טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentYaniv Orit
SubjectResistance of Pseudomonas aeruginosa to Aminoglycosides:
Regulation Studies of APH(3')-llb Enzyme and MexXY
Efflux Pump
DepartmentDepartment of Biotechnology
Supervisor Professor Timor Baasov


Abstract

Aminoglycoside antibiotics represent miracle drugs in antipseudomonal chemotherapy implicated in the treatment of a variety of infections, particularly pulmonary infections in cystic fibrosis (CF) patients. Such infections are difficult to treat because of the bacterial resistance to multiple antibiotics. Resistance to aminoglycosides typically results from decreased permeability, expression of aminoglycoside-inactivating enzymes, ribosomal alterations and overexpression of several multidrug efflux systems.

         The aph(3')-IIb is a chromosomal phosphotransferase gene in Pseudomonas aeruginosa, which encodes the aminoglycoside modifying enzyme APH(3')-IIb. Its regulation and importance for clinical resistance to P. aeruginosa strains have not yet been investigated. Although at least five efflux systems are involved in antibiotic resistance in P. aeruginosa, only the MexXY efflux pump has been shown to cause tolerance to aminoglycosides.

         The goal of this study was to investigate the effects of aminoglycosides on the regulation of the enzyme APH(3')-IIb in P. aeruaginosa, at the transcriptional, translational and post translational levels, along with the contribution of the APH(3')-IIb and MexXY efflux pump to aminoglycoside resistance in aminoglycoside-resistant clinical isolates of P. aeruginosa from CF patients.

The amounts of aph(3')-IIb and mexY transcripts were measured by quantitative real time-PCR. Induction experiments under sub MIC levels of several aminoglycosides revealed that mRNA levels of mexY were induced between three- to ten-fold by aminoglycosides. In contrast, the mRNA levels of the aph(3')-IIb were not induced significantly by any of the tested aminoglycosides. Western blot analysis indicated that protein expression levels of APH(3‘)-IIb were not changed significantly by the presence of Kanamycin and Neomycin, which serve substrates of the APH(3')-IIb enzyme. However, post translational regulation study of the recombinant APH(3’)-IIb, revealed that the protein undergoes post translational modification, most likely a phosphorylation, even though such modification seems not affecting the enzymatic activity. All of the resistant strains isolated from the CF patients showed low basal levels of APH(3’)-IIb both at the mRNA and protein level, suggesting that in these strains APH(3’)-IIb is not the main resistance mechanism against aminoglycosides. In contrast, the data obtained in this study demonstrate that the MexXY efflux system is overproduced in the CF isolated strains and plays an important role in their resistance profile.