IAP-24-109

Elucidating the function of lasso peptides in bacterial interspecies interactions

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 (1,2,3). Microcin J25 is the best studied lasso peptide that may interfere with iron uptake during bacterial growth by hijacking TonB-dependent Fe-siderophore receptors (1,4). Only two outer membrane (OM) receptors, FhuA and PupB, are known for lasso peptides. Inner membrane transport is carried out by YddA, TonB, ExbB, ExbD, and SbmA or similar ABC transporter. Once inside the bacterial cell, the known cytoplasmic target of lasso peptides is RNA polymerase (1,5). Despite of this highly conserved molecular target, antibacterial activity is known to be species or even strain specific and have guilt-by-association approach (6,7). Cellular uptake via interaction with specific membrane receptors may determine the narrow spectrum activity of individual lasso peptides. Moreover, OM receptors may be subjected to iron-mediated transcriptional repression (5). 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 peptide’s function under specific environmental conditions and to better understand the structure-receptor interactions. This will shed light into the specificity of lasso peptides.

The project benefits from previous work done on identification, expression and bioactivity studies of a novel lasso peptide, which the PhD student can develop further. They can express lasso peptides as maltose binding protein (MBP) fusion as well as via entire cloned lasso peptide biosynthetic gene cluster (BGC) using existing MBP fusion protocol as well as develop heterologous expression of the lasso peptide BGC using Streptomyces. Initial bioactivity studies done on expressed novel lasso peptide revealed effect on growth of significant pathogen Staphylococcus aureus. The PhD student will expand antibacterial activity assays to a panel of S. aureus strains including significant antibiotic-resistant strains and test under specific growth conditions.

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 will investigate this further by undertaking bioactivity assays with bacterial strains deficient in polyphosphate metabolising enzymes (polyphosphate kinase, PKK, and polyphosphatase, PPX). The PhD student can also expand the bioactivity assays to include physiology and survival studies on mutants deficient in biofilm formation.

They will undertake/investigate the following objectives/hypotheses:

1. Analysis of lasso peptide(s) and their producer organism(s) from environmental samples using high-throughput analytical techniques such as metagenomics and metaproteomics
2. Analysis of yield and congeners of novel lasso peptide(s) produced by MBP-tagging vectors/engineered strains of bacteria using mass spectrometry
3. Novel lasso peptide interferes with bacterial physiology and survival of specific bacterial species/strains
4. Iron-mediated transcriptional repression of OM receptors affects lasso peptide’s activity
5. The lasso peptide has a novel mode of action via binding/interference with polyphosphate reserves of bacteria.

Subject to progress in identification, expression and bioactivity studies, the PhD student can continue to undertake further bioinformatics and/or structural and biochemical investigation of the novel lasso peptide(s). They can also expand to deciphering the transport pathways of the novel lasso peptide(s) by undertaking phenotype-guided comparative genomics investigation and/or by including protein level interrogation using immunoprecipitation to the transport pathways analysis. It is also possible to include transcriptomics analysis on S. aureus challenged with novel lasso peptide.

The project will contribute to our understanding of lasso peptide’s 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 interspecies interactions of bacteria used in natural environment with potential applications benefiting the public health.

Identification and Expression studies: The student will collect suitable environmental and/or simplified ecosystem samples and analyse them using metagenomics and metaproteomics techniques. The student will express novel lasso peptide BGC(s) using existing MBP-tagging vector protocol as well as develop cloning and heterologous expression with host, Streptomyces coelicolor M1154.
Training and oversight: LK/Origin Peptides Ltd

High-throughput analytical techniques: The student will assess yield and congeners of novel lasso peptide(s) 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 and the University of Dundee. Additional training in chemical analyses (e.g. HPLC, MS) is provided in Institute of Chemical Sciences at Heriot-Watt University, Origin Peptide Ltd and by attending external training courses.

Training and oversight: PC/LK/HY/Origin Peptides Ltd

Bioactivity studies: The student will undertake bacterial fitness and survival studies using selected bacteria, including strains deficient in biofilm formation and polyphosphate metabolism The student will assess the antimicrobial activity of the peptides and the inhibitory action under different growth conditions. The student will involve standard in vitro techniques but also in vivo efficacy in Galleria mellonella larvae.

Training and oversight: LK/PC

Bioinformatics: The student will analyse proteomics 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/Origin Peptides Ltd

Year 1

12 months at Heriot-Watt Univ (Oct-Sept):

– First 3 months: Literature review on lasso peptides, bacterial interspecies interaction, bioactivity assays, Initial laboratory training
– Followed by 6-9 months: Metagenomic studies of environmental samples, MBP-tagged 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 will attend relevant data analysis training courses on bioinformatics.

Year 2

Three months in Origin Peptides Ltd (Oct-Dec) and 3 months at Heriot-Watt (Jan-Mar) and 6 months at St Andrews Univ (Apr-Sept):

– Analyse metaproteomics data and the yield and congeners of novel lasso peptide produced by engineered strains of bacteria using mass spectrometry
– Continue with bioactivity assays
– Undertake bioinformatics analysis of lasso peptide BGC for evaluating potential transport pathways for example.

Presentation of research at national conference(s)

Year 3

Continue and 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).
Further career planning.

The PhD student will acquire the following training & skills:

Interdisciplinary work including microbiology, genomics, proteomics, 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.