IAP2-23-141

Elucidating the function of lasso peptides in bacterial cell-cell communication

Lasso peptides are low molecular weight peptides believed to be used by bacteria for colonization and survival. They are a diverse subclass of ribosomally synthesized and post-translationally-modified natural products with wide range of bioactivities such as enzyme inhibition and receptor antagonistic as well as antimicrobial activities. Microcin J25 is the best studied lasso peptide that can interfere with iron uptake during bacterial growth by hijacking siderophore receptors and preventing competitor from scavenging iron from the environment. Only two outer membrane (OM) receptors, FhuA and PupB, are known for lasso peptides and one receptor, YddA, for inner membrane. Once inside the bacterial cell, the known cytoplasmic target of lasso peptides is RNA polymerase. Cellular uptake via interaction with specific receptors may determine the narrow spectrum activity of individual lasso peptides. Poor understanding and limited strategies for elucidating lasso peptides role in nature is hindering efforts to develop lasso peptide compounds to tackle antimicrobial resistance. This project aims to elucidate lasso peptides role in nature to better understand the structure-receptor interactions and shed light into the specificity of lasso peptides.

The project benefits from previous work done on expression studies of a novel lasso peptide biosynthetic gene cluster (BGC), which the PhD student can develop further. They can express the lasso peptide BGC using existing maltose binding protein (MBP) protocol as well as develop heterologous expression of the lasso peptide BGC using Streptomyces. Initial bioactivity studies done on MBP expressed novel lasso peptide revealed effect on growth of Staphylococcus aureus. PhD student can expand the bioactivity assays further to include fitness and survival studies on selected bacteria including E. coli MG1655 and set of relevant KEIO collection mutants deficient in motility/biofilm formation.

The novel lasso peptide’s amino acid sequence revealed a strong positive charge that is likely to bind (poly)phosphates via electrostatic interaction. Polyphospates are known to have pleiotropic roles in bacteria including fitness/survival. The PhD student is to investigate this further by undertaking bioactivity assays with bacterial strains deficient in polyphosphate metabolising enzymes (polyphosphate kinase, PKK, and polyphosphatase, PPX).

They will undertake/investigate the following objectives/hypothesis:

1. Analysis of yield and congeners of novel lasso peptide produced by MPB-tagging vectors/engineered strains of bacteria using high-throughput analytical techniques
2. Novel lasso peptide interferes with bacterial fitness of specific bacterial species
3. The lasso peptide has a novel mode of action via binding/interference with polyphosphate reserves of bacteria

Subject to progress in bioactivity studies, PhD student can continue to undertake further bioinformatics and/or structural and biochemical investigation of the novel lasso peptide. They can also expand to deciphering the transport pathways of the novel lasso peptide by undertaking phenotype-guided comparative genomics investigation. Alternatively, they may choose to pursue expression/bioactivity studies of other novel lasso peptide(s).

The project will contribute to our understanding of lasso peptides role in nature by aiming to reveal novel mode of action in bacterial interactions. It will shed light into bacterial behaviour under specific (iron limited) environmental conditions. This work aligns with NERC disciplinary remit by unravelling the complex cell-cell communication of bacteria used in natural environment with potential applications benefiting the health of the public.

Methodology

Expression studies: The student will express novel lasso peptide BGC using existing MBP-tagging vector protocol as well as develop cloning and heterologous expression with host, Streptomyces coelicolor M1154.
Training and oversight: LK

High-throughput analytical techniques: The student will assess yield and congeners of novel lasso peptide using techniques such as high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS). The student will have access to an excellent mass spectrometry and proteomics facility at the University of St Andrews. Additional training in chemical analyses (e.g. HPLC, MS) is provided in Institute of Chemical Sciences at Heriot-Watt University, and by attending external training courses.
Training and oversight: PC/LK/HY

Bioactivity studies: The student will undertake bacterial fitness and survival studies using selected bacteria, including strains deficient in motility, biofilm formation, polyphosphate metabolism.
Training and oversight: LK/PC

Bioinformatics: The student will analyse proteomics/metabolomics and DNA sequencing data using bioinformatics tools with cloud-based computing CLIMB MRC. Additional training is provided by viewing taught material online and by attending external training courses.
Training and oversight: LK

Project Timeline

Year 1

First 12 months in Heriot-Watt (Oct-Sept):

– First 3 months: Literature review on lasso peptides, bacterial cell-cell communication, bioactivity assays, Initial laboratory training
– Followed by 6-9 months: MBP-tagged expression as well as cloning and heterologous expression of novel lasso peptide, Initial bioactivity assays
– At the end of the first year, the student is to submit first year review report and attend major review

In addition to this the student is to attend relevant data analysis training courses on bioinformatics.

Year 2

6 months in St Andrews (Oct-March) and 6 months in Heriot-Watt (April-Sept):

Continue with bioactivity assays

Analyse the yield and congeners of novel lasso peptide produced by MPB-tagging vectors/engineered strains of bacteria using high-throughput analytical techniques

Undertake bioinformatics analysis of lasso peptide BGC for evaluating potential transport pathways for example.

Presentation of research at national conference(s)

Year 3

Finalise laboratory experiments and bioinformatics analyses and submit manuscript(s) for publication. Prepare/write up data/results for dissertation.

Presentation of research at national conference(s)

Year 3.5

Finalise dissertation and manuscript(s) for publication

Presentation at national conference(s)

Apply for jobs

Training
& Skills

The PhD student will obtain the following training & skills:

Interdisciplinary work including microbiology, analytical chemistry and bioinformatics. Scientific research and communication skills e.g. critical evaluation of scientific literature, academic integrity, study design and laboratory analysis/analytical skills, data handling, statistical analysis and data storage, scientific report writing and presentation. In additional to this the student can engage with teaching at undergraduate level as a demonstrator during practical courses.

References & further reading

Do T, Thokkadam A, Leach R, Link AJ. Phenotype-Guided Comparative Genomics Identifies the Complete Transport Pathway of the Antimicrobial Lasso Peptide Ubonodin in Burkholderia. ACS Chem Biol. 2022 Aug 19;17(8):2332-2343. doi: 10.1021/acschembio.2c00420. Epub 2022 Jul 8. PMID: 35802499; PMCID: PMC9454059.

Hegemann JD, Zimmermann M, Xie X, Marahiel MA. Lasso peptides: an intriguing class of bacterial natural products. Acc Chem Res. 2015 Jul 21;48(7):1909-19. doi: 10.1021/acs.accounts.5b00156. Epub 2015 Jun 16. PMID: 26079760.

Mathavan I, Zirah S, Mehmood S, Choudhury HG, Goulard C, Li Y, Robinson CV, Rebuffat S, Beis K. Structural basis for hijacking siderophore receptors by antimicrobial lasso peptides. Nat Chem Biol. 2014 May;10(5):340-2. doi: 10.1038/nchembio.1499. Epub 2014 Apr 6. PMID: 24705590; PMCID: PMC3992131.

Wang M, Fage CD, He Y, Mi J, Yang Y, Li F, An X, Fan H, Song L, Zhu S, Tong Y. Recent Advances and Perspectives on Expanding the Chemical Diversity of Lasso Peptides. Front Bioeng Biotechnol. 2021 Sep 24;9:741364. doi: 10.3389/fbioe.2021.741364. PMID: 34631682; PMCID: PMC8498205.

Xiu H, Wang M, Fage CD, He Y, Niu X, Han M, Li F, An X, Fan H, Song L, Zheng G, Zhu S, Tong Y. Discovery and Characterization of Rubrinodin Provide Clues into the Evolution of Lasso Peptides. Biochemistry. 2022 Apr 5;61(7):595-607. doi: 10.1021/acs.biochem.2c00029. Epub 2022 Mar 17. PMID: 35298141.

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