tuberculosisH37Rv clones were grown for 3 days in the absence and presence of 2 g/ml pristinamycin IA prior to rifampin exposure

tuberculosisH37Rv clones were grown for 3 days in the absence and presence of 2 g/ml pristinamycin IA prior to rifampin exposure. ofM. tuberculosis, indicating that SigF does not affect rifampin tolerancein vivo. The treatment of drug-sensitiveMycobacterium tuberculosisinfections is typically carried out using a cocktail of four drugs (isoniazid, rifampin, pyrazinamide, and ethambutol), for a 2-month period followed by 4 months of treatment with rifampin and isoniazid. Pharmacodynamic analysis of the sputum CFU following the start of this drug regimen typically shows a rapid decrease (1 to 2 2 logs) in bacterial burden in the first week of treatment. This early bactericidal stage is, however, followed Difluprednate by a far lower rate of bacterial elimination, thought to be due to the persistence of a bacterial subpopulation that is Difluprednate phenotypically less drug susceptible and often termed drug tolerant. Understanding the mechanisms underlying this drug tolerance is essential for efforts to shorten and improve tuberculosis drug therapy. Numerousin vitromodels have been proposed that potentially mimic conditions that give rise to the phenotypically drug-tolerant bacterial population foundin vivo; these include nutrient starvation (3), growth in a hypoxic environment (28,29), or nitric oxide challenge (12). These models all involve driving the bacteria into a nonreplicating state which consequently often decreases their drug susceptibility. This is especially true for those drugs that target the biogenesis of the bacterial cells wall (i.e., isoniazid and ethambutol), as this process is essential when the cells are actively growing. Intriguingly, inhibition of RNA polymerase (RNAP) by Mouse monoclonal to BCL-10 the currently most effective antitubercular drug, rifampin, is also subject to drug tolerance, with stationary-phase bacteria being less rifampin susceptible (11). Transcription is an essential process, regardless of whether the bacteria are in log phase or in stationary phase. Interestingly, the addition of very high concentrations of rifampin to stationary-phase bacteria has revealed that transcription still occurs, though at a somewhat lower rate (11).M. tuberculosismust therefore have an alternative mechanism to circumvent rifampin inhibition and allow for continued transcription. Recent studies have revealed that some accessory proteins such as GroEL1 and RbpA can bind RNAP and prevent rifampin inhibitionin vitro(8,19). Here, we investigate whether altering the sigma factor usage can have a similar impact on the affinity of rifampin for RNAP, allowing transcription in its presence. Rifampin binds to the subunit of RNAP and forms a physical barrier that preventsde Difluprednate novoRNA from elongating out of the RNAP complex (18). It has been speculated that rifampin is also able to interact directly with domain 3.2 of the housekeeping sigma factor A (SigA) that inserts deeply into RNAP (1). In response to external stress factors,M. tuberculosiscan utilize any of the 13 sigma factors (SigA to SigM) to alter its transcriptome. Interestingly, SigF is one of only three sigma factors with a domain 3.2 (24) (the others being SigA and B), and it has been implicated in the entry of mycobacteria into stationary phase (7). In addition, studies conducted in a different system revealed that changing sigma factor usage inEscherichia colifrom Sig70 to an alternative sigma factor, Sig32, dramatically impacts rifampin susceptibility (30). A role for SigF in rifampin tolerance was first suggested when it was shown that deletion ofsigFfrom anM. tuberculosisstrain (CDC1551) resulted in increased susceptibility to rifampin (5). That work, however, did not investigate the mechanism underlying this change in sensitivity to rifampin, a question that we seek to investigate in this report. The aim of this study is to determine if SigF can directly cause allosteric alterations in RNAP that will affect rifampin binding and therefore its activity. This is investigated by determining the rifampin inhibition of SigA- and SigF-specificin vitrotranscription. Second, we sought to determine whether increasedsigFexpression led to increased bacterial tolerance to rifampin and to confirm that deletion ofsigFaffected rifampin’s bactericidal activity. == MATERIALS AND METHODS == == Reagents. Difluprednate == All PCRs were performed using Phusion DNA polymerase (Finnzymes) with primers from Microsynth (Balgach, Switzerland). Restriction enzymes were purchased from New England Biolabs, pUC19 and chemically competent Top10E.colifrom Invitrogen (Basel, Switzerland), and chemically competent.