Abstract Rationale Mycobacterium abscessus (MABS) exists as two morphotypes - the environmental, smooth infecting form and the host-adapted rough morphotype. Six smooth and six rough MABS isolates from soil and wells were studied including four matched smooth-rough pairs plus two additional smooth and two additional rough isolates. All isolates were spread onto 7H10 plates and imaged using the Keyence VHX-7000 microscope. Figure 1 shows high-resolution images of smooth and rough colonies that illustrate smooth-to-rough morphotype transition via apparent loss of extracellular matrix (ECM) and colony dome collapse, releasing exposed bacilli to create a broadened rough fringe. We hypothesize that loss of ECM among smooth MABS destabilizes the domed colony structure, releasing bacilli, increasing colony size and reducing colony circularity. Methods To understand the transcriptomic and genomic changes among smooth and rough MABS, isolates were grown in liquid culture for 7 days, RNA extracted, and RNAseq performed to identify differentially expressed genes. To add context, four respiratory smooth-rough pairs isolated from people with cystic fibrosis were included to elucidate factors driving morphotype transition. DNA was extracted and whole genome sequenced using Illumina NovaSeq and Oxford Nanopore technologies. Short and long reads were combined to create a de novo hybrid genome assembly, with pangenome comparison using Roary. Results By microscopy, smooth colonies showed an external coating resembling ECM and beginning stages of biofilm formation (Figure 1) which was absent among rough colonies. Generally, isolate gene expression clustered by morphotype. Multiple genes were upregulated in roughs compared to smooths, including resA₁, acg, eccE3, argB, and sigB, all of which are implicated in thiol reductive stress induced biofilm formation, which is characterized by an ECM containing high levels of cellulose and polysaccharides, increased iron sequestration, and higher survival in oxidative stress and hypoxic conditions, such as in human macrophages. Smooth isolates, however, had upregulation of papA3₁, involved in the biosynthesis of polyacyltrehalose and development of air-exposed biofilms containing an ECM saturated in lipids and mycolic acids. Combined analysis of Illumina short-read and Nanopore long-read data revealed isolates tended to genomically cluster by pair rather than morphotype. Loss of the cwsA, ppiA, and glpG genes were observed for respiratory rough isolates; already, glpG has roles in biofilm formation for M. smegmatis and was lost most often (n = 3/4 isolates). Conclusion Rough MABS isolates were found to have loss of canonical ECM material, increased expression of stress-related genes, and loss of genes related to biofilm formation. This abstract is funded by: National Science Foundation #1743587
Honda et al. (Fri,) studied this question.