Molecular characterization of a novel pyocin and its immunity protein in pseudomonas aeruginosa
Bahareh Haji Rasouliha
Date of Issue2014
School of Chemical and Biomedical Engineering
Agency for Science, Technology and Research (A*STAR)
Pseudomonas aeruginosa, a Gram-negative bacterium poses a serious health threat, particularly in immunocompromised patients. Due to its inherent resistance towards many antibiotics, there is a demand for developing an effective strategy to specifically target and treat P. aeruginosa infections. P. aeruginosa is able to secrete antibacterial bacteriocins, called pyocins. To date, three types of pyocin, namely the F, R, and S types, have been discovered. These pyocins are secreted by P. aeruginosa strains, and effective to kill the same or related species. However, pyocin-producing strains are resistant to their own bacteriocins, theoretically due to the presence of probable immunity proteins. Based on the mechanisms of pyocin-killing and pyocin-resistance, pyocins could be applied to attack P. aeruginosa. Furthermore, applying pyocins in therapeutic purposes can be advanced due to the specificity of these antibacterial peptides. Pyocins bind to specific receptors on cell surface. In line with above advantages and the urgent demand of therapeutic strategies against P. aeruginosa, this thesis first aimed to elucidate the mechanisms of pyocin S5-killing and S5-resistance. To elucidate the killing mechanisms of pyocin S5, pyocin S5 gene from P. aeruginosa PAO1 was heterologously expressed in Escherichia coli, followed by the assay of killing activity against clinical isolates of P. aeruginosa and the study of killing mechanism against the clinical isolate DWW3. It was elucidated that pyocin S5 shows the highest killing activity against DWW3 among a range of P. aeruginosa isolates; pyocin S5 is a pore-forming bacteriocin capable of damaging DWW3 cell membrane, which was evidenced by ATP and nucleic acid leakage, as well as morphological changes including cell shape distortion. To elucidate pyocin S5-resistance mechanism in S5-producing strains of P. aeruginosa such as PAO1 (as the second goal of this work), the role of a putative S5I gene in conferring resistance to pyocin S5 was identified using fluorescent and FESEM microscopy on DWW3 (a Pyocin S5-sensitive strain) and DWW3-expressing S5I strains before and after exposure to pyocin S5. It was shown that following pyocin S5 exposure, morphological changes such as cell shape distortion, occurred in DWW3, while no such cell disturbance appeared in the DWW3 expressing S5I. This result indicated that S5I conferred pyocin S5 resistance through the prevention of membrane damage by pyocin S5. Furthermore, this study aimed to explore virulence factor regulation in P. aeruginosa, which can help to improve the approaches used to inhibit the pathogenicity, invasion, and antibiotic resistance of this bacterium in patients. Therefore, the regulatory role of mvaU gene on a virulence factor (pyoverdine, PVD) of P. aeruginosa was illustrated by examining the transcriptome profiling of the mvaU mutants vs. wild-type PAO1 strain using DNA microarray. The microarray results showed that in the mvaU mutants, three genes involved in PVD biosynthesis—pvdF, pvdJ, and pvdG—have been significantly up-regulated. Hence, It was hypothesized that mvaU might be responsible for the regulation of these three genes. This hypothesis was verified by qRT-PCR analysis and PVD measurements in wild type and mutant strains. The PVD measurements demonstrated high concentration of PVD in mutant strain compared to the wild type. This result beckons further investigations in genome profiling and the possible regulation mechanism of PVD upon mvaU in P. aeruginosa. In summary, this thesis verified the antimicrobial mechanism of pyocin S5 against clinical isolates of P. aeruginosa from cystic fibrosis patients. Moreover, the efficacy and the mode of action of S5 immunity gene were elucidated, which renders the host resistance to pyocin S5. In addition, transcriptome profiling revealed that mvaU, a global regulatory gene, is involved in pyoverdine regulation. The findings in this study, including the relationship between PVD-biosynthesis genes and mvaU regulatory gene, and the affirmation of S5 and S5I role, provide valuable insights in understanding virulence gene of P. aeruginosa and further expanding therapeutic approaches that can be taken against pathogenicity and antibiotic resistance properties of this bacterium.