Mycobacterium Tuberculosis Persister And Application

Mycobacterium tuberculosis (M tuberculosis, MTB) remains one of the most significance human pathogens since its discovery in 1882. An estimated 1.5 million people died from tubercle bacillus (TB) in 2006. The recent emergence of the Multidrug-resistant TB (MDR-TB), extensively drug-resistant TB (XDR-TB), HIV-associated TB and weak health systems are major challenges. China remains one of the High M/XDR-TB Burden Countries.MDR-TB and XDR-TB, which occur due to non-adherence to therapy and misuse of TB drugs, have caused a great deal of public concern. The current TB and drug-resistant TB problem is at least partly attributable to inefficient or suboptimal TB therapy. The goals of TB therapy are to kill tubercle bacilli rapidly, prevent the emergence of drug resistance, eliminate persistent bacilli to prevent relapse, and minimize disease transmission. But these goals are not appropriately met with current TB therapy. Standard TB therapy today, as part of the directly observed treatment, directly observed treatment short-course (DOTS) strategy, takes at least 6 months, is a regimen that consists of four drugs:isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB) taken daily for two months during the'intensive' treatment phase, followed by two drugs:INH and RIF taken daily for four months in the "continuation" or "sterilizing" phase. This therapy, when taken strictly, has a cure rate of 85%. However, this lengthy treatment is often associated with significant side effects and poor patient compliance, to emergence of drug resistance. Moreover, DOTS may not work in areas with high incidence of HIV or MDR-TB, where DOTS-plus, which is DOTS plus second-line TB drugs, is recommended. Treatment of MDR-TB takes up to 2 years and is not only costly but has significant toxicity. The mechanism underlying lengthy TB therapy is not well understood. But the presence of persistent and dormant TB bacteria is thought to be the cause for the lengthy TB chemotherapy, since the current TB drugs are not effective in eliminating persistent or dormant bacilli. The unique feature of M. tuberculosis is persisting in a latent state without causing disease. It is important to understand the mechanism of this phenomenon and devise therapeutic strategies targeting the persistent or dormant organisms. The goals of new TB drug development are to shorten therapy, treat MDR-TB, shorten treatment for latent TB infection (LTBI), and be used safely with antiretroviral therapy. In addition to the risk associated with any new drug development, developing new TB drugs has been a challenge to scientists for decades.M. tuberculosis, a facultative parasitic bacterium, spreads by aerosol and infects its host through the airways. The bacterium is phagocytosed by resident macrophages in the lung, and when successful is able to replicate inside these cells, which are actually designed to kill invading microbes. Macrophages are capable of inducing programmed cell death through apoptosis when infected with an intracellular pathogen, as a type of "emergency exit" to kill the persistent or dormant bacilli. This can function as a direct deprivation of the replication niche of the persistent or dormant organisms, as well as a signal to the remaining immune system, which is thought to enhance killing of intramacrophage mycobacteria. Thus, apoptosis has been assigned a role in the immune response against M. tuberculosis, and prevention of macrophage apoptosis by virulent M. tuberculosis appears to be a mechanism of virulence.Professional phagocytes have a vast and sophisticated arsenal of microbicidal features, such as reactive oxygen, low pH, hypoxia, lack of nutrition and essential elements, antimicrobial proteins and peptides, lysozymes and proteases and so on. The pathogenicity of M. tuberculosis is largely attributed to its ability to survive within macrophages dependent on its construction and regulation. Mycobacteria have a very complex cell wall, which largely consists of mycolic acids linked to arabinogalactan, which is attached to the peptidoglycan, not only protects the bacteria from toxic compounds within the macrophages, but also contains components with important immunomodulatory function. Mycolic acids, the most striking component of the cell wall, it's a long-chainα-aldyl-β-hydroxy fatty acids. Furthermore, this component only found in mycobacteria, and plays an important role in the taxonomy.The synthesis of mycolic acid in M. tuberculosis is mainly through the co-actions of two pathways including fatty acid synthase type I (FAS-I) pathway and type II (FAS-II) pathway. A large number of fatty acid synthases and their regulators are involved in these two pathways. The fatty acid metabolism regulator (FadR) studied in this paper is a very important one. FadR in Escherichia coli (E. coli) regulates negatively in saturated fatty acid degradation, while functions positively in the synthesis of unsaturated fatty acids. And there is no relevant report about FadR in M. tuberculosis. Researchers have classified the possible regulators of fatty acid metabolism in M. tuberculosis and Mycobacteria smegmatis (M. smegmatis), and predicted several regulators close to those in E. coli. In this paper, the GntR family in Mycobacteria is classified, consequently the FadR subfamily in GntR family is determined, also Rv0494 is further determined as the potenial FadR encoding gene. Ultimately, Rv0494 is certified to adjust the fatty acid levels by way of degradation of fatty acids 14:0 iso 3OH to producing a new one.The previous study found that in E. coli, the mutation of FadR would up-regulate the expression of aceBAK, the encoding gene of operon consisting of the key enzyme in glyoxylate cycle bypass. In the harsh environments of macrophages, M. tuberculosis obtains the energy for its growth mainly depending on glyoxylate cycle bapass. Once the bypass is blocked, the survival rate of M. tuberculosis would be greatly reduced. FadR regulate the expression of aceBAK indirectly, through regulating isocitrate lyase regulator (IclR) expression.Isocitrate lyase (ICL; EC 4.1.3.1) plays a key role in the pathogenesis of M. tuberculosis, this enzyme allows M. tuberculosis to grow on acetate or fatty acids as the sole carbon sources since the glyoxylate bypass provides a source of carbon that can enter the Krebs cycle. So ICL can enhance bacterial survival in inflammatory macrophages to promote the persistence of infection. A high-throughput screen developed to screen active extracts derived from traditional Chinese medicines (TCMs) for inhibition of ICL has been done in our previous studies. Meanwhile, we find that ICL is regulated by IclR and FadR in E. coli through many references. Some of them announced that the control of the glyoxylate bypass operon (aceBAK) is mediated by two regulatory proteins, IclR and FadR. IclR is a repressor protein which has previously been shown to bind to a site which overlaps the aceBAK promoter. FadR is a repressor/activator protein which participates in control of the genes of fatty acid metabolism. A sequence just upstream of the IclR promoter bears a striking resemblance to FadR binding sites found in the fatty acid metabolic genes. In E. coli, the main player in transcription regulation of fatty acid metabolism is the FadR, which is involved in negative regulation of fatty acid degradation and in positive and negative regulation of the cellular processes related to it, as well as in positive regulation of the biosynthesis of unsaturated fatty acids in a concerted manner with negative regulation of FadR, but the result has ever not been reported in M. tuberculosis. Our research is focus on the function of FadR in M. tuberculosis, and expectation to find the potential drug target.Transfer-messenger RNA (abbreviated tmRNA, also known as 10Sa RNA and by its genetic name SsrA) is a bacterial RNA molecule, and has dual tRNA-like and messenger RNA-like properties. In trans-translation, tmRNA and its associated proteins bind to bacterial ribosomes which have stalled in the middle of protein biosynthesis, for example when reaching the end of a messenger RNA which has lost its stop codon. Trans-translation is essential in some bacterial species, whereas other bacteria require tmRNA to survive when subjected to stressful growth conditions. Through sequencing of Mycobacterium tuberculosis ssrA, researcher found that the 3' end of tmRNA is similar to the T stem-loop of tRNA. In E. coli, we constructed an SsrA deletion mutant, and found that its inactivation leads to higher susceptibility than that of the parent strain to antibiotics. The SsrA deletion mutant phenotype could be complemented by a functional SsrA gene. The SsrA gene may be an ideal drug target for designing new drugs that kill persister bacteria.