ABSTRACT The complex cell wall and biofilm matrix (ECM) act as key barriers to antibiotics in mycobacteria. Here, the ECM-proteins of Mycobacterium marinum ATCC927, a non-tuberculous mycobacterial model, was monitored over three months by label-free proteomics and compared with cell-surface proteins on planktonic cells to uncover pathways leading to virulence, tolerance, and persistence. We show that ATCC927 forms pellicle-type (PBFs) and submerged-type (SBFs) biofilms after two weeks and two days of growth, respectively, and that the increased CelA1 synthesis in this strain prevents biofilm formation and leads to reduced rifampicin tolerance. The proteomic data suggests that specific changes in mycolic acid synthesis (cord factor), Esx1-secretion, and cell-wall adhesins explain the appearance of PBFs as ribbon-like cords and SBFs as lichen-like structures. A subpopulation of cells resisting the 64 × MIC rifampicin (persisters) were detected in both biofilm subtypes, and already in one-week-old SBFs. The key forces boosting their development could include subtype-dependent changes in asymmetric cell division, cell wall biogenesis, tricarboxylic acid/glyoxylate cycle activities, and energy/redox/iron metabolisms. The effect of varying ambient oxygen tensions on each cell type and non-classical protein secretion are likely factors explaining majority of the subtype-specific changes. The proteomic findings also imply that Esx1-type protein secretion is more efficient in PL and PBF cells, while SBF may prefer both the Esx5- and non-classical pathways to control virulence and prolonged viability/persistence. In conclusion, this study reports a first proteomic insight into aging mycobacterial biofilm-ECMs and indicates biofilm subtype-dependent mechanisms conferring increased adaptive potential and virulence on non-tuberculous mycobacteria. IMPORTANCE Mycobacteria are naturally resilient and mycobacterial infections are notoriously difficult to treat with antibiotics, with biofilm formation being the main factor complicating the successful treatment of TB. The present study shows that non-tuberculous Mycobacterium marinum ATCC927 forms submerged- and pellicle-type biofilms with lichen- and ribbon-like structures, respectively, as well as persister cells under the same conditions. We show that both biofilm subtypes differ in terms of virulence-, tolerance- and persistence-conferring activities, highlighting the fact that both subtypes should be targeted to maximize the power of antimycobacterial treatment therapies.

Surface-Shaving Proteomics of Mycobacterium marinum Identifies Biofilm Subtype-Specific Changes Affecting Virulence, Tolerance and Persistence