New Insights into the Methylation of Mycobacterium tuberculosis Heparin Binding Hemagglutinin Adhesin Expressed in Rhodococcus erythropolis.

In recent years, knowledge of the role that protein methylation is playing on the physiopathogenesis of bacteria has grown. In Mycobacterium tuberculosis, methylation of the heparin binding hemagglutinin adhesin modulates the immune response, making this protein a subunit vaccine candidate. Through its C-terminal lysine-rich domain, this surface antigen interacts with heparan sulfate proteoglycans present in non-phagocytic cells, leading to extrapulmonary dissemination of the pathogen. In this study, the adhesin was expressed as a recombinant methylated protein in Rhodococcus erythropolis L88 and it was found associated to lipid droplets when bacteria were grown under nitrogen limitation. In order to delve into the role methylation could have in host-pathogen interactions, a comparative analysis was carried out between methylated and unmethylated protein produced in Escherichia coli. We found that methylation had an impact on lowering protein isoelectric point, but no differences between the proteins were found in their capacity to interact with heparin and A549 epithelial cells. An important finding was that HbhA is a Fatty Acid Binding Protein and differences in the conformational stability of the protein in complex with the fatty acid were observed between methylated and unmethylated protein. Together, these results suggest that the described role for this mycobacteria protein in lipid bodies formation could be related to its capacity to transport fatty acids. Obtained results also provide new clues about the role HbhA methylation could have in tuberculosis and point out the importance of having heterologous expression systems to obtain modified proteins.

New Isoflavonoids from the extract of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC. and their antimycobacterial activity.

ETHNOPHARMACOLOGY RELEVANCE: The evaluation of the antimycobacterial activity of extracts of medicinal plants used by Mayos against tuberculosis and respiratory problems, allowed the identification of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC (Fabaceae) as the best candidate to find new antimycobacterial compounds. AIM OF THE STUDY: To isolate and characterize the compounds of R. precatoria responsible for the inhibitory and bactericidal activity against Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis ATCC 700084. To determine antimycobacterial synergistic effect of pure compounds and their selectivity index towards Vero cells. MATERIALS AND METHODS: A total of six flavonoids were purified by silica gel column chromatography. Structural elucidation of the isolated compounds was achieved by using 1D and 2D NMR spectroscopy techniques. The configuration at the C-3 chiral center was established by quantum mechanical calculation of the electronic circular dichroism (ECD) spectrum. In vitro inhibitory and bactericidal activity against M. tuberculosis and M. smegmatis were determined with the redox indicator Alamar Blue (resazurin). Synergy was determined by X/Y quotient. Cytotoxicity was measured by MTT assay. RESULTS: The isolated compounds were identified as precatorin A (1), precatorin B (2), precatorin C (3), lupinifolin (4), cajanone (5) and lupinifolinol (6). Compounds 1-3 are new. Compounds 1 to 5 inhibited the growth of M. tuberculosis (MIC ≥31.25µg/mL); compounds 1, 2, 4 and 5 killed the bacteria (MBC ≥31.25µg/mL) and also inhibited M. smegmatis (MIC ≥125µg/mL), while 1 and 4 also resulted bactericidal (MBC ≥125µg/mL). Compounds 4 and 5 presented synergistic effect (X/Y quotient value <0.5) at a concentration of 1/2 MIC of each compound in the combination. Cytotoxicity in murine macrophages (RAW 264.7 cells) gave IC50 values of 13.3-46.98µM, for compounds 1-5. CONCLUSIONS: In this work we isolated two new isoflavanones (1 and 2), and one new isoflavone (3) with a weak antimycobacterial activity. The (3R) absolute configuration was assigned to 1 by computational analysis of its ECD spectrum and to 2 and 5 by similarity of their ECD spectra with that of 1. We are also reporting by first time, activity against virulent strain of M. tuberculosis for compounds 4 and 5 and their antimycobacterial synergistic effect.

Antimycobacterial activity of medicinal plants used by the Mayo people of Sonora, Mexico.

ETHNOPHARMACOLOGICAL RELEVANCE: Tuberculosis (TB) is an infectious disease mainly caused by Mycobacterium tuberculosis (Mtb), which generates 9 million new cases worldwide each year. The Mayo ethnicity of southern Sonora, Mexico is more than 2000 years old, and the Mayos possess extensive knowledge of traditional medicine. AIMS OF THE STUDY: To evaluate the antimycobacterial activity levels of extracts of medicinal plants used by the Mayos against Mtb and Mycobacterium smegmatis (Msm) in the treatment of TB, respiratory diseases and related symptoms. MATERIALS AND METHODS: A total of 34 plant species were collected, and 191 extracts were created with n-hexane, dichloromethane, ethyl acetate (EtOAc), methanol and water. Their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined against Mtb H37Rv using the microplate alamar blue assay (MABA) and against Msm using the resazurin microplate assay (REMA) at 6 and 2 days of exposure, respectively, and at concentrations of 250-1.9µg/mL (n-hexane extracts) and 1000-7.81µg/mL (extracts obtained with dichloromethane, EtOAc, methanol and water). RESULTS: Rhynchosia precatoria (Willd.) DC. (n-hexane root extract), Euphorbia albomarginata Torr. and A. Gray. (EtOAc shoot extract) and Helianthus annuus L. (n-hexane stem extract) were the most active plants against Mtb H37Rv, with MICs of 15.6, 250, 250µg/mL and MBCs of 31.25, 250, 250µg/mL, respectively. R. precatoria (root) was the only active plant against Msm, with MIC and MBC values of ≥250µg/mL. None of the aqueous extracts were active. CONCLUSIONS: This study validates the medicinal use of certain plants used by the Mayo people in the treatment of TB and related symptoms. R. precatoria, E. albomarginata and H. annuus are promising plant sources of active compounds that act against Mtb H37Rv. To our knowledge, this is the first time that their antimycobacterial activity has been reported. Crude extracts obtained with n-hexane, EtOAc and dichloromethane were the most active against Mtb H37Rv.

Reynosin and santamarine: two sesquiterpene lactones from Ambrosia confertiflora with bactericidal activity against clinical strains of Mycobacterium tuberculosis.

CONTEXT: Tuberculosis is primarily caused by Mycobacterium tuberculosis (Mtb). Previous studies have shown that the dichloromethanic extract of Ambrosia confertiflora DC (Asteraceae) inhibited Mtb. OBJECTIVE: To isolate the compounds responsible for the mycobactericidal activity against clinical Mtb strains. MATERIALS AND METHODS: The dichloromethanic extract of aerial parts of A. confertiflora was separated using chromatography columns. Mycobactericidal activity of the isolated compounds was evaluated using the Alamar Blue bioassay (128-16 µg/mL, 7 days). Cytotoxicity was tested against normal cell line L929 using the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium]) assay (100-3.125 µg/mL, 48 h). Compound structures were elucidated by 1H and 13C uni- and bidimensional NMR. RESULTS: Two sesquiterpene lactones (SQLs) with mycobactericidal activity were identified: santamarine and reynosin. Reynosin was the most active compound, with a minimal bactericidal concentration (MBC) of 128 µg/mL against the H37Rv, 366-2009 and 104-2010 Mtb strains and a minimal inhibitory concentration (MIC) of 64, 64, 128, 128 and 128 µg/mL against the H37Rv, 104-2010, 63-2009, 366-2009 and 430-2010 Mtb strains, respectively. Santamarine had MBCs of 128 µg/mL against the H3Rv and 104-2010 Mtb strains and MICs of 128 µg/mL against the H37Rv, 366-2009 and 104-2010 Mtb strains. We also isolated 1,10-epoxyparthenolide but only showed mycobacteriostatic activity (MIC 128 µg/mL) against the Mtb strain. Compounds were tested against the L929 cell line and the calculated selectivity index was <1. DISCUSSION AND CONCLUSIONS: This is the first report of the mycobactericidal activity of these compounds against clinical Mtb strains. It is also the first report of the isolation of 1,10-epoxyparthenolide from A. confertiflora. The anti-mycobacterial activity of A. confertiflora was attributed to the SQLs identified.

In vitro anti-mycobacterial activity of nine medicinal plants used by ethnic groups in Sonora, Mexico.

BACKGROUND: Sonoran ethnic groups (Yaquis, Mayos, Seris, Guarijíos, Pimas, Kikapúes and Pápagos) use mainly herbal based preparations as their first line of medicinal treatment. Among the plants used are those with anti-tuberculosis properties; however, no formal research is available. METHODS: Organic extracts were obtained from nine medicinal plants traditionally used by Sonoran ethnic groups to treat different kinds of diseases; three of them are mainly used to treat tuberculosis. All of the extracts were tested against Mycobacterium tuberculosis H37Rv using the Alamar Blue redox bioassay. RESULTS: Methanolic extracts from Ambrosia confertiflora, Ambrosia ambrosioides and Guaiacum coulteri showed minimal inhibitory concentration (MIC) values of 200, 790 and 1000 µg/mL, respectively, whereas no effect was observed with the rest of the methanolic extracts at the concentrations tested. Chloroform, dichloromethane, and ethyl acetate extracts from Ambrosia confertiflora showed a MIC of 90, 120 and 160 µg/mL, respectively. CONCLUSIONS: A. confertiflora and A. ambrosioides showed the best anti-mycobacterial activity in vitro. The activity of Guaiacum coulteri is consistent with the traditional use by Sonoran ethnic groups as anti-tuberculosis agent.For these reasons, it is important to investigate a broader spectrum of medicinal plants in order to find compounds active against Mycobacterium tuberculosis.