Biochemical characterization of cellulose isolated from mycobacterial biofilms

Mycobacterium tuberculosis (Mtb) forms cellulose-enriched biofilms. These biofilms play a crucial role in the tuberculosis pathogenesis and drug tolerance exhibited by Mtb in vivo. However, the biophysical characteristics of the cellulose remain poorly explored. In the current study, the Updegraff method was employed to enrich acid-hydrolysis-resistant polysaccharides containing cellulose from three distinct models of biofilms: pellicle, submerged, and macrocolony biofilms of slow-growing Mtb and fast-growing M. smegmatis. The isolated material was subjected to Elemental analysis, Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (PXRD), thermogravimetric analysis, and field-emission scanning electron microscopy (FESEM) analysis. PXRD analysis confirmed the presence of cellulose in this biomaterial. PXRD-derived crystallinity index for cellulose isolated from these biofilms was in the range of 52% to 61%, indicating that cellulose in mycobacterial biofilms is amorphous, rather than crystalline. Furthermore, the spectral peak shift in FTIR spectra, combined with a decrease in the absorption band at 1430 cm^-1 (also known as the crystallinity band), and a shift in the thermogravimetric spectra, further confirms the presence of amorphous cellulose. These are the first pieces of evidence to indicate that cellulose isolated from mycobacterial biofilms is amorphous in nature. Amorphous cellulose is found in the biofilms of E. coli, Salmonella, Sarcina, and Rhodobacter sphaeroides, while crystalline cellulose is found in the biofilms of K. xylinus. The nature of cellulose affects several features of biofilms, including retention of water, biofilm architecture etc. This study also conducted the first comprehensive and comparative analyses of the glycosyl composition and linkage of acid hydrolysis-resistant material obtained from pellicle, submerged, and macrocolony biofilms of Mtb and M. smegmatis. This analysis suggests that the presence of large quantities of cellulose in mycobacterial biofilms. Glycosyl composition and linkage analyses also revealed the presence of yet unidentified polysaccharides, including those rich in arabinose and mannose. The exact biochemical nature and role of these polysaccharides in mycobacterial biofilms and the pathogenesis of tuberculosis remain unknown. In summary, these results expand the current understanding of the nature of EPS in mycobacterial biofilms, which can be further targeted to disrupt biofilm integrity and improve antibiotic efficacy.