cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG

• Published in: Microbiology (Society for General Microbiology) Vol. 147; no. 8; pp. 2293 - 2305
• Main Authors: Li, Ming-Shi, Monahan, Irene M, Waddell, Simon J, Mangan, Joseph A, Martin, Steve L, Everett, Martin J, Butcher, Philip D
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.08.2001; Society for General Microbiology
• Subjects: Acyltransferases - chemistry; Acyltransferases - genetics; Acyltransferases - metabolism; Amino Acid Sequence; Bacterial diseases; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Base Sequence; Biological and medical sciences; DNA Primers; DNA, Complementary; Experimental bacterial diseases and models; fadD28 gene; Fundamental and applied biological sciences. Psychology; furB gene; Gene Expression Regulation, Bacterial; Genetics; Genomic Library; groEL-2 gene; Humans; Infectious diseases; Macrophages - microbiology; mas gene; Medical sciences; Microbiology; Molecular Sequence Data; Mycobacterium bovis; Mycobacterium bovis - enzymology; Mycobacterium bovis - genetics; Mycobacterium bovis - growth & development; Mycobacterium tuberculosis; Mycobacterium tuberculosis - genetics; mycocerosic acid synthase; Nucleic Acid Hybridization - methods; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Messenger - metabolism; rplE gene; Transcription, Genetic; Tumor Cells, Cultured; Facilitation; Mycobacterium bovis; Enzyme; Host agent relation; Host; Mycobacterial infection; Hybridization; Synthase; Infection; Tuberculosis; Complementary DNA; Gene; Mycobacterium tuberculosis; Mycobacteriales; Ribosomal RNA; Bacteriosis; Mycobacteriaceae; Bacteria; Actinomycetes; Population dynamics; Intracellular; Cell; Phagocytosis; Macrophage
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-147-8-2293

Department of Medical Microbiology, St George’s Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK 1 Glaxo Wellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK 2 Author for correspondence: Philip D. Butcher. Tel: +44 20 8725 5721. Fax: +44 20 8672 0234. e-mail: butcherp{at}sghms.ac.uk Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell–intracellular pathogen interactions. To identify such genes a cDNA–total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase ( mas ). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB , groEL-2 , rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA–RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis. Keywords: high-density gridded genomic library, macrophage, mycobacterial mRNA microarray Abbreviations: MBN, mung-bean nuclease a Present address: Department of Paediatrics, Imperial College School of Medicine, St Mary’s Hospital, Norfolk Place, London W2 1PG, UK.