Detection of mRNA Transcripts and Active Transcription in Persistent Mycobacterium tuberculosis Induced by Exposure to Rifampin or Pyrazinamide

Abstract

Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.Tuberculosis is an important infectious disease, with over 1 billion people subclinically infected and the 3 million new cases each year resulting in 7% of the total number of deaths (20). Case finding and treatment are the main control measures, but chemotherapy takes 6 months to achieve a cure because bacteria persist during chemotherapy and cause relapse (2 to 5% in 5 years) after the end of treatment (14). These persistent bacteria are usually drug sensitive at relapse, and so their resistance to chemotherapy is phenotypic (17) (or tolerant) rather than genetic. It is suggested that an altered physiological state of persistent Mycobacterium tuberculosis accounts for its tolerance to drugs as well as the ability to survive in the host for many years. Persistence is likely to be a combined effect of both the immune system and bacterial physiology, resulting in what is generally referred to as a latent state (1). A key question is whether the metabolism of persistent bacteria is switched off with no cell division (spore-like) or, alternatively, is active (30).The first major advance in the detection of quiescent M. tuberculosis was the development of the Cornell mouse model (25). In this model, animals are infected with M. tuberculosis and treated for 3 months with chemotherapy which renders the tissues sterile; then steroids are administered, which leads to relapse in a high proportion of the mice. Immediately after the end of antibiotic therapy, the persistent bacteria are invisible by microscopy and are uncultivable, but about 10 5 genome equivalents of M. tuberculosis DNA per ml of tissue in the organs have been detected by DNA amplification (4). The presence of DNA could be indicative of dead bacilli or, possibly, of live bacilli which can multiply when the host immune system becomes weakened by steroids. Since DNA can be detected in dead bacteria (9,16,24), targeting genomic DNA is not suitable for the ...

The RNA polymerase‐binding protein RbpA confers basal levels of rifampicin resistance on Streptomyces coelicolor

“…Furthermore, the differential sensitivity of σ R -RNAP and σ HrdB -RNAP to Rif might have important implications for how M. tuberculosis responds to Rif treatment. Transcriptional activity has been detected in M. tuberculosis even following extensive treatment with Rif, including heat-induced expression of DnaK (Hu et al, 2000). It is known from other work that DnaK is controlled by the σ R homologue, σ H , in M. tuberculosis (Raman et al, 2001), which suggests that like σ R -RNAP, σ H -RNAP is resistant to Rif.…”

Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts

“…Alternatively, nonreplicating cells may contain some unresolved FtsZ-associated structures. Recently it has been shown that dormant cultures of M. tuberculosis exhibit both transcriptional and metabolic activity (Hu et al, 2000). It is unknown whether such transcriptional activity extends to the ftsZ gene.…”

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations

“…Thus, noncolony-forming Mtb were phenotypically tolerant to combination chemotherapy, and their viability was demonstrated by their ability to cause relapse. More recent studies of cfu-negative organ samples from Mtb-infected mice treated with other drug regimens suggested the presence of DD Mtb by detection of Mtb mRNA transcripts (49) or demonstrated the presence of DD Mtb by recovery of Mtb in liquid medium supplemented with CF from log-phase Mtb (50). The ability to use non-agar-based culture methods to grow DD Mtb from mice (22) or human sputum (13,14) makes the term "nonculturable" inappropriate.…”