Kirsi Savijoki,#a Henna Myllymäki,#b,* Hanna Luukinen,b Lauri Paulamäki,b Leena-Maija Vanha-aho,b Aleksandra Svorjova,b Ilkka Miettinen,a Adyary Fallarero,a,* Teemu O. Ihalainen,b Jari Yli-Kauhaluoma,c Tuula A. Nyman,d and Mataleena Parikkacorresponding authorb


"aDrug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
bFaculty of Medicine and Health Technology, Tampere University, Tampere, Finland
cDrug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
dInstitute of Clinical Medicine, Department of Immunology, Rikshospitalet, Oslo University Hospital, Oslo, Norway
corresponding authorCorresponding author.
#Contributed equally.
Mataleena Parikka: [email protected]
*Present address: Henna Myllymäki, UoE Centre for Inflammation Research, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom; Adyary Fallarero, Thermo Fischer Scientific, Vantaa, Finland.
Kirsi Savijoki and Henna Myllymäki contributed equally to this work. Author order was determined in order of increasing seniority.
Citation Savijoki K, Myllymäki H, Luukinen H, Paulamäki L, Vanha-aho L-M, Svorjova A, Miettinen I, Fallarero A, Ihalainen TO, Yli-Kauhaluoma J, Nyman TA, Parikka M. 2021. Surface-shaving proteomics of Mycobacterium marinum identifies biofilm subtype-specific changes affecting virulence, tolerance, and persistence. mSystems 6:e00500-21.




The complex cell wall and biofilm matrix (ECM) act as key barriers to antibiotics in mycobacteria. Here, the ECM and envelope proteins of Mycobacterium marinum ATCC 927, a nontuberculous mycobacterial model, were monitored over 3 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 ATCC 927 forms pellicle-type and submerged-type biofilms (PBFs and SBFs, respectively) after 2 weeks and 2 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 suggest 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 64× MIC rifampicin (persisters) was detected in both biofilm subtypes and already in 1-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 various ambient oxygen tensions on each cell type and nonclassical protein secretion are likely factors explaining the majority of the subtype-specific changes. The proteomic findings also imply that Esx1-type protein secretion is more efficient in planktonic (PL) and PBF cells, while SBF may prefer both the Esx5 and nonclassical pathways to control virulence and prolonged viability/persistence. In conclusion, this study reports the first proteomic insight into aging mycobacterial biofilm ECMs and indicates biofilm subtype-dependent mechanisms conferring increased adaptive potential and virulence of nontuberculous mycobacteria.



In situ imaging of the SBFs was conducted with Nikon FN1 upright epifluorescence microscope equipped with a 20×/0.8 dry lens objective, Hamamatsu ORCA-Flash4.0 V3 digital complementary metal-oxide-semiconductor (CMOS) camera, and CoolLED pE-4000 light source.

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The pE-4000 Universal Illumination System offers 16 selectable wavelengths from 365 - 770 nm, making it a highly flexible illuminator covering a wide variety of fluorophores

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