| Since Robert Koch identified Mycobacterium tuberculosis as the causative agent of TB in1882, a breakthrough in the fighting against this deadly pathogen, the global Tb epidemic seems unmitigated substantially. About a record9.8million new cases were noted in2011. This serious situation highlights the emergency of the newly and effective treatment strategies for TB. Although the anti-TB chemotherapy had lasted for half a century, one-third of the world's population still asymptomatically harbors a dormant or latent form of M. tuberculosis, with a life long risk of disease reactivation. The reactivation of latent TB represents high risk for other abnormalities, especially for the patient infected with human immunodeficiency viruses, or with diabetes and other disease need anti-tumour necrosis factor therapy. In recently years, the TB epidemic has been further fuelled by the multi and extensively drug resistant tuberculosis (MDR-TB and XDR TB), the classical anti-TB therapy is also undergoing acid test.The first-line anti-tuberculous drug including isoniazid(INH, H), rifampicin(RIF, R), pyramiznamide(PZA, Z) and ethambutol (EMB, E). The MDR-TB is defined as tuberculosis that is resistant at least to isoniazid (INH) and rifampicin (RIF), the two most powerful first-line anti-TB drugs. The treatment of MDR-TB largely relies on the second line anti-TB drugs, including aminoglycosides, polypeptides, thioamides and fluoroquinolones. The fluoroquinolones have bactericidal activity against M.tuberculosis, excellent oral bioavailability, favorable safety profiles, it's an important second line anti-TB drug. Data from phase â…¡ trials of fluoroquinolones-containing regimens for shortening the duration of treatment for pulmonary TB are encouraging and phase â…¢ trials are currently underway, also highlighted the importance of fluoroquinolones in anti-TB therapy.The target of fluoroquinolones in bacteria is DNA gyrase and topoisomerase â…£. M.tuberculosis is unusual in possessing only one type â…¡ topoisomerase, DNA gyrase. The amino acid substitutions within the quinolone resistance-determining region(QRDR) of GyrAB can dramatically reduce susceptibility to fluoroquinolones. Although a variety of mutation types have been reported in several countries and regions, epidemiologic information on fluoroquinolone-resistant M. tuberculosis in mainland China remains obscure. To investigate the mutation types of the genes encoding the A and B subunits of DNA gyrase (gyrA and gyrB, respectively) in ofloxacin-and levofloxacin-resistant M.tuberculosis strains prevalent in mainland China,177clinical drug-resistant isolates collected by the National Tuberculosis Reference Laboratory of China were analysed. The GyrB single mutations (Glu498and Gly551) and double mutation (Thr539Asn-Gly551Arg) as well as a GyrA double mutation (Asp94Asn-Gly112His) were reported to be involved in fluoroquinolone resistance for the first time. To simplify quantification of the contribution of each mutation type and mutation site to overall fluoroquinolone resistance, a mathematical method was established by assigning each resistance allele a numerical score between5and50(the larger the number, the higher the resistance level). The score of double mutants is the sum of the scores for the two single alleles. The double mutation types, including Asn538Ile(GyrB)-Asp94Ala(GyrA), Ala543Val(GyrB)-Asp94Asn(GyrA) and Ala543Val(GyrB)-Asp94Gly(GyrA) scored relatively high by this methodology, serve as a cautionary tale that it is imperative to use fluoroquinolones rationally.During millions of years of evolution, bacteria must have encountered a variety of stressful environment, including exposure to antibiotics. Thus, it is not surprising if bacteria have acquired genetic and biochemical systems that protect cells from antimicrobial lethality. Bacterial genes defining intrinsic resistance to antibiotics encode proteins that can be targeted by antibiotic potentiators. Fluoroquinolones is a type of second line anti-tuberculosis drugs, have been widely used in tuberculosis therapy. To find the intrinsic resistance related genes, a transposon inerstion library of Mycoabcterium.smegmatis was screened with subinhibitory concentration of moxifloxacin to find supersusceptible mutants. Transposon insertions3genes were found to cause at least twofold to8folds to moxifloxacin. These included mutants with disruption of genes encoding proteins involved in DNA damage repair (RuvA, Rv2593), FHA proteint(GarA, Rv1827) as well as a proteasome accessory factor (pafC,Rv2095). Electroproration of recombinant plasmids Palace+Rv2592-Rv2593, Palace+Rv1827to M136and M625respectively, partly reverse the fluoroquinolones resistance. The complement results help validate RuvAB as a potential antibiotic combination target for Mycobacterium, other hypersensitive phenotype related genes need further validation.Although the target of quinolones is clear, the killing pathways of different quinolones are strikingly different, distinguished by whether bactercidal effect are effected by the protein synthesis inhibitor(chloramphenicol) or the hypoxic condition. First-generation compounds, such as nalidixic and oxolinic acids, are not lethal in the presence of chloramphenicol or during anaerobic growth; the second-generation agent norfloxacin fails to kill bacterium in the presence of chloramphenicol, but at high concentrations it kills cells growing anaerobically. Ciprofloxacin, a third-generation compound, kills under both conditions but requires higher concentrations during anaerobiosis; the lethal activity of fourth generation C-8-methoxy derivatives, such as moxifloxacin, is affected little by chloramphenicol or anaerobic growth. The early experiment validated the bactericidal process of quinolone is a two steps characteristic. Step1:formed Quinolone-Topoisomerase-DNA complexes, rapid inhibition of DNA replication, bring secondary damage that might contribute to slowly quinolone-mediated cell death; Step2:broken DNA released from Quinolone-Topoisomerase-DNA complex, chromosome fragmentation that contribute to rapid cell death. Researchers are doubt there presence a sucide factor in step2that lead the cleaved complex collapsed. In our experiment, RuvAB mutant strain showed an obvious higher survival rate after moxifloxacin treatment when the protein synthesis depend killing pathway was blocked, suggested the helicase RuvAB is an important factors for protein independent killing pathway of quinolones.The high biosafety level III required to tackle its causative agent Mycobacterium tuberculosis seriously hinders the exploration of its biology and new countermeasures. M. smegmatis is a widely recognized good surrogate of M.tuberculosis, due to their conserved transcriptional machinery, sigma factors and two-component systems. However, their distinct lifestyles often confound the explanation of the results. M.tuberculosis leads both parasitic and free life, while M.smegmatis is largely saprophyte. To make full advantage of this model, it is helpful to discover the genome features associated with M. smegmatis unique niches, such as its saprophytic life, high salt tolerance and relative short generation time. We employed the gene ontology enrichment analysis to characterize the unique lifestyle of M.smegmatis. Gene ontology enrichment analysis provided24terms, most are relevant to the special lifestyle of M. smegmatis, especially the saprophytic niche, high salt tolerance adaptation and short generation time. In-depth functional characterization of these genes will shed new lights on the genetic basis of M.smegmatis saprophytic life and hasten the understanding of the unique biology of M.tuberculosis.
|
PDF Full Text Request