TY - JOUR
T1 - Surface-shaving proteomics of mycobacterium marinum identifies biofilm subtype-specific changes affecting virulence, tolerance, and persistence
AU - Savijoki, Kirsi
AU - Myllymäki, Henna
AU - Luukinen, Hanna
AU - Paulamäki, Lauri
AU - Vanha-Aho, Leena Maija
AU - Svorjova, Aleksandra
AU - Miettinen, Ilkka
AU - Fallarero, Adyary
AU - Ihalainen, Teemu O.
AU - Yli-Kauhaluoma, Jari
AU - Nyman, Tuula A.
AU - Parikka, Mataleena
N1 - Funding Information:
This work received financial support from the Academy of Finland (project no. 322010 to A.F., J.Y.-K., and M.P. and no. 326674 to M.P.), the Jane and Aatos Erkko Foundation (to M.P.), the Sigrid Jusélius Foundation (to M.P.), the Foundation of the Finnish Anti-Tuberculosis Association (Suomen Tuberkuloosin Vastustamisyhdistyksen Säätiö; to H.L., L.-M.V.-A., and K.S.), and the Tampere Tuberculosis Foundation (to M.P., K.S., H.M., H.L., and L.-M.V.-A.). Mass spectrometry-based proteomic analyses were performed by the Proteomics Core Facility, Department of Immunology, University of Oslo/Oslo University Hospital, which is supported by the Core Facilities program of the South-Eastern Norway Regional Health Authority. This core facility is also a member of the National Network of Advanced Proteomics Infrastructure (NAPI), which is funded by the Research Council of Norway INFRASTRUKTUR-program (project number 295910 to T.A.N.).
Publisher Copyright:
© 2021 American Society for Microbiology. All rights reserved.
PY - 2021/6
Y1 - 2021/6
N2 - 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. 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 tuberculosis (TB). The present study shows that nontuberculous Mycobacterium marinum ATCC 927 forms submergedand 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.
AB - 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. 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 tuberculosis (TB). The present study shows that nontuberculous Mycobacterium marinum ATCC 927 forms submergedand 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.
KW - Biofilm matrix
KW - Biofilms
KW - cell surface proteomics
KW - Mycobacterium marinum
KW - Persistence
KW - Tolerance
U2 - 10.1128/mSystems.00500-21
DO - 10.1128/mSystems.00500-21
M3 - Article
AN - SCOPUS:85109327941
VL - 6
IS - 3
M1 - e00500-21
ER -