Probing Differences in Gene Essentiality Between Mycobacteroides abscessus Smooth and Rough Morphotypes During Infection - PROJECT SUMMARY Several pathogens utilize morphological changes to improve their fitness in a particular environment, but these changes often coincide with broad physiological reprogramming. Mycobacteroides abscessus (MAB) is one such pathogen, presenting as a smooth colony (MABS) in the environment and host, which can transition to a rough (MABR) morphotype following an unknown stimulus during infection. MAB is an emerging pathogen among immunocompromised and immunocompetent individuals causing a wide variety of infections, including respiratory, skin abscesses, soft tissue infection, and bacteremia. Several groups have shown that these two colony morphotypes are phenotypically distinct in vitro and in the host, yet the underlying genetic components contributing to their behavioral differences are still unknown. We have found that MABS and MABR require unique genes for survival indicating that they are molecularly distinct despite having a nearly identical genome. There still remains a significant gap in knowledge in the molecular genetic mechanisms underlying physiology that controls the phenotypic differences. To address this, in Aim 1, I will use transposon insertion (Tn-seq) libraries in both MABS and MABR to examine genes required for survival in a murine abscess model and seven infection relevant conditions (e.g., low oxygen, nitric oxide, metal limitation, abscess infection). The data gathered from individual infection relevant conditions will then be leveraged to determine which antimicrobial mechanisms the two morphotypes face in the host and if they use similar pathways to respond. I have already generated an ordered transposon library which will allow us to confirm our findings of select mutants. In Aim 2, I will investigate uniquely essential genes, MAB_2726c (unique to MABS) and MAB_3329c (unique to MABR) by assaying survival following knocked down with CRISPRi. These genes are transcriptional regulators which likely have broad effects; therefore, I will define their regulome using CHiPSeq and confirm our findings using RNAseq following repression by CRISPRi and controlling for differences in growth using a chemostat. I hypothesize that M. abscessus MABS and MABR have different essential genes when exposed to stress, during infection, and differential essentiality of transcriptional regulators contribute to broad physiological changes that confer phenotypic differences. Due to the lack of effective therapies and a vaccine, this fellowship aims to build a research portfolio that will address the urgent need for further understanding of this pathogen. Tn-seq offers a method for quick and broad identification of genes essential for survival in various environments and public availability of the data generated not only benefits my study of MAB as a postdoc but also as an independent scientist and the field at large.
