Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS)

Abstract

A widely applicable, positive cDNA selection method was developed to identify RNAs synthesized by Mycobacterium tuberculosis in response to phagocytosis by cultured human primary macrophages. cDNAs for sigE and sigH (alternative sigma factors), aceA (isocitrate lyase), ponA (class I penicillin-binding protein), pks2 (polyketide synthase), uvrA (UvrABC endonuclease), and ctpV (putative cation transporter) were obtained from macrophage-grown bacteria. cDNAs for ORFs Rv3070, Rv3483c, Rv0903c (encoding a putative bacterial two-component transcriptional activator), and Rv0170 of the mce1 virulence operon also were obtained from phagocytized bacilli. cDNAs for these genomic regions were not obtained from approximately 1, 000-fold more bacteria grown in laboratory broth. Methods described here, which have identified M. tuberculosis genes expressed in response to host interaction, will allow the study of gene expression in a variety of microorganisms, including expression resulting from interaction with human tissues in natural disease states.

Figure 1

Identification of microbial genes transcribed in vivo by SCOTS. Normalized microbial cDNA is obtained directly from limited samples of infected host cells or tissue (A). cDNAs corresponding to genes preferentially expressed in environment I relative to environment II are obtained by differential cDNA hybridization (B).

Figure 2

Bacterial cDNA obtained from intracellular (48-h infection) or broth-grown M. tuberculosis H37Rv. cDNAs were prepared as described in the text and used as probes against a Southern blot of H37Rv chromosomal DNA digested with PstI. The probe for lane 1 was the cloned rrnA operon of M. tuberculosis. Lane 2 was probed with the PCR-amplified total cDNA prepared from infected macrophages as described. Probes for lanes 3, 4, and 5 were bacterial cDNA obtained from infected macrophages by one, two, and three rounds of SCOTS, respectively. Lane 6 was probed with bacterial cDNA obtained by three rounds of SCOTS from a broth culture grown to an OD (A₆₀₀) of 0.4.

Figure 3

Identification of sigma factor cDNAs obtained by SCOTS. cDNA probes normalized by three rounds of SCOTS obtained from broth cultures grown to an OD (A₆₀₀) of 0.15 (B) or 0.54 (C) or from infected macrophages 18 h (D) or 48 h (E) after phagocytosis were used in Southern hybridization assays with previously described (22) agarose gel-purified, PCR-amplified DNA fragments (A) containing sigA (160 bp), sigB (106 bp), sigC (85 bp), sigD (113 bp), sigE (115 bp), sigF (92 bp), sigG (91 bp), sigH (81 bp), or rpoB (418 bp). mig (150 bp) is an M. avium DNA fragment not present in H37Rv.

Figure 4

Transcription of M. tuberculosis H37Rv genes during growth in Middlebrook 7H9 broth (A) or human macrophages (B) as detected by cDNA probes prepared by SCOTS. Bacterial cDNA probes prepared by three rounds of SCOTS (see text) from broth cultures grown to an OD (A₆₀₀) of 0.4 or from intracellular bacteria 48 h after infection were hybridized to PCR-amplified plasmid fragments containing the indicated H37Rv genes (lanes 1–4) or cloned bacterial cDNA fragments isolated from macrophage-grown bacilli. mig (150 bp) is an M. avium DNA fragment not present in M. tuberculosis. The H37Rv genome coordinates of specific sequences are available on request.

Figure 5

Modulation of M. tuberculosis H37Rv transcript levels during growth in human macrophages as detected by cDNA probes prepared by SCOTS. Bacterial cDNA probes prepared by three rounds of SCOTS (see text) from intracellular bacteria 18 (A), 48 (B), and 110 h (C) after infection were hybridized to PCR-amplified plasmid fragments containing the indicated H37Rv genes (lanes 1–4) or cloned bacterial cDNAs isolated from phagocytized bacilli (48 h).