Rv1717 Is a Cell Wall - Associated β-Galactosidase of Mycobacterium tuberculosis That Is Involved in Biofilm Dispersion

Abstract

Understanding the function of conserved hypothetical protein (CHP)s expressed by a pathogen in the infected host can lead to better understanding of its pathogenesis. The present work describes the functional characterization of a CHP, Rv1717 of Mycobacterium tuberculosis (Mtb). Rv1717 has been previously reported to be upregulated in TB patient lungs. Rv1717 belongs to the cupin superfamily of functionally diverse proteins, several of them being carbohydrate handling proteins. Bioinformatic analysis of the amino acid sequence revealed similarity to glycosyl hydrolases. Enzymatic studies with recombinant Rv1717 purified from Escherichia coli showed that the protein is a β-D-galactosidase specific for pyranose form rather than the furanose form. We expressed the protein in Mycobacterium smegmatis (Msm), which lacks its ortholog. In MsmRv1717, the protein was found to localize to the cell wall (CW) with a preference to the poles. MsmRv1717 showed significant changes in colony morphology and cell surface properties. Most striking observation was its unusual Congo red colony morphotype, reduced ability to form biofilms, pellicles and autoagglutinate. Exogenous Rv1717 not only prevented biofilm formation in Msm, but also degraded preformed biofilms, suggesting that its substrate likely exists in the exopolysaccharides of the biofilm matrix. Presence of galactose in the extracellular polymeric substance (EPS) has not been reported before and hence we used the galactose-specific Wisteria floribunda lectin (WFL) to test the same. The lectin extensively bound to Msm and Mtb EPS, but not the bacterium per se. Purified Rv1717 also hydrolyzed exopolysaccharides extracted from Msm biofilm. Eventually, to decipher its role in Mtb, we downregulated its expression and demonstrate that the strain is unable to disperse from in vitro biofilms, unlike the wild type. Biofilms exposed to carbon starvation showed a sudden upregulation of Rv1717 transcripts supporting the potential role of Rv1717 in Mtb dispersing from a deteriorating biofilm.

Mycobacterial Adhesion: From Hydrophobic to Receptor-Ligand Interactions

“…The presence of cellulose in the biofilm extracellular matrix was also reported for M. avium, Mycobacterium fortuitum, and M. smegmatis [65,66]. The detection of mannose and galactose in the composition of M. ulcerans [43] and M. smegmatis [67] biofilm extracellular matrices, respectively, in addition to glucose, may hint towards the presence of yet unidentified exopolysaccharides. Importantly, cellulose was also detected in mycobacterial biofilms occurring during infection [65], pointing to a role for biofilm extracellular matrix exopolysaccharides in pathophysiology.…”

Unique Features of Mycobacterium abscessus Biofilms Formed in Synthetic Cystic Fibrosis Medium

“…Alternatively, cellulose was recently reported to be a structural component of NTM biofilms grown in laboratory medium under thiol reductive stress (Chakraborty et al, 2021) and, along with capsular α-D-glucan (Ortalo-Magné et al, 1995), may represent a source of ECM glucose. The fact that 3 to 4 times more arabinose and galactose were found in the ECM of biofilmgrown cells compared to that of planktonically grown cells is either suggestive of the release of arabinogalactan from lysed cells within the biofilm or of the presence of as yet uncharacterized arabinan-and galactan-based exopolysaccharide(s) in the biofilm matrix of Mabs as recently proposed in M. smegmatis and M. tuberculosis (Bharti et al, 2020).…”

c-di-AMP Accumulation Regulates Growth, Metabolism, and Immunogenicity of Mycobacterium smegmatis

“…It has been reported that smooth or rough/wrinkled colony morphology was associated with the biofilm formation in several bacteria ( Friedman and Kolter, 2004 ; Uhlich et al, 2006 ; Bharti et al, 2020 ). Further, the ability of Δ cnpB strain to form biofilm was determined in Sauton’s medium at 48, 72, and 120 h respectively, and biomass was quantified by the crystal violet assay.…”