Proteomic analysis of streptomycin resistant and sensitive clinical isolates of Mycobacterium tuberculosis

Abstract

Background: Streptomycin (SM) is a broad spectrum antibiotic and is an important component of any anti-tuberculosis therapy regimen. Several mechanisms have been proposed to explain the emergence of resistance but still our knowledge is inadequate. Proteins form a very complex network and drugs are countered by their modification/efflux or over expression/modification of targets. As proteins manifest most of the biological processes, these are attractive targets for developing drugs, immunodiagnostics or therapeutics. The aim of present study was to analyze and compare the protein profile of whole cell extracts from Mycobacterium tuberculosis clinical isolates susceptible and resistant to SM.

Results: Two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was employed for analyzing the protein profiles. Homology and in silico characterization for identified proteins was assessed using BLAST, InterProScan and KEGG database searches. Computational studies on the possible interactions between SM and identified proteins were carried out by a battery of online servers and softwares, namely, CLUSTALW (KEGG), I-TASSER, VMD, PatchDock and FireDock. On comparing 2DE patterns, nine proteins were found consistently overexpressed in SM resistant isolates and were identified as Rv0350, Rv0440, Rv1240, Rv3075c, Rv2971, Rv3028c, Rv2145c, Rv2031c and Rv0569. In silico docking analysis showed significant interactions of SM with essential (Rv0350, Rv0440 and Rv2971) and non essential (Rv1240, Rv3075c and Rv2031c) genes.

Conclusions: The computational results suggest high protein binding affinity of SM and suggested many possible interactions between identified proteins and the drug. Bioinformatic analysis proves attributive for analysis of diversity of proteins identified by whole proteome analysis. In-depth study of the these proteins will give an insight into probable sites of drug action other than established primary sites and hence may help in search of novel chemotherapeutic agents at these new sites as inhibitors.

Figure 1

Composite images of 2 D gels of proteins extracted from M. tuberculosis clinical isolates: (a) SM susceptible, (b) SM resistant. Spots indicated by arrow were excised and analyzed by MS.

Figure 2

Magnified regions of 2 D gels showing the overexpressed proteins (a) sensitive isolate; (b) SM resistant isolate.

Figure 3

3D models of overexpressed proteins showing docking with SM. Residues constituting interacting site, active site and conserved site are represented as space filled models with rest of the structure represented by cartoon structures. (a) Rv0350: Nine motifs are marked by numerals, docking cavity and SM are indicated by arrows and motifs 4, 5 & 7 are interacting with SM. (b) Rv0440: Red coloured SM, green coloured cavity and orange coloured conserved domain has been marked. Conserved domain is in the close vicinity of interacting site. (c) Rv1240: SM (green) is interacting with active site residues (blue) and other residues in the close vicinity (yellow) in the cavity. (d) Rv3075c: SM (yellow) interacting clearly with the central cavity residues (blue) of the globular protein. (e) Rv2971: SM (green) binding at the opposite side (brown) from conserved site of aldo/keto reductase (light blue) in the protein. Central cavity is present in the middle of complete β-barrel. (f) Rv2031c: SM (green) interacting with the outer part (yellow) of the protein in place of conserved HSP20-like chaperone domain.

Figure 4

3D models of proteins found non-interacting with SM represented by cartoon structures. (a) Rv3028c: Protein contains one α-subunit motif and one β-subunit interacting motif and one conserved site in the α-subunit motif. (b) Rv2145c: Whole protein consists of only α-helix and contains DivIVA domain indicating its role in cell division/cell shape. (c) Rv0569: Domain of unknown function (DUF1918) is present in the β-barrel of protein.