12pm – 1.30pm GMT, 26 March 2021 ‐ 1 hour 30 mins
Presentation
Fighting Antimicrobial Resistance with machine learning, omics and big data analysis - Professor Tania Dottorini
These talks are led by professionals in their field and are an opportunity to explore research conducted beyond your institution or organisation. This session is themed around biotechnology.
ECS talks are an opportunity for scientists who are beginning their career to showcase their recent work. The project being presented does not have to be complete, but it’s a great way to practice presenting and answering audience questions in a friendly, supportive environment.
To understand regulation of the key transcriptional enzyme, RNA polymerase, in Streptomyces. Also, to investigate the influence of RNA polymerase levels on antibiotic production and expression of silent gene clusters, that are not expressed in this organism under standard growth conditions and potentially encode novel antibiotics.
Analysis of RNA -seq data from S. coelicolor and S. venezuelae revealed that the rpoBC operon, which encodes the large β and β’ subunits of RNA polymerase, is subjected to a largely unstudied form of gene regulation known as reiterative transcription (RT) at the transcriptional start site. Expression level from wildtype and mutant rpoBC promoter regions were tested using qRT-PCR before and after the induction of the stringent response to determine effects of RT on transcript level during both exponential growth and in response to stress.
RpoBC is downregulated quickly after the induction of the stringent response in Streptomyces. RT is confirmed to occur at the transcriptional start site of the rpoBC operon and is hypothesised to contribute to this drop in expression observed in response to stress.
The Streptomyces genus are well known to be prolific producers of useful secondary metabolites, including over half of all clinically relevant antibiotics. Enabling the production of previously uncharacterised compounds may allow the production of new antibiotics that can contribute to overcoming the current antimicrobial resistance crisis.
Bentley, S., Chater, K., Cerdeño-Tárraga, A.-M., Challis, G. L., Thomson, N. R., James, K. D., Harris, D. E., Quail, M. a, Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C. W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., … Hopwood, D. a. (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature, 417(6885), 141–147. https://doi.org/10.1038/417141a
Hesketh, A., Chen, W. J., Ryding, J., Chang, S., & Bibb, M. (2007). The global role of ppGpp synthesis in morphological differentiation and antibiotic production in Streptomyces coelicolor A3(2). Genome Biology, 8(8). https://doi.org/10.1186/gb-2007-8-8-r161
Küster, C., Piepersberg, W., & Distler, J. (1998). Cloning and transcriptional analysis of the rplKA-or f31-rplJL gene cluster of Streptomyces griseus. Mol Gen Genet, 257(2), 219–229. http://www.ncbi.nlm.nih.gov/pubmed/9491081
Ochi, K., & Hosaka, T. (2013). New strategies for drug discovery: Activation of silent or weakly expressed microbial gene clusters. Applied Microbiology and Biotechnology, 97(1), 87–98. https://doi.org/10.1007/s00253-012-4551-9
Turnbough, C. L. (2011). Regulation of gene expression by reiterative transcription. Current Opinion in Microbiology, 14(2), 142–147. https://doi.org/10.1016/j.mib.2011.01.012
The University of Nottingham
Professor Tania Dottorini is an Associate Professor in Bioinformatics at the Nottingham School of Veterinary Medicine and Science (since June, 2016). Previously, a research fellow at the Imperial College London (2011 - 2016), Professor Dottorini's research themes include the development of novel bioinformatics and machine learning solutions to understand and diagnose infectious diseases in humans and animals.
Professor Dottorini's current interests are in the development of original methods and algorithms to gain deeper insight in biological problems related to human and animal health. To this purpose their research aims to merge different disciplines and knowledge/skills, including: bioinformatics and machine learning to develop predictive models and solve data mining tasks, in particular in scenarios involving large-scale data analysis from omics technologies (genomics, transcriptomics, proteomics, etc).
This study aimed to investigate the potential of phage depolymerases to prevent and treat catheter-associated urinary tract infections (CAUTIs) caused by Proteus mirabilis. The main objectives of the project were:
1. Identification and sequencing of bacteriophages demonstrating strong ability to depolymerise exopolysaccharides (EPS) produced by P. mirabilis;
2. Recombinant production of phage enzymes with such activities;
3. Screening of expressed recombinant proteins for potential antimicrobial activity against P .mirabilis isolates and
4. In vitro testing of the isolated depolymerases as anti-biofilm agents.
A number of P. mirabilis bacteriophages were isolated using standard methods from environmental and agricultural sources. Subsequent screening for the presence of zones of matrix depolymerisation (halos) surrounding phage plaques formed on P. mirabilis lawns resulted in identification of several candidate phages. These were sequenced and their genomes annotated. Based on the results of sequence comparisons to experimentally verified depolymerases, a gene of a tail-spike protein of a new Proteus phage PM (isolated and characterised in this study), was selected for molecular cloning. The vector construct containing the tail-spike gene was successfully used for heterologous expression in E. coli and the resulting recombinant tail-spike protein was purified and concentrated. The ability of the protein to degrade EPS matrix of P. mirabilis B2000 (the host of PM phage), was confirmed by standard spot tests. In the next phase, in vitro tests were conducted with the recombinant tail spike protein, which demonstrated its ability to reduce the adherence of P. mirabilis to surfaces, thus hindering normal biofilm formation. Finally, the stability of the depolymerase against one of the major virulence factors of uropathogenic P. mirabilis, the 54-kDa extracellular metalloprotease ZapA, was investigated. Despite partial degradation, the tail-spike protein retained its depolymerase activity.
With P. mirabilis being a common cause of difficult-to-treat CAUTIs, novel antimicrobials are required to treat such infections, especially in light of the increasing antibiotic resistance. We have shown the ability of Proteus bacteriophages isolated in this study to significantly reduce microbial load on indwelling catheters. In this study we have isolated and characterised the first phage-encoded depolymerase specific to P. mirabilis and demonstrated the ability of this phage-derived enzyme to delay the formation of biofilms by reducing the number of bacteria attaching to surfaces. This work highlights the potential for phages and their respective enzymes to act as effective antimicrobials, particularly in the treatment of P. mirabilis CAUTIs.
With antimicrobial resistance at an all timeall-time high, the need for novel therapeutics is essential to tackle such issues. Bacteriophages have demonstrated remarkable potential as novel therapeutic agents against infections caused by various bacterial pathogens, including multi- and pan-drug-resistant ones, but our knowledge of the advantages, disadvantages and the range of applications of phage therapy is incomplete. As P. mirabilis via its urease production causes the build up of biofilm crystals and subsequent blockage of urinary catheters, this presents a serious problem, particularly for immunocompromised patients, as bacteraemia can occur, usually resulting in death. We for the first time isolated a depolymerase enzyme from a Proteus phage and demonstrated its ability to control biofilms formed by Proteus. As phage depolymerases do not kill bacteria, acting as anti-virulence agents instead, the use of phage depolymerases could become a promising therapeutic option in treatment of CAUTIs, helping to reduce the use of antibiotics and contributing to tackling the antibiotic resistance crisis.
1. Macleod SM, Stickler DJ. Species interactions in mixed-community crystalline biofilms on urinary catheters. J Med Microbiol. 2007;56(11):1549-1557. doi:10.1099/jmm.0.47395-0
2. Alves DR, Nzakizwanayo J, Dedi C, et al. Genomic and Ecogenomic Characterization of Proteus mirabilis Bacteriophages. 2019;10(August):1-14. doi:10.3389/fmicb.2019.01783
3. Milo S, Thet NT, Liu D, Nzakizwanayo J, Jones B V., Jenkins ATA. An in-situ infection detection sensor coating for urinary catheters. Biosens Bioelectron. 2016;81:166-172. doi:10.1016/j.bios.2016.02.059
4. Nicolle LE. Infections associated with urinary catheters. Clin Infect Dis Second Ed. 2015:722-727. doi:10.1017/CBO9781139855952.122
5. Clokie MRJ, Kropinski AM. Bacteriophages Methods and Protocols Volume 2: Molecular and Applied Aspects. Methods Mol Biol. 2009;502:xxii, 373 p. doi:10.1007/978-1-60327-565-1