Investigating the mechanism used by Pyocin S3 to target and kill susceptible Pseudomonas aeruginosa strains
Publication Date
March 15, 2023
Creator
Abstract
Pseudomonas aeruginosa is an opportunistic pathogen that causes infections with a high mortality and morbidity rate. P. aeruginosa produces bacteriocins (known as pyocins) that kill strains of the same species. Therefore, pyocins may have potential as alternatives to antibiotics as novel narrow-spectrum antimicrobials. Pyocin S3 (PyoS3), contains a DNase domain that kills targeted prey bacteria. The DNase domain of PyoS3 is interesting because it does not have an H-N-H fold and does not share sequence homology with type E colicins and other DNase S-type Pyocins. Little is known about the mechanisms that PyoS3 uses to enter prey cells or the structure of its domains. PyoS3 has been suggested to utilise the Type 2 pyoverdine (PvDII) receptor FpvAII to target prey cells. FpvAII uses the TonB1 system for translocation, whereas some colicins that share sequence homology with S-type pyocins (type A and E9 colicins) use the Tol system for translocation. Thus, we hypothesized that PyoS3 utilises either TonB or Tol for translocation.
Genetic engineering was performed to separately overexpress TonB1, TolAIII, and two versions of PyoS3: the receptor and translocation domains (R+T), and the full-length protein with its cognate immunity protein (immS3) to prevent the producing cells from undergoing suicide. These proteins were purified via metal chelation affinity chromatography and size-exclusion chromatography (SEC). Analytical SEC was performed with the aim of detecting potential interactions between these proteins. Evidence for formation of a complex between PyoS3 R+T and TonB1 was obtained, but no complex was evident between PyoS3 R+T and TolAIII. This result supports the hypothesis that PyoS3 utilises FpvAII for uptake, since FpvAII is a TonB-dependent transporter.
To identify genes involved in the uptake and translocation of PyoS3 into prey bacteria, Tn5 transposon mutagenesis was conducted with the PSA 892 PyoS3-sensitive P. aeruginosa strain. Three mutants were analysed, mutants #44, #107 and #362. In comparison to the PSA 892 parental strain, mutant 44 and mutant 362 showed smaller and larger PyoS3 growth inhibition zones, respectively. The transposon insertions in mutant 44 is located in a gene encoding the LuxR family transcriptional regulator VqsR and in the gene encoding a short-chain dehydrogenase reductase (SDR) family oxidoreductase in mutant 362. Both of these genes regulate quorum-sensing (QS) in P. aeruginosa; thus, QS may play a role in the uptake of PyoS3. Mutant 107 was completely resistant to PyoS3-mediated killing and contains a transposon insertion within the pvdM gene. PvdM is an protein that delivers ferribactin into the periplasm to the PvdQ protein for PvD maturation, then the PvdQ delivers the PvD to PvdRT-OpmQ efflux pump to release it out form the cell. In addition, PvdM shares sequence homology with TonB-dependent transporters, which might explain why the PvdM mutant was fully resistant to PyoS3.
Finally, we attempted to determine the crystal structure of PyoS3-Imm3. However the resulted crystal analysis using the Cambridge Structural Database determined the crystals were an imidazole salt. Furthermore, Crystals were successfully grown in a new set of screens; however, X-ray diffraction analysis could not be conducted due to Covid-19 restrictions. Future work could analyse the newly grown crystals to determine if they are proteinaceous and generate X-ray diffraction patterns to solve the structure of PyoS3.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
72228
UoN Repository URI
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Faisal-albaqami-14290936-PhD-thesis-revised.pdf
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Examined. Investigating the mechanism used by Pyocin S3 to target and kill susceptible Pseudomonas aeruginosa strains
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