Abstract
Analysis of the transcriptional profiles of Mycobacterium tuberculosis after treatment with antimycobacterial compounds has improved our understanding of the ways mycobacteria respond to antibiotic stress, and revealed new insights into the mode of action of different antimycobacterial compound classes. RNA profiling of drug-induced changes has become an important tool in multiple stages of the antibacterial drug development process from target elucidation, to identifying target drift, and ultimately to revealing drug resistance mechanisms. The transcriptional response of M. tuberculosis to antimycobacterial compounds may be determined in isolation, in comparison with other compound classes, or between drug-sensitive and drug-resistant mycobacterial isolates. Additional information confirming the growth state of mycobacteria on addition of the antibacterial compound, and the effect that this compound has on mycobacterial growth, is essential for interpreting the transcriptional signatures acquired. This chapter describes the methods required for the extraction of representative total mycobacterial RNA, the subsequent hybridisation of this RNA to an M. tuberculosis complex microarray, and the analysis strategies employed to interpret the transcriptional data generated.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wilson M, DeRisi J, Kristensen HH et al (1999) Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization. Proc Natl Acad Sci USA 96(22):12833–12838
Boshoff HI, Myers TG, Copp BR, McNeil MR, Wilson MA, Barry CE III (2004) The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action. J Biol Chem 279(38):40174–40184
Waddell SJ, Stabler RA, Laing K, Kremer L, Reynolds RC, Besra GS (2004) The use of microarray analysis to determine the gene expression profiles of Mycobacterium tuberculosis in response to anti-bacterial compounds. Tuberculosis (Edinb) 84(3–4):263–274
Betts JC, McLaren A, Lennon MG et al (2003) Signature gene expression profiles discriminate between isoniazid-, thiolactomycin-, and triclosan-treated Mycobacterium tuberculosis. Antimicrob Agents Chemother 47(9):2903–2913
Caoili JC, Mayorova A, Sikes D, Hickman L, Plikaytis BB, Shinnick TM (2006) Evaluation of the TB-Biochip oligonucleotide microarray system for rapid detection of rifampin resistance in Mycobacterium tuberculosis. J Clin Microbiol 44(7):2378–2381
Fu LM, Shinnick TM (2007) Understanding the action of INH on a highly INH-resistant Mycobacterium tuberculosis strain using Genechips. Tuberculosis (Edinb) 87(1):63–70
Wayne LG, Sramek HA (1994) Metronidazole is bactericidal to dormant cells of Mycobacterium tuberculosis. Antimicrob Agents Chemother 38(9):2054–2058
Mangan JA, Monahan IM, Butcher PD (2002) Gene expression during host-pathogen interactions: approaches to bacterial mRNA extraction and labeling for microarray analysis. In: Wren BW, Dorrell N (eds) Functional microbial genomics. Academic, London, pp 137–151
Stewart GR, Wernisch L, Stabler R et al (2002) Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148(10):3129–3138
Hinds J, Laing K, Mangan JA, Butcher PD (2002) Glass slide microarrays for bacterial genomes. In: Wren BW, Dorrell N (eds) Functional microbial genomics. Academic, London, pp 83–99
Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95(25):14863–14868
Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98(9):5116–5121
Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Statist Soc Ser B57:289–300
Cole ST, Brosch R, Parkhill J et al (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393(6685):537–544
Allen BW (1998) Mycobacteria: general culture methodology and safety considerations. In: Parish T, Stoker NG (eds) Mycobacteria protocols. Humana, Totowa, pp 15–30
Talaat AM, Hunter P, Johnston SA (2000) Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis. Nat Biotechnol 18(6):679–682
Fu LM (2006) Exploring drug action on Mycobacterium tuberculosis using affymetrix oligonucleotide genechips. Tuberculosis (Edinb) 86(2):134–143
Acknowledgments
SJW was funded by an EU Sixth Framework Programme “New Medicines for Tuberculosis”, NM4TB (project number 018923). SJW and PDB would like to acknowledge the Wellcome Trust and its Functional Genomics Resources Initiative for funding the multi-collaborative microbial pathogen microarray facility at St. George’s (BμG@S), and thank Jason Hinds and Kate Gould at BμG@S for helpful suggestions and the supply of M. tuberculosis microarrays. M. tuberculosis H37Rv genomic DNA was generously provided by TB Vaccine Testing and Research Materials, Colorado State University (HHSN266200400091C).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Waddell, S.J., Butcher, P.D. (2010). Use of DNA Arrays to Study Transcriptional Responses to Antimycobacterial Compounds. In: Gillespie, S., McHugh, T. (eds) Antibiotic Resistance Protocols. Methods in Molecular Biology, vol 642. Humana Press. https://doi.org/10.1007/978-1-60327-279-7_6
Download citation
DOI: https://doi.org/10.1007/978-1-60327-279-7_6
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-278-0
Online ISBN: 978-1-60327-279-7
eBook Packages: Springer Protocols