| Author: | Liu, Yang |
| Title: | Characterization of novel factors in the interactions of pseudomonas aeruginosa biofilms with caenorhabditis elegans |
| Advisors: | Chua, Song Lin (ABCT) |
| Degree: | Ph.D. |
| Year: | 2022 |
| Subject: | Microorganisms -- Behavior Microbial ecology Host-bacteria relationships Infection Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | 158 pages : color illustrations |
| Language: | English |
| Abstract: | Bacteria-host interaction is an important research field in microbiology to understand the adhesion and infection of the pathogenic bacteria to the host organisms. One convenient whole-animal model of infection is the Pseudomonas. aeruginosa bacteria- Caenorhabditis. elegans nematode model, where the bacteria establish an intestinal infection within the host after the animal feeds on the bacteria. C. elegans is often used to studying bacterial virulence, especially for intestinal infections. P. aeruginosa is a major human opportunistic Gram-negative pathogen, which accounts for many lung, intestinal and urinary tract infections. With a strong ability to form multicellular biofilms encased in self-secreted matrix, P. aeruginosa employs the c-di-GMP secondary messenger signaling system which is found in most bacterial species, to colonize host surfaces and cause biofilm-mediated infections. Iron is an important nutrient for biofilm formation, so P. aeruginosa employs pyoverdine, a fluorescent iron siderophore for scavenging iron from the environment. Till date, most omics methods, especially proteomics focus on studying either the bacteria or host, but there is a lack of suitable approaches which can evaluate both host and bacteria simultaneously. Here, I developed a label-free SILAC proteomics method, which is mostly used in mammalian cell studies, to evaluate bacterial and host factors during the infection (6 h.p.i and 24 h.p.i) simultaneously, with high sensitivity and accuracy. This SILAC approach can be employed for other in vivo infection research involving host-pathogen interactions. Based on the proteomics results, novel factors from P. aeruginosa such as AlgR and PvdA, and the AlgR protein expression and correspondingly higher PvdA expression by 24 h.p.i were identified. AlgR is a protein which regulates the pathway alginate biosynthesis, which is part of Glycan biosynthesis (Little Alexander, Okkotsu et al. 2018). Protein PvdA catalyzes the conversion of L-ornithine to N5-hydroxyornithine, the first step in the biosynthesis of all hydroxamate-containing siderophores, such as pyoverdine (Minandri, Imperi et al. 2016). This also corroborated with a recent study that downregulation of AlgR will lead to upregulation of PvdA, the first enzyme in the synthesis pathway of pyoverdine. From the point view of host, C. elegans correspondingly upregulated its novel factor, FTN-2, which is ferritin, a host iron scavenging protein known for physiological iron homeostasis. It's showed that, during the interactions, P. aeruginosa upregulated pyoverdine production and secretion, in response to C. elegans's ferritin, indicating the iron competition is a novel process between P. aeruginosa and C. elegans. This showed the robustness of the SILAC approach in revealing host-pathogen interactions and indicating the role of FTN-2 in iron competition with pathogens during infections. To interfere with the iron competition and tilt the favor against the pathogen, I next identified and evaluated an inhibitor which downregulate pyoverdine production. This is achieved by using Galangin as a novel PvdA inhibitor identified by structure-based virtual-screening, where Galangin reduced pyoverdine synthesis and biofilm formation. The combinatorial treatment of Galangin and ciprofloxacin antibiotic reduced P. aeruginosa's ability to establish effective infection and promoted animal survival. My study shows the SILAC method designed in this study is a robust proteomics method with reproducibility and reliability, and results provide insights into the mechanisms detailing host-pathogen interactions and offer novel strategies for anti-infective treatment. |
| Rights: | All rights reserved |
| Access: | open access |
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