–412.