Streptomycin induced protein expression analysis in Mycobacterium tuberculosis by two-dimensional gel electrophoresis & mass spectrometry.
OBJECTIVE
The resistance of Mycobacterium tuberculosis to streptomycin, a core drug for treatment of category II tuberculosis (TB) has posed a major challenge to the health providers as well as research workers worldwide and has severely compromised the therapeutic options. A significant proportion of streptomycin resistant M. tuberculosis isolates failed to show mutations in conventional targets like rpsL and rrs. Although efflux, permeability, etc. are also known to contribute, yet a substantial proportion of isolates remains resistant suggesting involvement of other unknown mechanism. A resistant isolate may show altered gene as well as protein expression under drug induced conditions and a whole cell proteome analysis under induced conditions might help in further understanding the mechanisms of drug resistance. The present study was therefore designed with the objective to identify proteins related to streptomycin resistance in M. tuberculosis isolate grown in presence and absence of streptomycin (SM).


METHODS
A clinical isolate of M. tuberculosis from Mycobacterial Repository Centre at the Institute (NJIL & OMD), Agra was grown in Sauton's medium for 36 h with/without subinhibitory concentration of the drug (2 μg/ml) and the cell lysate of isolates was prepared by sonication and centrifugation. Two-dimensional (2D) gel electrophoresis was employed to study the protein profile. The selected proteins were finally identified by MALDI-TOF mass spectrometry.


RESULTS
Our study revealed eight inducible proteins (DnaK, fabG4, DNA-binding, hypothetical, two 14 kDa antigen and two 10 kDa chaperonin) that were upregulated in the presence of drug.


CONCLUSIONS
This preliminary study has thrown light on whether or not and how the resistant isolate responds to streptomycin at its non-toxic but sub-inhibitory concentration. An in-depth study of the upregulated proteins will give an insight into probable sites of drug action other than established primary sites.
Department of Biochemistry, National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), Agra, India.
Prashant
P
Sharma
Bhavnesh
B
Kumar
Neelja
N
Singhal
Vishwa Mohan
VM
Katoch
Krishnamurthy
K
Venkatesan
Devendra Singh
DS
Chauhan
Deepa
D
Bisht
D001426:Bacterial Proteins / Q000378:metabolism*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013307:Streptomycin / Q000494:pharmacology*; D014238:Trichloroacetic Acid; D014357:Trypsin
D001426:Bacterial Proteins; D014238:Trichloroacetic Acid; D014357:Trypsin; D013307:Streptomycin