It is estimated that nearly 5 million patients who die annually have at least one antimicrobial-resistant infection and these infections directly cause 1.3 million deaths globally. This is only expected to increase over the coming decades. With this tremendous problem in mind, scientists from VIB in Belgium and Inscripta partnered to perform an experiment using the Onyx™ platform to map protein function and stability systematically across three essential genes E. coli. Their goal was to use these maps both as a guide to antibiotic development and also as a window into how microbes become resistant to these compounds. At Inscripta we pride ourselves in adapting to our customers’ needs and being a research partner. VIB was part of an early access program, their efforts were led by Liselot Dewachter, a postdoc in Jan Michiels’s lab along with the Inscripta applications development team. The results were recently published in bioRxiv, below is a summary of their methods and results.

The experimental design was to generate what’s called a “saturation editing” library for three essential genes involved in cell envelope synthesis in E. coli. A saturation editing library changes each amino acid in a protein at each position to every other amino acid with the goal of identifying key positions in the protein. This is usually associated with plasmid-based editing but for more meaningful results the edits were made in their native genomic locations. If you’ve ever worked with essential genes, you can appreciate how difficult they are to modify because this process often leads to cell death. The three genes of interest were FabZ, LpxC, and MurA, all involved in cellular envelope synthesis. The three genes varied in size, FabZ, for example, is 151 residues and required ~3000 mutants whereas MurA is 419 residues and required ~8300 mutants. The final library created contained ~17,000 variants total, all generated using saturation editing or “deep mutational scanning”.

Another novel aspect of this experiment was that large numbers of edits are generally tracked using barcodes, but VIB wanted to observe the edits directly on the genome which further showcases the adaptability of the Onyx platform. Since the entire library focused on a small number of genomic loci, it was possible to directly survey edits using targeted genomic amplification. Genomic amplicons for each gene, each approximately 2kb in size, were created by targeted PCR amplification followed by sequencing to read out the edit status. Every time an edit was detected it was confirmed to actually be on the genome, not on the barcode.

Analysis showed that not all essential genes were equivalent in terms of tolerance to amino acid substitutions. MurA was fairly intolerant to substitutions (around 50%), but FabZ was more tolerant, showing 83% of all attempted edits were successful and observed in the assay. It was reassuring to see patterns of substitution tolerance that make sense biologically as there were certain positions in the protein that are intolerant to substitutions. There is a high level of overlap with these areas and known functional sites in the proteins that have been shown previously to be intolerant to modification. To better understand the biological function of these results, the tolerance scores were mapped against the protein structure. For each gene, this analysis revealed interesting patterns; the intolerant amino acids were clustered in specific regions, typically around active sites of the enzyme and other functionally important regions.

It was decided to extend the analysis and combine the Onyx data and orthogonal data for an even more meaningful result. VIB used a measurement termed relative solvent accessibility which is a measure of how deeply embedded an amino acid is in a protein’s hydrophobic core. When accessibility is 0 there is generally also 0 tolerance for substitutions. The authors speculated that these amino acids may be required for protein folding stability and are buried deep inside and not able to be changed. Other amino acids found to have higher solvent accessibility and therefore are closer to the periphery of the protein that still exhibited low tolerance are likely important for overall protein function.

The end goal was to connect these protein function landscapes to the development of antibiotic resistance and, ultimately, identification of new and more robust compounds. To investigate whether the Onyx libraries also contain edits that enable resistance to existing antimicrobials, the libraries were grown in the presence of common antibiotics to find resistance clones. This was accomplished by plating the library out on media containing several different antibiotics at several times the minimum inhibitory concentration. They were then assessed for the emergence of clones that survived relative to the baseline spontaneous mutation rate. It was observed that cells edited by Onyx also have a much higher rate of colony formation. Thus, Onyx libraries not only tell us which residues are most essential for the protein but also indicate which mutations may lead to escape of proteins from suppression with an antibiotic. The edits that survive antimicrobial activity are also clustered in very specific regions. This gives a better understanding of essential genes and how they interact with compounds and can help lead to development of new compounds which are more robust to development of resistance. The authors concluded that, among the three proteins studied, MurA is the superior antimicrobial target due to its low mutational flexibility which decreases the chance of acquiring resistance-conferring mutations that simultaneously preserve MurA function.

You can read the full publication here.

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^References:

^{2022, January; The latest estimates of global anti-microbial resistance show urgent policy action is needed to save lives, IHME, https://www.healthdata.org/new…}