Phenotypic profiling of mycobacterial mutant libraries, through chemical-genomic screening

Sullivan, Rudi (2024). Phenotypic profiling of mycobacterial mutant libraries, through chemical-genomic screening. University of Birmingham. Ph.D.

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Abstract

The genus Mycobacterium encompasses numerous important human and environmental pathogens including the causative agent of tuberculosis, Mycobacterium tuberculosis, which is responsible for around 1.5 million deaths each year. Although curable, the treatment for M. tuberculosis is extensive and antibiotic resistance is an ongoing concern. Therefore, there is a demand for the development of new anti-mycobacterial drugs. Functional characterisation of mycobacterial genomes could lead to the discovery of new drug targets and help to characterise drug resistance pathways. However, mycobacterial genomes are poorly characterised with ~25% of the M. tuberculosis genome remaining functionally unannotated. Therefore, new methods are required for the characterisation of the outstanding genome.

Ordered mutant libraries are extremely valuable resources that not only provide a catalogue of mutants for further study but can also be combined with functional genomics to help characterise bacterial genomes. As Mycobacterium abscessus genetic resources are limited, we constructed an ordered M. abscessus mutant library. The library was constructed via random transposon mutagenesis using the phage delivery vector, ΦMycoMarT7, in which 18,432 mutants were manually ordered into 96-well plates. Although we were unable to finish the deconvolution of the library, once complete, this library will represent the largest collection of M. abscessus mutants in existence.

To demonstrate the utility of ordered libraries for functionally characterising bacterial genomes, we optimised a method for the chemical-genomic screening of mycobacteria using a M. bovis BCG mutant library. We first condensed the M. bovis BCG mutant library so that it was compatible with high-throughput screening. To increase experimental throughput and reduce human error, we implemented robotics to develop the chemical genomics method. In this method, M. bovis BCG liquid source plates were replicated in a 384-colony format onto solid agar replica plates containing sub-inhibitory concentrations of different perturbations. Colony morphology and mutant fitness was then assessed via IRIS and ChemGAPP. To validate the method, we conducted a pilot screen using 2 library plates and 22 conditions and discovered 76 mutants had significant fitness changes in response to at least one condition, many of which had insertions in genes that had not been previously characterised. Through clustering analysis, we also identified various uncharacterised genes that may fulfil roles in important biological pathways.

Lastly, we developed a pipeline for validating the screening hits in which markerless mycobacterial gene deletion mutants were generated via ORBIT and phenotypically characterised via microscopy, growth kinetics and chemical screening. This research contributed towards the discovery of two cell-wall associated enzymes, the D,Dcarboxypeptidase, LpqF, and the α-1,6 mannanase, MSMEG_0740 (Rv0365c).

Therefore, taken together, this work outlines a method for characterising mycobacterial genomes through the generation of ordered mutant libraries, functional genomics and reverse-genetic phenotyping. Using this pipeline, we successfully generated an ordered M. abscessus mutant library which will represent a useful resource for future study, we conducted a pilot functional genomic screening to phenotypically profile mutants of numerous previously uncharacterised genes and through reverse-genetics, we also discovered two new enzymes involved in the synthesis and recycling of the mycobacterial cell wall.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):