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.
