Figure 9: Effect of ECM-degrading enzymes on the development of Mtb biofilms.

(a) Mtb cells overexpressing GFP were treated with 6 mM DTT to induce TRS. After 3 h of DTT exposure, the cultures were treated with proteinase K, cellulase, α-amylase, lipase or DNase. After 29 h, the effects of various enzymes on the formation of Mtb biofilms were observed through CLSM. Cellulase and proteinase K inhibited the Mtb biofilm formation, suggesting that cellulose fibres and unidentified structural proteins play a critical role in the early stages of biofilm attachment. In contrast, amylase, lipase and DNase had no effect on the structural integrity of biofilm initiation and maturation. (b) The biomass of biofilms developed in the presence of enzymes capable of degrading the ECM was estimated using COMSTAT. (c) CV assays of Mtb biofilms developed despite the presence of cellulase, lipase, proteinase K and DNase. Cellulase and proteinase K inhibited biofilm development, whereas lipase, DNase and α-amylase had no effect on the biofilm formation and hence on the CV staining. The data presented in b,c are expressed as the mean (±s.e.m.). Statistical significance was determined using Student’s t-test. *P<0.05. (b,c) Representative of at least three independent biological experiments performed in triplicate.