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.
Highlights
Mycobacterium tuberculosis (Mtb) caused nearly 10.0 million new tuberculosis (TB) cases and 1.5 million TB deaths in the year 2018 (WHO, 2019)
Refolding of the protein to a stable native structure was confirmed by far-UV circular dichroism (Supplementary Figure S1)
According to the Carbohydrate Active Enzymes database or CAZy (Lombard et al, 2014) definition, glycosyl hydrolases (GHs/glycosidases) are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon
Summary
Mycobacterium tuberculosis (Mtb) caused nearly 10.0 million new tuberculosis (TB) cases and 1.5 million TB deaths in the year 2018 (WHO, 2019). Mtb is a highly successful pathogen because of its ability to persist in the host despite a functional immune system. Development of Mtb persisters, despite extensive research, is still inadequately understood. Characterizing the HPs of Mtb and discovering their role in its virulence or persistence will guide the development of novel strategies to counter this deadly pathogen. Identification and characterization of the proteins involved in persistence will aid developing novel drugs for shorter anti-tubercular regimens. Analysis of Mtb transcription pattern in serial sputa helps to shortlist those proteins that are upregulated after 14 days of treatment. These proteins could be important for the bacilli to persist amidst therapy
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