Qualitative anti-tubercular activity of synthetic ethyl 7-acetyl2-substituted-3-(4-substituted benzoyl) indolizine-1-carboxylate analogues

Both the emergence of multidrug-resistant and extensively drug-resistant tuberculosis (TB) are currently the majorchallenges in the treatment of TB. Only delamanid and bedaquiline have been recently approved as anti-TB drugs inthe past 40 years. In an attempt to search for active anti-TB compounds against the sensitive strain of Mycobacteriumtuberculosis, H37Rv—a series of synthetic ethyl 7-acetyl-2-substituted-3-(4-substituted benzoyl)indolizine-1-carboxylates (2a–r)—have been screened for in vitro qualitative anti-TB activity using an agar dilution method. Itwas found that compounds 2a, 2b, 2c, 2f, 2g, 2i, 2j, 2l, 2o, 2p, and 2r, which have various functional groups on theindolizine nucleus, were active against the H37Rv strain.

In silico dissection of Type VII Secretion System components across bacteria: New directions towards functional characterization.

Type VII Secretion System (T7SS) is one of the factors involved in virulence of Mycobacteriun tuberculosis H37Rv. Numerous research efforts have been made in the last decade towards characterizing the components of this secretion system. An extensive genome-wide analysis through compilation of isolated information is required to obtain a global view of diverse characteristics and pathogenicity-related aspects of this machinery. The present study suggests that differences in structural components (of T7SS) between Actinobacteria and Firmicutes, observed earlier in a few organisms, is indeed a global trend. A few hitherto uncharacterized T7SS-like clusters have been identified in the pathogenic bacteria Enterococcus faecalis, Saccharomonospora viridis, Streptococcus equi, Streptococcuss gordonii and Streptococcus sanguinis. Experimental verification of these clusters can shed lights on their role in bacterial pathogenesis. Similarly, verification of the identified variants of T7SS clusters consisting additional membrane components may help in unraveling new mechanism of protein translocation through T7SS. A database of various components of T7SS has been developed to facilitate easy access and interpretation of T7SS related data.

Effect of Mcl-1 signaling pathway blockers on apoptosis of mouse macro-phages infected with Mycobacterium tuberculosis H37Rv / 中国病理生理杂志

[ ABSTRACT] AIM: To explore the effects of Mcl-1 signal pathway blockers on Mcl-1 expression, macrophage apoptosis and Mycobacterium tuberculosis in the model of mice infected with Mycobacterium tuberculosis H37Rv.METH-ODS:A mouse infection model was established by intraperitoneal injection of H37Rv suspension.The signaling pathway blockers AG490, PD98059 and LY294002 for JAK/STAT, MAPK and PI3K, respectively, were intraperitoneally injected into the mice infected with H37Rv.Cell acid-fast staining was used to observe whether the mouse peritoneal macrophages infected with H37Rv were successfully established.Immunocytochemical method was employed to detect Mcl-1 expression in the mouse peritoneal macrophages infected with H37Rv.The apoptotic rate in each group was measured by flow cytomer-ty.The scavenging capacity of apoptotic macrophages against H37Rv was determined by Mycobacterium tuberculosis colony counting.RESULTS:The result of cell acid-fast staining revealed the existence of dispersive arrangement of red short anti-acid Mycobacterium tuberculosis within infected macrophages.The result of cell immunocytochemistry showed strongly posi-tive expression of Mcl-1 protein in H37Rv infection group, AG490 treatment group and LY294002 treatment group, weakly positive expression of Mcl-1 protein in PD98059 treatment group, and negative expression of Mcl-1 protein in control group. The result of flow cytometry found that the macrophage apoptotic rate in H37Rv infection group was higher than that in con-trol group, while that in PD98059 treatment group was high than that in other groups with statistically significant differences (P<0.05).The result of Mycobacterium tuberculosis colony counting showed that PD98059 treatment had the most signifi-cant inhibitory effect on H37Rv strain.CONCLUSION: Mcl-1 signaling pathway blockers increase the apoptotic rate of macrophages infected with Mycobacterium tuberculosis H37Rv and inhibit the growth of Mycobacterium tuberculosis by inhibi-ting the signaling pathways of JAK/STAT, MAPK and PI3K, among which the MAPK has the most obvious interfering effect on Mcl-1, and leads to the highest apoptotic rate of infected macrophages and the strongest bacteriostasis.

Actividad antituberculosa in vitro de los extractos etanólicos y agliconas de flavonoides de las especies: Grindelia boliviana Rusby (ch'iri ch'iri) y Chenopodium incisum Poir (Arq'a paiqo) sobre cepas de Micobacterium tuberculosis H37Rv / In vitro antituberculous activity of the ethanolic extracts and flavonoid aglycones of the species: Bolivian grindelia Rusby (ch'iri ch'iri) and Chenopodium incisum Poir (Arq'a paiqo) on strains of Mycobacterium tuberculosis H37Rv

El presente trabajo de investigación fue realizado con el propósito de determinar la actividad antituberculosa in vitre de los extractos etanólicos y agliconas de flavonoides de las especies Grindelia boliviana Rusby (Ch'iri ch'iri) y Chenopodium incisum Poir. (Arq'a paiqo) frente a la cepa de Micobacterium tuberculosis H37Rv. La investigación inicia con la recolección de las plantas, secado y molienda de las partes aéreas (hojas, tallos, flores y semillas) para luego ser extraída con solución de etanol al 70% por baño ultrasónico. Los extractos obtenidos se filtran y se depositan en un balón para ser concentrados, en el rotavapor a 40°C individualmente, posteriormente se realizaron las pruebas de solubilidad, identificación cualitativa de los metabolitos secundarios y porcentaje de extracción. Para la extracción de agliconas de flavonoides se tomó material vegetal seco de cada especie, realizándose la extracción con alcohol al 30% en baño ultrasónico, luego se filtró y centrifugó obteniéndose la fase acuosa de interés, los extractos así obtenidos fueron sometidos a una hidrólisis ácida con HCI 6N a la temperatura de 100°C a ebullición. El precipitado obtenido fue purificado en una columna de C-18, obteniéndose las agliconas de flavonoides los cuales fueron !dentificados por cromatografía líquida de alta resolución. Utilizando el método de proporciones de CANETTI, RIST Y GROSSET 1963, se procedió a comprobar la actividad antituberculosa in vitre de los extractos etanólicos y las agliconas de flavonoides, los cuales fueron incorporados al medio Lowenstein Jensen (MU) partiendo desde una concentración mínima hasta una concentración máxima. Luego se realizó el sembrado de las diluciones 10-3 mg/mL, 10-5 mg/mL y 10-6 mg/mL de Micobacterium tuberculosis H37Rv, a los tubos con los medios de Lowenstein Jensen de los extractos etanólicos, como de las agliconas de flavonoides. Finalmente se lleva a la incubadora a 37°c y se espera los 42 días para hacer las lecturas respectivas. Los resultados mostraron: Para los extractos etanólicos. El extracto etanólico de Grindelia boliviana Rusby (Ch'iri ch'iri) presentó una buena actividad antituberculosa a partir de la concentración de 33 mg/mL que sería la CMI, respecto al Chenopodium incisum Poir (Arq'a paiqo) presentó una mejor actividad antimicobacteriana con una CMI de 29 mg/mL, lo cual le confiere una significativa actividad antituberculosa. Para las agliconas de flavonoides. Las agliconas de flavonoides de Grindelia boliviana Rusby (Ch'iri ch'iri) presentó una CMI de 2.8 mg/MI, dicha actividad podría deberse a la presencia de las agliconas de flavonoides que no fueron identificadas. Respecto a las agliconas de Chenopodium incisum Poir (Arq'a paiqo) también presentó una CMI en 2.8 mg/mL. En el análisis por HPLC se logró identificar la aglicona de flavonoide (kaempferol) por comparación con su respectivo estándar, probablemente este metabolito secundario sería el responsable de la actividad antituberculosa. A partir de los resultados presentados se realizó la prueba estadística de ANOVA y POS ANOVA (TUKEY) teniendo como variable independiente las diferentes concentraciones de los extractos y como variable dependiente la actividad antituberculosa cuya significancia es 0.00, el cual indica que existe una diferencia significativa entre las distintas concentraciones

Establishment of a guinea pig model for biological diagnostic reagent of tuberculosis / 中国比较医学杂志

Objective To establish a guinea pig model for diagnostic reagent of tuberculosis.Methods By single or multiple subcutaneous injection of heat-killed H37 Rv in different doses in the groin of guinea pigs to establish a model of positive response to 0.1 mL (5 IU) standard tuberculin ( TB-PPD) skin test.Results Three doses of heat-killed H37 Rv ( 0.2 mg/mL, 0.3 mg/mL and 0.5 mg/mL ) could be used to generate the model of biological diagnosis of tuberculosis.After 24 and 48 hours, the diameter of red spot by TB-PPD skin test was 15.4 ±2.3 mm when a dose of 0.2 mg/mL heat-killed H37 Rv was administered for immunizing and allergizing the guinea pigs.The biggest red spot was induced at doses of 0.3 mg/mL and 0.5 mg/mL.The test results showed that the immune response induced by multiple njection to immunizing and allergizing guinea pigs was not significantly different than that induced by single immunizing injection, and the first skin test was better than the second, third and fourth skin test (P≤0.05).In addition, the body weight of the guinea pigs was still increasing after infection with heat-killed H37 Rv, and ulcers occurred in the injection sites in some guinea pigs.Conclusions A single subcutaneous injection of 0.2 mg/mL heat-killed H37 Rv in guinea pigs can be used well to establish a reliable model for biological diagnostic reagent of tuberculosis.Increasing the sensitizing dose and multiple sensitization can not increase the intensity of the delayed-type hypersensitivity ( DTH) response.

Consistency of standard laboratory strain Mycobacterium tuberculosis H37Rv with ethionamide susceptibility testing.

Drug susceptibility pattern of standard Mycobacterium tuberculosis strain H37Rv showed discrepancy in minimum inhibitory concentration method for ethionamide and consistent results were obtained for the other second line drugs namely, kanamycin and ofloxacin. It is, therefore, necessary to revisit the susceptibility testing method for ethionamide for effective clinical management of patients with drug resistant tuberculosis.

Molecular modeling of 2-nitropropane dioxygenase domain of Mycobacterium tuberculosis H37Rv and docking of herbal ligands.

The 3D structure of enoyl reductase (ER) domain generated by the SWISS MODEL server contains the 2-nitropropane dioxygenase (2NPD) structure displaying the TIM barrel fold. Though TIM barrel fold is made up of both main and inserted domains, in our study, we could only predict the structure of the main domain, which had central barrel of eight β-strands surrounded by eight α-helices. Superimposition of the 2NPD region of ER domain of Mycobacterium tuberculosis H37Rv on to the corresponding region of 2UVA_G revealed a good structural alignment between the two, suggesting this template to be a good structural homologue. Among various herbal ligands that were screened as inhibitors, daucosterol was found to bind in closest proximity to the flavin mono nucleotide (FMN) binding site with the lowest docking energy

Antimycobacterial neolignans isolated from Aristolochia taliscana

Tuberculosis (TB - Mycobacterium tuberculosis) is an ancient infectious disease that has appeared once again as a serious worldwide health problem and now comprises the second leading cause of death resulting from a single infection. The prevalence of multidrug resistance (MDR) TB is increasing and therapeutic options for treatment are not always accessible; in fact, some patients do not respond to the available drugs. Therefore, there is an urgent need to develop novel anti-TB agents. The aim of the present study was to screen extracts of Aristolochia taliscana, a plant used in traditional Mexican medicine to treat cough and snake bites, for antimycobacterial activity. The hexanic extract of A. taliscana was tested by microdilution alamar blue assay against Mycobacterium strains and bioguided fractionation led to the isolation of the neolignans licarin A, licarin B and eupomatenoid-7, all of which had antimycobacterial activity. Licarin A was the most active compound, with minimum inhibitory concentrations of 3.12-12.5 ìg/mL against the following M. tuberculosis strains: H37Rv, four mono-resistant H37Rv variants and 12 clinical MDR isolates, as well as against five non-tuberculous mycobacteria (NTM) strains. In conclusion, licarin A represents a potentially active anti-TB agent to treat MDR M. tuberculosis and NTM strains.

Experimental evidence for dual infection with two genetically distinguishable mycobacterium tuberculosis strains using a guinea pig model.

Background: Infection with Mycobacterium tuberculosis is thought to be induced by a single strain, and the presence of two strains within the same tuberculosis (TB) patient can rarely be considered. Aims: The present experimental study was done to investigate the phenomenon of mixed infection with the H37Rv and Kurono strains of Mycobacterium tuberculosis, which can be distinguished genetically from each other by IS6110 restriction fragment length polymorphism (RFLP). Methods: The guinea pigs were infected with the two strains simultaneously, or initially with H37Rv or Kurono followed by the other strain seven days later or 30 days later, via the airborne route. Results: Two strains were recognized in individual guinea pigs only when the guinea pigs were infected initially with H37Rv and later infected with Kurono strain. The coinfecting strains were unequally distributed in the lung, liver, spleen and lymph nodes depending on number of the colony-forming unit (CFU) and order of infection. Although pulmonary lesions were diminished significantly (incomplete protection against M. tuberculosis) 7 weeks after guinea pigs were infected with H37Rv initially and with Kurono strain 30days later, Kurono strain was recognized in the lungs and H37Rv was recognized in the liver, spleen and pulmonary hilar lymph nodes. Conclusions: These results suggest that prior exposure with a particular strain may lead to partial protection or altered immunity which affects the subsequent exposure and that multiple infections may not be so rare clinically and occur in high-incidence regions of tuberculosis.

Analysis of the Cell Lysate Proteome of a Korean Mycobacterium tuberculosis Isolate K01 with H37Rv and H37Ra Strains

Despite recent economic prosperity, Korea still has high prevalence of tuberculosis. Molecular biologic characterization of Korean Mycobacterium tuberculosis strains might provide a deeper understanding of the forces contributing to the spread of tuberculosis in Korea. Therefore, we analyzed the cell lysate proteome of a representative Korean Mycobacterium tuberculosis isolate (K01) in comparison with laboratory reference strains H37Rv and H37Ra. Seven spots were strongly expressed only in K01 strain compared with M. tuberculosis H37Rv and H37Ra. Through continuous MALDI-MS analysis, these spots were identified as hypothetical protein Rv3849, secreted immunogenic protein Mpt64, Acetyl/propionyl-CoA Carbpxylase (AccD1), alkyl hydroperoxide reductase C (AhpC), N-acetylmuramyl-L-alanine amidase, a putative UDP glucose epimerase, and a transposase. A deeper study of these proteins may provide a clue in the development of effective new anti-tuberculosis vaccines against Korean M. tuberculosis isolates.

Effect of oral exposure of Mycobacterium avium intracellular on the protective immunity induced by BCG.

The relative protective efficacy of oral administration of mycobacteria as compared to the conventional intradermal route of vaccination has been assessed in guinea pigs. Skin test reactivity to partially purified protein derivative and protective immunity to challenge with virulent Mycobacterium tuberculosis were used as parameters of protective immunity. Oral immunisation of guinea pigs either with BCG or with Mycobacterium avium intracellulare induces skin test reactivity and protective immunity comparable to that induced by intradermal route of vaccination. Oral exposure of Mycobacterium avium intracellulare prior to oral or intradermal dose of BCG did not interfere with the protective immunity induced by BCG in guinea pigs challenged with Mycobacterium tuberculosis H37Rv.