Aims: This study aimed to evaluate the antimicrobial activity of naturally derived phenylpropanoids from Alpinia conchigera (A. conchigera) Griff. and its synthetic analogues, as well as interactions between selected compounds with first-line tuberculosis (TB) drug, rifampicin, against Mycobacterium smegmatis, a potential opportunistic nontuberculous mycobacterium (NTM) and a surrogate organism for TB. Methodology and results: Twelve phenylpropanoids of A. conchigera were evaluated for antimicrobial activity against M. smegmatis (ATCC 14468). The phenylpropanoid compound from A. conchigera with the lowest minimum inhibitory concentration and bactericidal (MIC, MBC) values were selected for checkerboard tetrazolium microplate assay (TEMA) with rifampicin to determine drug interactions. A majority of the compounds had antimicrobial activity, however, purified natural compound 1'S-1'-acetoxychavicol acetate (ACA) showed the highest antimicrobial activity with an MIC value of 62.5 µg/mL against M. smegmatis. The combination of ACA and rifampicin produced indifferent interaction with fractional inhibition concentration (FIC) index of 1.5, while the combination of rifampicin and ACA synthetic analogue 4-allyl-2,6- methoxyphenyl isobutyrate produced a synergistic interaction effect with FIC index of 0.5. None of the compounds tested were bactericidal but appear to be bacteriostatic. Conclusion, significance and impact of study This study presents the first report on the antimicrobial potential of natural A. conchigera-derived ACA against M. smegmatis as well as the synergistic interaction of 4-allyl-2,6- methoxyphenyl isobutyrate with rifampicin which warrants further investigation.
Anti-Infective Agents ; Alpinia ; Mycobacterium smegmatis

Tuberculosis (TB) patients are normally treated with a combination of antibiotics. However, with improper or incomplete treatment of antibiotics, the disease may progress to multidrug-resistant TB (MDR-TB). The treatment of MDR-TB is very costly and inefficient. Therefore, there is a great demand of new therapeutic approaches for MDR-TB such as photodynamic therapy. In this study, we tried to optimize the conditions for photodynamic inactivation of TB using methylene blue as a photosensitizer. Different combinations of methylene blue concentrations and light doses were tested for their photodynamic effects to A549 cells or Mycobacterium smegmatis (M. smegmatis). We also tested the effect of photodynamic therapy on ciprofloxacin-resistant M. smegmatis. Methylene blue treatment alone did not affect the survival rates of A549 cells or bacteria up to 5 µg/ml. When the A549 and M. smegmatis cells treated with methylene blue were irradiated with laser light (wavelength, 630 nm), photodynamic inactivation of cells was increased in methylene blue concentration- and light dose-dependent manners. Interestingly, the ciprofloxacin-resistant M. smegmatis exhibited higher level of susceptibility to methylene blue-mediated photodynamic inactivation. This study suggests that photodynamic therapy at 3.6 J/cm2 in the presence of 5 µg/ml methylene blue may be an appropriate range for therapy due to the high bactericidal activity against high level of ciprofloxacin-resistant M. smegmatis and the low damaging effect to mammalian cells. This study demonstrates that photodynamic therapy could be a potential alternative for MDR-TB treatment.
Anti-Bacterial Agents ; Bacteria ; Ciprofloxacin ; Humans ; Methylene Blue ; Mycobacterium smegmatis* ; Mycobacterium* ; Photochemotherapy* ; Survival Rate ; Tuberculosis

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.
Antitubercular Agents/pharmacology* ; Bacterial Proteins/metabolism* ; Drug Resistance ; Mycobacterium smegmatis/metabolism* ; Peptide Elongation Factor G/pharmacology*

OBJECTIVETo study the effect of Mycobacterium smegmatis vaccine on the level of nitric oxide (NO) produced by peritoneal macrophages in immunized mice.

METHODSBalb/c mice were randomized into low-dose, middle-dose, and high-dose groups (injected with different doses of Mycobacterium smegmatis vaccine) and a control group (injected with normal saline). Then the peritoneal macrophages were cultured with lipopolysaccharide in vitro. The supernatants were collected and the concentrations of NO were analyzed through the reaction with Griess reagents.

RESULTSThe levels of NO produced by the peritoneal macrophages in the control group, low-dose group, middle-dose group, and high-dose group were (3.50 +/- 3.11), (16.63 +/- 6.47), (13.97 +/- 6.20), and (7.55 +/- 2.26) ng/ml, respectively. The levels of NO in all dosing groups were significantly different from that in control group (P < 0.01).

CONCLUSIONMycobacterium smegmatis vaccine can promote the peritoneal macrophages to produce NO in mice.

Animals ; Bacterial Vaccines ; therapeutic use ; Lipopolysaccharides ; Macrophages, Peritoneal ; metabolism ; Mice ; Mice, Inbred BALB C ; Mycobacterium smegmatis ; Nitric Oxide ; metabolism

Adaptor protein ClpS is an essential regulator of prokaryotic ATP-dependent protease ClpAP, which delivers certain protein substrates with specific amino acid sequences to ClpAP for degradation. However, ClpS also functions as the inhibitor of the ClpAP-mediated protein degradation for other proteins. Here, we constructed the clpS-overexpression Mycobacterium smegmatis strain, and showed for the first time that overexpression of ClpS increased the resistance of M. smegmatis to rifampicin that is one of most widely used antibiotic drugs in treatment of tuberculosis. Using quantitative proteomic technology, we systematically analyzed effects of ClpS overexpression on changes in M. smegmatis proteome, and proposed that the increased rifampicin resistance was caused by ClpS-regulated drug sedimentation and drug metabolism. Our results indicate that the changes in degradation related proteins enhanced drug resistance and quantitative proteomic analysis is an important tool for understanding molecular mechanisms responsible for bacteria drug resistance.
ATP-Dependent Proteases ; metabolism ; Drug Resistance, Bacterial ; Endopeptidase Clp ; metabolism ; Mycobacterium smegmatis ; drug effects ; metabolism ; Proteolysis ; Proteomics ; Rifampin ; pharmacology

Cryoelectron Microscopy ; Mycobacterium smegmatis ; genetics ; ultrastructure ; RNA, Ribosomal ; ultrastructure ; Ribosomal Proteins ; genetics ; isolation & purification ; Ribosomes ; genetics ; ultrastructure

ADP-ribosyltransferase (ADPRT) catalyzes the reaction in which the ADP-ribose moiety of beta-NAD+ is transferred to specific amino acid residues in target proteins. The ADPRT of Mycobacterium smegmatis has been known to inactivate rifampin through ADP-ribosylation. However, the enzymatic characteristics and functions of the enzyme have not been elucidated yet. In this study, the ADPRT-glutathione S-transferase (GST) fusion protein was expressed in Escherichia coli and enzymatic characteristics of the fusion protein were investigated. ADPRT-GST fusion protein was an ADPribosyltransferase that had no NAD glycohydrolase activity. ADPRT-GST fusion protein showed no self-inactivation phenomenon that is a universal nature for all NAD glycohydrolases and is important in regulating its activity. ADPRT activity of the enzyme was decreased by novobiocin and isonicotinic acid hydrazide. These results suggest that Mycobacterium smegmatis ADPRT could be regulated by a different way from other NADases and involved in bacterial physiological process through a post-translational modification of cytosolic proteins.
Adenosine Diphosphate Ribose ; ADP Ribose Transferases* ; Cytosol ; Escherichia coli ; Isoniazid ; Mycobacterium smegmatis* ; Mycobacterium* ; NAD+ Nucleosidase ; Novobiocin ; Physiological Processes ; Protein Processing, Post-Translational ; Rifampin

The on-site labeling and localization tracking of membrane proteins in pathogenic bacteria are tedious work. In order to develop a novel protein labeling technology at super resolution level (nanometer scale) using the photoactivatable localization microscopy (PALM), the chimeric protein of the outer membrane protein A (OmpA) of Mycobacterium tuberculosis and the photoactivatable mEos2m protein were expressed in the non-pathogenic Mycobacterium smegmatis. The recombinant bacteria were fixed on slide, activated by 405 nm laser and subject to PALM imaging to capture photons released by the fusion protein. Meanwhile, colony and cell morphology were visualized under regular fluorescent stereomicroscope and upright fluorescent microscope to characterize fluorescence conversion and protein localization. The fusion proteins formed a "belt"-like structure on cell membrane of M. smegmatis under PALM, providing direct evidence of on-site imaging of membrane proteins. Expression of fusion protein did not compromise the localization properties of OmpA. Thus, mEos2m could be used as a labeling probe to track localizations of non-oligomer oriented membrane proteins. This indicates non-pathogenic M. smegmatis could be served as a model strain to characterize the function and localization of the proteins derived from pathogenic M. tuberculosis. This is the first report using PALM to characterize localization of membrane proteins.
Bacterial Outer Membrane Proteins ; analysis ; chemistry ; Fluorescent Dyes ; Light ; Microscopy ; methods ; Mycobacterium smegmatis ; chemistry ; Mycobacterium tuberculosis ; chemistry ; Nanotechnology ; methods ; Staining and Labeling ; methods ; Stochastic Processes

9α-hydroxy-4-androstene-3,17-dione (9-OH-AD) is an important intermediate in the steroidal drugs production. 3-ketosteroid-9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the transformation of 4-androstene-3,17-dione (AD) into 9-OH-AD specifically. In the present study, the putative KshA and KshB genes were cloned from Mycobacterium smegmatis mc(2)155 and Gordonia neofelifaecis NRRL B-59395 respectively, and were inserted into the expression vector pNIT, the co-expression plasmids of kshA-kshB were obtained and electroporated into Mycobacterium sp. NRRL B-3805 cells. The recombinants were used to transform steroids, the main product was characterized as 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), showing that kshA and kshB were expressed successfully. Different from the original strain Mycobacterium sp. NRRL B-3805 that accumulates 4-androstene-3,17-dione, the recombinants accumulates 9α-hydroxy-4-androstene-3,17-dione as the main product. This results indicates that the putative genes kshA, kshB encode active KshA and KshB, respectively. The process of biotransformation was investigated and the results show that phytosterol is the most suitable substrate for biotransformation, kshA and kshB from M. smegmatis mc(2)155 seemed to exhibit high activity, because the resultant recombinant of them catalyzed the biotransformation of phytosterol to 9-OH-AD in a percent conversion of 90%, which was much higher than that of G. neofelifaecis NRRL B-59395. This study on the manipulation of the ksh genes in Mycobacterium sp. NRRL B-3805 provides a new pathway for producing steroid medicines.
Androstenedione ; analogs & derivatives ; biosynthesis ; Bacterial Proteins ; genetics ; metabolism ; Biotransformation ; Ketosteroids ; Mixed Function Oxygenases ; genetics ; metabolism ; Mycobacterium ; metabolism ; Mycobacterium smegmatis ; enzymology ; Plasmids

To construct the secretive prokaryotic shuttle expression plasmid pBCG-SP-HSP65, the signal peptide sequence of antigen 85B amplified from Bacillus Calmette-guérin (BCG) genome by PCR and the whole HSP65 DNA sequence of human M. tuberculosis obtained from the plasmid pCMV-MTHSP65 by PCR were cloned into the plasmid pBCG-2100 under the control of the promoter of Heat Shock Protein 70 (HSP70) from human M. tuberculosis. Recombinants were electroporated into Mycobacterial smegmatis and induced by heating. Results of the induced expression were detected by SDS-PAGE and the biological activity of the expressed protein was tested by Western-blot analysis. Results showed pBCG-SP-MTHSP65 was constructed successfully and confirmed by restriction endonuclease analysis, PCR detection and DNA sequencing analysis. After it was electroporated into Mycobacterial smegmatis and induced by heating, the percentage of expressed 65kD protein in Mycobacterial smegmatis detected by SDS-PAGE was 20% in total bacterial protein. But the percentage of expressed 65kD protein in recombibinant Mycobacterial smegmatis was up to 34.46% in total bacterial protein and 68.56% in the total protein of cell lysate supernants, Which demonstrated the recombinant HSP65 gene could express in recombinant with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive proteins could specially combine with antibody against human M. tuberculosis HSP65. Orally, pBCG-SP-HSP65 was successfully constructed; HSP65 gene could express in Mycobacterial smegmatis with high efficiency via it. And the expressed proteins possess the biological activity. So it provids experimental evidence for the application of the recombinant Mycobacterial smegmatis and the development of the vaccine against tuberculosis.
Bacterial Proteins ; biosynthesis ; genetics ; Chaperonin 60 ; Chaperonins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Mycobacterium smegmatis ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics