Clomiphene Citrate Shows Effective and Sustained Antimicrobial Activity against Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) causes chronic pulmonary infections and is the most difficult non-tuberculous mycobacteria (NTM) to treat due to its resistance to current antimicrobial drugs, with a treatment success rate of 45.6%. Thus, novel treatment drugs are needed, of which we identified the drug clomiphene citrate (CC), known to treat infertility in women, to exhibit inhibitory activity against M. abscessus. To assess the potential of CC as a treatment for M. abscessus pulmonary diseases, we measured its efficacy in vitro and established the intracellular activity of CC against M. abscessus in human macrophages. CC significantly inhibited the growth of not only wild-type M. abscessus strains but also clinical isolate strains and clarithromycin (CLR)-resistant strains of M. abscessus. CC's drug efficacy did not have cytotoxicity in the infected macrophages. Furthermore, CC worked in anaerobic non-replicating conditions as well as in the presence of biofilm. The results of this in vitro study on M. abscessus activity suggest the possibility of using CC to develop new drug hypotheses for the treatment of M. abscessus infections.

Development of potent chemical antituberculosis agents targeting Mycobacterium tuberculosis acetohydroxyacid synthase.

Mycobacterium tuberculosis acetohydroxyacid synthase (MTB-AHAS) has been suggested as a crucial target for antibacterial agents. High-throughput screening of a chemical library was performed to identify potent new inhibitors of MTB-AHAS. Among the 6800 tested compounds, 15 were identified as potent inhibitors, exhibiting >80-90% inhibition of in vitro MTB-AHAS activity at a fixed concentration of 20 µM. Five compounds belonging to the triazolopyrimidine structural class showed greater inhibition potency, with a half-maximum inhibition concentration (IC50 value) in the low micromolar range (0.4-1.24 µM). Furthermore, potent inhibitors demonstrated non-competitive, uncompetitive or mixed-competitive inhibition. Molecular docking experiments with these potent chemicals using a homology model of MTB-AHAS indicated hydrophobic and hydrogen bond interactions with some key herbicide binding site residues with binding energies (ΔG) of -8.04 to -10.68 Kcal/mol, respectively. The binding modes were consistent with inhibition mechanisms, as the chemicals were oriented outside the active site. Importantly, these potent inhibitors demonstrated significant growth inhibition of various clinically isolated multidrug-resistant and extensively drug-resistant M. tuberculosis strains, with 50% minimum inhibitory concentrations (MIC50 values) ranging from 0.2 µg/mL to 0.8 µg/mL, which resemble the MICs of conventional drugs for tuberculosis (isoniazid, 0.1 µg/mL; rifampicin, 0.4 µg/mL). Thus, the identified potent inhibitors show potential as scaffolds for further in vivo studies and might provide an impetus for the development of strong antituberculosis agents targeting MTB-AHAS.

Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001 nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36 µg/ml and 6.24 µg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents.

Expression, purification and improved antigenicity of the Mycobacterium tuberculosis PstS1 antigen for serodiagnosis.

The phosphate-specific transport substrate binding protein-1 (PstS1) is a potential antigen used for the serological diagnosis of tuberculosis. For a highly specific diagnostic result, it is important that the recombinant PstS1 be highly pure and correctly folded. In this study, the PstS1 was expressed as fusion protein with glutathione-S-transferase (PstS1-GST) and Escherichia coli trigger factor (PstS1-TF) and their immunodiagnostic potentials were evaluated. The insoluble PstS1-GST was denatured and refolded to the native conformation by a step-gradient dilution, followed by purification with affinity chromatography on immobilized glutathione whereas the soluble PstS1-TF was directly purified by Ni-NTA affinity and size-exclusion chromatographies. The levels of antibody responses to PstS1-TF and PstS1-GST were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of 22 tuberculosis patients with smear-positive and culture-positive tuberculosis as well as 20 healthy individuals; the antigenicities of the samples were evaluated in terms of sensitivity and specificity. To determine the diagnostic accuracy, receiver operation characteristic (ROC) curves were constructed and then the areas under the ROC curves (AUC) were calculated; the AUC values for PstS1-TF and PstS1-GST were 0.971 and 0.877 with 95% confidence intervals (CI) of 0.927-1.000 and 0.768-0.986, respectively. The specificity of PstS1-TF was reduced from 89.5% to 84.2%, but in case of PstS1-GST it dropped drastically from 78.9% to 26.3% when the sensitivity was raised from 86.4% up to 95.5%. These results indicate that PstS1-TF is capable of producing more accurate and consistent serodiagnostic results than PstS1-GST, possibly due to its conformation being closer to the native state.

Complete Genome Sequence of Mycobacterium bovis BCG Korea, the Korean Vaccine Strain for Substantial Production.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis, and the strains used worldwide represent a family of daughter strains with distinct genotypic characteristics. Here, we report the complete genome sequence of M. bovis BCG Korea, the strain that will be actually used in Korea for vaccine production.

Deciphering the proteome of the in vivo diagnostic reagent "purified protein derivative" from Mycobacterium tuberculosis.

Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.

Correlation of the phenotypic ethambutol susceptibility of Mycobacterium tuberculosis with embB gene mutations in Korea.

The phenotypic resistance to ethambutol (EMB) in Mycobacterium tuberculosis with embB gene mutations is still unclear. This study was designed to better understand EMB resistance due to embB gene mutation. Sequencing analysis of the embB gene was performed for 124 EMB-susceptible and 93 EMB-resistant M. tuberculosis strains isolated from South Korea. The MIC was determined for EMB-susceptible M. tuberculosis strains with the embB mutation and wild-type on Löwenstein-Jenson (LJ) solid medium in duplicate. Two (2.8 %) of 72 pan-susceptible, two (9.1 %) of 22 any-drug-resistant but EMB-susceptible, nine (30.0 %) of 30 multidrug-resistant (MDR) but EMB-susceptible and 84 (90.3 %) of 93 EMB-resistant M. tuberculosis strains possessed embB mutations at various codons including 306, 319, 354, 360, 399, 405, 406, 459 and 497. Strains with embB mutations at codons 306, 354, 399, 405 and 497 had highly pronounced EMB resistance, while strains with mutations at codons 319 and 406 mutations were moderately resistant and those with an embB459 mutation were EMB-susceptible at the critical concentration (2.0 µg ml(-1)) on LJ solid medium. However, the mean MIC for strains with embB mutations (1.42 µg ml(-1)) was higher than that for strains without the embB mutation (1.0 µg ml(-1)) in EMB-susceptible M. tuberculosis isolates (P = 0.0052). Three novel embB mutations at codons 399, 405 and 459 were identified in this study. These results support the hypothesis that embB mutation except for a few specific mutation types may be the main cause of EMB resistance.

Inhibitory mechanism of novel inhibitors of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae.

Bacterial UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolphyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first step of bacterial cell wall synthesis. We identified thimerosal, thiram, and ebselen as effective inhibitors of Heamophilus influenzae MurA by screening a chemical library that consisted of a wide range of bioactive compounds. When MurA was preincubated with these inhibitors, their 50% inhibitory concentrations (IC50s) were found to range from 0.1 to 0.7 microM. In particular, thimerosal suppressed the growth of several different Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium at a concentration range of 1-2 microg/ml. These inhibitors covalently modified the cysteine residue near the active site of MurA. This modification changed the open conformation of MurA to a more closed configuration, which may have prevented the necessary conformational change from occurring during the enzyme reaction.

Spread of nontuberculous mycobacteria from 1993 to 2006 in Koreans.

In Korea, the prevalence of nontuberculous mycobacterial (NTM) pulmonary disease has risen, observed primarily in immunocompetent patients with or without preexisting lung disease. The purpose of this study was to determine the frequency of various species of NTM isolates from respiratory specimens in a single institution over a 14-year period in Korea. All samples referred to our reference laboratory over a 14-year period in Korea were analyzed. From 1993 to 2000 our laboratory used conventional NTM identification methods, and from 2001 we adapted PCR-restriction fragment length polymorphism analysis(PRA). A total of 17,915 isolates were collected from 1993 to 2006. The most frequently isolated organisms were M. avium complex (n=11,705, 65%), M. abscessus (n=2,076, 11.59%), M. fortuitum complex (n=1,279, 7.14%). M. chelonae complex (n=1,134, 6.33%), M. kansasii (n=762, 4.25%), M. szulgai (n=139, 0.78%), M. celatum (n=87, 0.49%), M. scrofulaceum (n=18, 0.10%) and M. marium (n=11, 0.06%).

Comparative proteomic analysis of virulent Korean Mycobacterium tuberculosis K-strain with other mycobacteria strain following infection of U-937 macrophage.

In Korea, the Mycobacterium tuberculosis K-strain is the most prevalent clinical isolates and belongs to the Beijing family. In this study, we conducted comparative porteomics of expressed proteins of clinical isolates of the K-strain with H37Rv, H37Ra as well as the vaccine strain of Mycobacterium bovis BCG following phagocytosis by the human monocytic cell line U-937. Proteins were analyzed by 2-D PAGE and MALDITOF-MS. Two proteins, Mb1363 (probable glycogen phosphorylase GlgP) and MT2656 (Haloalkane dehalogenase LinB) were most abundant after phagocytosis of M. tuberculosis K-strain. This approach provides a method to determine specific proteins that may have critical roles in tuberculosis pathogenesis.