MOLECULAR MECHANISMS OF AMINOGLYCOSIDE RESISTANCE

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DESCRIPTION: Since the introduction of antibiotics, bacteria kept developing resistance to all of them. Drug companies kept pace by developing new antimicrobial agents so when a pathogen became resistant to one of them there was another that would be effective. However, recently many multiple-drug resistance pathogenic bacteria have emerged towards which most antibiotics are ineffective. As a consequence, the race between development of resistance by pathogens and the pharmaceutical industry is becoming worse. As the problem of bacterial resistance increases, the availability of effective antimicrobial agents to treat infections gets curtailed and the costs grow higher. An approach to deal with the drug resistance problem is to study different aspects of the mechanisms of bacterial resistance to antibiotics and design rational drug strategies to overcome the problem based on the knowledge gained. Aminoglycosides are important for the treatment of serious Gram-negative infections. A very common mechanism of bacterial resistance to aminoglycosides is to eliminate the antibacterial activity through enzymatic inactivation by the action of modifying enzymes. A clinically important N-acetyltransferase enzyme is AAC(6')-Ib which confers resistance to several aminoglycosides including the semisynthetic amikacin, the most refractory to inactivating enzymes. The long term goal of this investigation is to understand at the molecular level the different events that result in bacterial resistance to aminoglycosides mediated by this enzyme. The specific aims for this project are: 1) Determination of the subcellular localization of AAC(6')-Ib. The location of an aminoglycoside-modifying enzyme is instrumental in the determination of the level of resistance of a bacterial cell to a certain aminoglycoside. The subcellular location of the enzyme AAC(6')-Ib will be determined by: a) treatment of spheroplasts with proteases, b) imunoelectron microscopy, and c) gene fusions. These approaches will complement those analysis already done using physical methods that release periplasmic proteins. 2) Identification of amino acids and regions critical for the function of AAC(6')-Ib. Not all amino acid residues and regions of a protein contribute equally to the function and structure of an enzyme. Thus, the goal of this specific aim is to identify regions and amino acids residues of the protein AAC(6')-Ib that are critical for its structure and enzymatic function. This will be done using random replacement mutagenesis, a technique that allows [one] to assess the importance of small portions of the protein. 3) Crystallization of AAC(6')-Ib. An important step in the characterization of biological processes is the correlation of biochemical and genetic data with the detailed structure of the molecules involved. Therefore, a long-term objective of this project is to determine the three dimensional structure of AAC(6')-Ib. The goal for this proposal is to generate crystals suitable for high-resolution X-ray diffraction analysis. This will be achieved by: a) purification of sufficient amounts of AAC(6')-Ib and b) establishment of the condition to produce the crystals.