This proposal targets a critical issue in the fight against the growing problem of antibiotic resistance: the search for strategies aimed at preserving the effectiveness of currently available antibiotics. Our model system is the aminoglycoside 6'-N-acetyltransferase type lb [AAC(6')-lb], an enzyme that mediates resistance to amikacin and other aminoglycosides. Our long term goal is to develop antisense oligonucleotides as pharmacological tools to selectively inhibit the expression of aac(6')-lb. To develop two kinds of antisense compounds that inhibit expression of aac(6')-lb by different mechanisms we designed specific aims 1 and 2: 1. Identification of nuclease-resistant oligodeoxynucleotide analogs that promote RNase H-mediated degradation of aac(6')-lb mRNA. We will design nuclease-resistant analogs and test their ability to mediate phenotypic conversion to amikacin susceptibility and determine the mechanism of action. 2. In vivo studies on RNase P-mediated degradation of aac(6')-lb mRNA by oligoribonucleotides and systematic analysis of the ability of nuclease-resistant oligoribonucleotide analogs to induce RNase P cleavage. We will design plasmids that code for selected oligoribonucleotides and test if they induce RNase P-mediated conversion to amikacin susceptibility. We will also carry out a systematic study on nuclease-resistant oligoribonucleotide analogs to determine which ones, if any, do not compromise RNase P-mediated cleavage of RNA. While achieving specific aims 1 and 2 will be an important step towards developing antisense compounds to preserve the efficacy of amikacin, many problems will remain to be solved. Two of these problems are: a) delivery methods to insure that antisense compounds reach the bacterial cell's cytoplasm are very limited; and b) the aac(6')-lb gene is often found in high copy number plasmids; as a consequence the large number of gene copies may make it very difficult to completely turn off expression.
Specific aims 3 and 4 have been designed to deal with these problems: 3. Development of liposome formulations capable of delivering oligonucleotides into the cell's cytosol. We will test the ability of several formulations of cationic liposome-encapsulated oligonucleotide analogs to reach the cytoplasm. The process of internalization will be characterized by fluorescence microscopy. 4. Search for peptide inhibitors of the AAC(6')-lb enzyme. Enzyme inhibitors could have a synergistic activity with antisense oligonucleotides by inhibiting the action of any residual AAC(6')-lb protein synthesized. Peptide inhibitors will be searched using phage display.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15AI047115-02A1
Application #
6895716
Study Section
Special Emphasis Panel (ZRG1-IDM-H (10))
Program Officer
Perdue, Samuel S
Project Start
2000-06-01
Project End
2009-05-31
Budget Start
2005-06-01
Budget End
2009-05-31
Support Year
2
Fiscal Year
2005
Total Cost
$209,115
Indirect Cost
Name
California State University Fullerton
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
106670755
City
Fullerton
State
CA
Country
United States
Zip Code
92831
Davies-Sala, Carol; Jani, Saumya; Zorreguieta, Angeles et al. (2018) Identification of the Acinetobacter baumannii Ribonuclease P Catalytic Subunit: Cleavage of a Target mRNA in the Presence of an External Guide Sequence. Front Microbiol 9:2408
Tran, Tung; Chiem, Kevin; Jani, Saumya et al. (2018) Identification of a small molecule inhibitor of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] using mixture-based combinatorial libraries. Int J Antimicrob Agents 51:752-761
Chiem, Kevin; Hue, Fong; Magallon, Jesus et al. (2018) Inhibition of aminoglycoside 6'-N-acetyltransferase type Ib-mediated amikacin resistance by zinc complexed with clioquinol, an ionophore active against tumors and neurodegenerative diseases. Int J Antimicrob Agents 51:271-273
Jani, Saumya; Jackson, Alexis; Davies-Sala, Carol et al. (2018) Assessment of External Guide Sequences' (EGS) Efficiency as Inducers of RNase P-Mediated Cleavage of mRNA Target Molecules. Methods Mol Biol 1737:89-98
Stietz, Maria S; Lopez, Christina; Osifo, Osasumwen et al. (2017) Evaluation of the electron transfer flavoprotein as an antibacterial target in Burkholderia cenocepacia. Can J Microbiol 63:857-863
Ramirez, Maria S; Tolmasky, Marcelo E (2017) Amikacin: Uses, Resistance, and Prospects for Inhibition. Molecules 22:
Jackson, Alexis; Jani, Saumya; Sala, Carol Davies et al. (2016) Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes. Biol Methods Protoc 1:
Chiem, Kevin; Jani, Saumya; Fuentes, Brooke et al. (2016) Identification of an Inhibitor of the Aminoglycoside 6'-N-Acetyltransferase type Ib [AAC(6')-Ib] by Glide Molecular Docking. Medchemcomm 7:184-189
Traglia, German Matías; Dixon, Chelsea; Chiem, Kevin et al. (2015) Draft Genome Sequence of Empedobacter (Formerly Wautersiella) falsenii comb. nov. Wf282, a Strain Isolated from a Cervical Neck Abscess. Genome Announc 3:
Davies-Sala, Carol; Soler-Bistué, Alfonso; Bonomo, Robert A et al. (2015) External guide sequence technology: a path to development of novel antimicrobial therapeutics. Ann N Y Acad Sci 1354:98-110

Showing the most recent 10 out of 44 publications