Development of a mycobacterial enzymatic lysis reagent

Abstract

Mycobacteria are responsible for significant mortality and morbidity worldwide. They have a complex, multi-layered cell wall including peptidoglycan, arabinogalactan and mycolic acid which contributes to their pathogenesis and antibiotic resistance. Diagnosis of mycobacterial infection and resistance profiles can be slow and inaccurate, especially in lower and middle-income countries where species like Mycobacterium tuberculosis are endemic. However lysis of these cells for DNA extraction which are crucial for diagnostics and tracking of these diseases often involve long incubation times, harsh mechanical lysis, or toxic chemicals such as phenol:chloroform. The aim of this thesis is to develop a low cost, rapid, enzymatic lysis reagent. In this investigation we characterise a founding member of a new family of glycoside hydrolases, DG02470, from the gut Bacteroidota species Dysgonomonas gadei capable of hydrolysing β-D-arabinofuranose linkages within mycobacterial arabinogalactan. Through biochemical and structural analysis, we have characterised the kinetic activity and structure of DG02470. Using DG02470 in combination with enzymes previously identified by the lab which target arabinogalactan, we have a developed a DNA extraction reagent using a modified GenElute™ Bacterial Genomic DNA Kit protocol. Enzymatic lysis extraction showed a significant increase in yield of gDNA when the protocol was used against M. smegmatis and M. abscessus when compared to extraction without the novel enzymes which target arabinogalactan and was comparable with bead beating extraction, which is not compatible with long read sequencing with Oxford Nanopore technology . We also show a significant increase in yield when the lysis reagent is utilised for genomic extraction from M. avium subsp. paratuberculosis, M. bovis BCG and Nocardia farcinica. Finally, we have shown we can obtain gDNA from the extracted DNA using the lysis reagent, which can be used for long read sequencing with Oxford Nanopore technology. Enzymatic lysis enabled complete genome assembly with fewer contigs when compared to bead beating. The enzymatic lysis reagent presented in this investigation has the potential to allow for a more accessible method of DNA extraction from mycobacteria, aiding in future research and diagnostics.