Expanding the repertoire of methodologies for CRISPR-based genetic manipulation and for hit compound discovery in Mycobacterium abscessus

Abstract

The recent years have seen a rise in infections caused by nontuberculous mycobacteria (NTM), especially by the highly drug-resistant Mycobacterium abscessus. Its large arsenal of intrinsic resistance mechanisms renders most antibiotics ineffective and current treatment regimens are suboptimal in curing M. abscessus infections despite prolonged use of antibiotic cocktails. Some success has been made to develop alternate drug combinations based on repurposing of approved antibiotics, but progress to advance new therapeutic options is hampered by conventional drug screening failing to yield sufficient hits for further development or identifying hits against the same targets such as MmpL3. Additionally, while transposon-sequencing (Tn-seq) has been the mainstay of genome-wide functionality studies, tools for genetic manipulation of M. abscessus have been tedious and inefficient in generating the desired engineered strains due to low rates of homologous recombination and high rates of background spontaneous antibiotic resistance.

In Chapters 2 and 3, we demonstrate analogous two-plasmid workflows that incorporate use of a fluorescent mCherry reporter to identify desired strains easily. We apply the recently developed mycobacterial CRISPR interference (CRISPRi) in Chapter 2 to generate CRISPRi hypomorph strains rapidly in M. abscessus, and subsequently validate through targeted gene silencing essentiality calls made by FiTnEss and HMM, two complementary analytical methods for Tn-seq datasets. In Chapter 3, we convert this CRISPRi platform into one that can perform CRISPR/Cas9-mediated genetic disruptions and establish an analogous two-plasmid workflow that can generate simple targeted single gene disruptions, and more complex multiple gene disruptions with 102-104 higher efficiency than reported for existing recombination-based methods. These complementary tools will greatly expedite targeted genetic manipulation to expand our understanding of the biology, pathogenesis, and drug resistance mechanisms of M. abscessus.

To address the limitations of whole-cell phenotypic screening, we first establish a multiplexed, target-based phenotypic screening platform in Chapter 2, using CRISPRi for the first time to evaluate chemical-genetic interactions in high throughput for the purposes of hit discovery. With our pilot screen, we show our method can predict chemical-genetic interactions with reasonable accuracy (65%) and can identify potential weakly active hit compounds while giving insight into their potential mechanisms of action. Using InhA inhibitors as an example, we hypothesize that InhA may represent a relatively overlooked target for M. abscessus, and that even intrinsic resistance to a well-known anti-tuberculosis drug, isoniazid, is complex and multi-factorial. We then show in Chapter 4 that screening a biased library of anti-tuberculosis bioactives can lead to the identification of new hit candidates with underexplored mechanisms of action, as seen with the discovery of novel compounds predicted to inhibit FadD32 and IlvB1 in M. abscessus. We thus propose two alternate approaches for improved hit discovery. By expanding the target genes included in the hypomorph screening pool, and by expanding curated libraries of anti-tuberculosis bioactives to include diverse mechanisms of action, we can potentially enrich screening hits for novel compounds representing a diverse range of mechanisms.