Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling

Abstract

SummaryThe search for new TB drugs that rapidly and effectively sterilize the tissues and are thus able to shorten the duration of chemotherapy from the current 6 months has been hampered by a lack of understanding of the metabolism of the bacterium when in a 'persistent' or latent form. Little is known about the condition in which the bacilli survive, although laboratory models have shown that Mycobacterium tuberculosis can exist in a non-growing, drug-resistant state that may mimic persistence in vivo. Using nutrient starvation, we have established a model in which M. tuberculosis arrests growth, decreases its respiration rate and is resistant to isoniazid, rifampicin and metronidazole. We have used microarray and proteome analysis to investigate the response of M. tuberculosis to nutrient starvation. Proteome analysis of 6-week-starved cultures revealed the induction of several proteins. Microarray analysis enabled us to monitor gene expression during adaptation to nutrient starvation and confirmed the changes seen at the protein level. This has provided evidence for slowdown of the transcription apparatus, energy metabolism, lipid biosynthesis and cell division in addition to induction of the stringent response and several other genes that may play a role in maintaining longterm survival within the host. Thus, we have generated a model with which we can search for agents active against persistent M. tuberculosis and revealed a number of potential targets expressed under these conditions.

Biochemical characterization of a chromosomal toxin–antitoxin system in Mycobacterium tuberculosis

“…tuberculosis Rv1991c gene was cloned into the NdeI-XhoI sites of pET28a, creating pET28a-(His) 6 Rv1991c for expression of (His) 6 Rv1991c in E. coli. The Rv1991a ORF was cloned into the NdeI-XhoI sites of pET28a, creating pET28a-(His) 6 Rv1991a for expression of (His) 6 Rv1991a in E. coli. Expression vector pACYCDuet (Novage) has two multi-cloning sites (MCS).…”

“…Native PAGE and Tricine SDS-PAGE (His) 6 Rv1991a and (His) 6 Rv1991c were mixed in binding buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 1 mM dithiothreitol, and 5% glycerol) at 4°C for 30 min. The compositions of the stacking gel and the separation gel are same as described previously [11].…”

“…The Rv0707 mRNA and era mRNA were prepared from these two DNA fragments respectively with the RiboMAXä T7 large scale RNA production system (Promega). RNA substrates were incubated with (His) 6 Rv1991c at 37°C for 15 min. Each reaction mixture (10 ll) contained 1.5 lg of RNA substrate, 100 pmol (His) 6 Rv1991c, 1 ll of RNase inhibitor, 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM DTT.…”

“…RNA substrates were incubated with (His) 6 Rv1991c at 37°C for 15 min. Each reaction mixture (10 ll) contained 1.5 lg of RNA substrate, 100 pmol (His) 6 Rv1991c, 1 ll of RNase inhibitor, 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM DTT. The reaction mixture was then subjected to 5% PAGE.…”

“…Rv1991c and Rv1991a form a complex (His) 6 Rv1991a and (His) 6 Rv1991c were expressed in E. coli BL21(DE3) and purified by affinity chromatography. (His) 6 Rv1991a and (His) 6 Rv1991c are referred as Rv1991a and Rv1991c in the following in vitro experiments. When the purified Rv1991a and Rv1991c were subjected to native PAGE respectively, Rv1991a was found as a band at the bottom of the gel (Fig.…”