Mycobacterium Smegmatis Mc Research Articles

The Extent of Antimicrobial Resistance Due to Efflux Pump Regulation.

A key mechanism driving antimicrobial resistance (AMR) stems from the ability of bacteria to up-regulate efflux pumps upon exposure to drugs. The resistance gained by this up-regulation is pliable because of the tight regulation of efflux pump levels. This leads to temporary enhancement in survivability of bacteria due to higher efflux pump levels in the presence of antibiotics, which can be reversed when the cells are no longer exposed to the drug. Knowledge of the extent of resistance thus gained would inform intervention strategies aimed at mitigating AMR. Here, we combine mathematical modeling and experiments to quantify the maximum extent of resistance that efflux pump up-regulation can confer via phenotypic induction in the presence of drugs and genotypic abrogation of regulation. Our model describes the dynamics of drug transport in and out of cells coupled with the associated regulation of efflux pump levels and predicts the increase in the minimum inhibitory concentration (MIC) of drugs due to such regulation. To test the model, we measured the uptake and efflux as well as the MIC of the compound ethidium bromide (EtBr), a substrate of the efflux pump LfrA, in wild-type Mycobacterium smegmatis mc2155, as well as in two laboratory-generated strains. Our model captured the observed EtBr levels and MIC fold-changes quantitatively. Further, the model identified key parameters associated with the resulting resistance, variations in which could underlie the extent to which such resistance arises across different drug-bacteria combinations, potentially offering tunable handles to optimize interventions aimed at minimizing AMR.

Complete Genome Sequences of Cluster A Mycobacteriophages Kenmech and Peterson and Cluster F Mycobacteriophage Rockne.

ABSTRACTKenmech, Peterson, and Rockne are bacteriophages that infect Mycobacterium smegmatis mc2 155. Both Kenmech and Peterson genomes are ~52 kbp long and contain 1 tRNA as well as 92 and 89 protein-coding genes, respectively. Rockne has a 56,704-bp genome with 108 protein-coding genes and no tRNA.

Synthesized Phosphonium Compounds Demonstrate Resistant Modulatory and Antibiofilm Formation Activities against Some Pathogenic Bacteria

A library of six compounds with new hybrids in a single molecule triazole ring attached to the phosphonium salts was synthesized. Click chemistry was, however, used to synthesize the 1-, 2-, and 3-triazole intermediates as a tether for the hybrid phosphonium salts. Their antibacterial activity against Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), and Mycobacterium smegmatis mc2155 was determined using the HT-SPOTi assay. Compound 2 showed the most effective antimicrobial activity as it inhibited the growth of Pseudomonas aeruginosa and Staphylococcus aureus at 0.0125 µg/mL and 31.25 µg/mL, respectively. From the FICI data, compounds 2ET-TOL (2) and RABYL-TOL (4) successfully modulated the activities of amoxicillin against Pseudomonas aeruginosa and Staphylococcus aureus. All the test compounds exhibited a concentration-dependent biofilm formation inhibition against S. aureus, except P-Z (compound 6). Compounds P-MEOXY (1) and 2ET-TOL (2) exhibited mild activity against P. aeruginosa with compound 4 showing antimycobacterial activity at 500 µg/mL.

Sulforaphane kills Mycobacterium tuberculosis H37Ra and Mycobacterium smegmatis mc2155 through a reactive oxygen species dependent mechanism.

Mycobacterium tuberculosis (M. tuberculosis) is a highly pathogenic intracellular pathogen that causes tuberculosis (TB), the leading cause of mortality from single infections. Redox homeostasis plays a very important role in the resistance of M. tuberculosis to antibiotic damage and various environmental stresses. The antioxidant sulforaphane (SFN) has been reported to exhibit anticancer activity and inhibit the growth of a variety of bacteria and fungi. Nonetheless, it remains unclear whether SFN exhibits anti-mycobacterial activity. Our results showed that the SFN against M. tuberculosis H37Ra exhibited bactericidal activity in a time and dose-dependent manner. The anti-tubercular activity of SFN was significantly correlated with bacterial reactive oxygen species (ROS) levels. In addition, SFN promoted the bactericidal effect of macrophages on intracellular bacteria in a dose-dependent manner, mediated by increasing intracellular mitochondrial ROS levels and decreasing cytoplasmic ROS levels. Taken together, our data revealed the previously unrecognized antimicrobial functions of SFN. Future studies focusing on the mechanism of SFN in macrophages against M. tuberculosis are essential for developing new host-directed therapeutic approaches against TB.

Improving high-throughput techniques for bacteriophage discovery in multi-well plates

Bacteriophages (also called phages) are viruses of bacteria that have numerous applications in medicine, agriculture, ecology, and molecular biology. With the increasing interest in phages for their many uses, it is now especially important to make phage discovery more efficient and economical.Using the host Mycobacterium smegmatis mc2155, which is a model organism for phage discovery research and is closely related to important pathogens of humans and other animals, we investigated three procedures that are an integral part of phage discovery: enrichment of environmental samples, phage isolation and detection (which can also be used for host range determination), and phage purification. Enrichment in 6-well plates was successful with most environmental samples, and enrichment in 24- and 96-well plates was successful with some environmental samples, demonstrating that larger sample volumes are preferred when possible, but smaller sample volumes may be acceptable if the starting concentration of phages is sufficiently high. Measuring absorbance in multi-well plates was at least as sensitive as the traditional plaque assay for the detection of phages. We also demonstrated a technique for the purification of single phage types from mixed cultures in liquid medium.Multi-well techniques can be used as alternatives or complementary approaches to traditional methods of phage discovery and characterization depending on the needs of the researcher in terms of time, available resources, host species, phage-bacteria matches, and specific goals. In the future, these techniques could be applied to the discovery of phages of aquatic mycobacteria and other hosts for which few phages have currently been isolated.

Mycobacteriophages: From Petri dish to patient.

Mycobacteriophages—bacteriophages infecting Mycobacterium hosts—contribute substantially to our understanding of viral diversity and evolution, provide resources for advancing Mycobacterium genetics, are the basis of high-impact science education programs, and show considerable therapeutic potential. Over 10,000 individual mycobacteriophages have been isolated by high school and undergraduate students using the model organism Mycobacterium smegmatis mc2155 and 2,100 have been completely sequenced, giving a high-resolution view of the phages that infect a single common host strain. The phage genomes are revealed to be highly diverse and architecturally mosaic and are replete with genes of unknown function. Mycobacteriophages have provided many widely used tools for Mycobacterium genetics including integration-proficient vectors and recombineering systems, as well as systems for efficient delivery of reporter genes, transposons, and allelic exchange substrates. The genomic insights and engineering tools have facilitated exploration of phages for treatment of Mycobacterium infections, although their full therapeutic potential has yet to be realized.

Streptocinnamides A and B, Depsipeptides from Streptomyces sp. KMM 9044.

The bacterium Streptomyces sp. KMM 9044 from a sample of marine sediment collected in the northwestern part of the Sea of Japan produces highly chlorinated depsiheptapeptides streptocinnamides A (1) and B (2), representatives of a new structural group of antibiotics. The structures of 1 and 2 were determined using nuclear magnetic resonance and mass spectrometry studies and confirmed by a series of chemical transformations. Streptocinnamide A potently inhibits Micrococcus sp. KMM 1467, Arthrobacter sp. ATCC 21022, and Mycobacterium smegmatis MC2 155.

Expression of a novel mycobacterial phosphodiesterase successfully lowers cAMP levels resulting in reduced tolerance to cell wall–targeting antimicrobials

cAMP and antimicrobial susceptibility in mycobacteriaAntimicrobial tolerance, the ability to survive exposure to antimicrobials via transient nonspecific means, promotes the development of antimicrobial resistance (AMR). The study of the molecular mechanisms that result in antimicrobial tolerance is therefore essential for the understanding of AMR. In gram-negative bacteria, the second messenger molecule 3′’,5′’-cAMP has been previously shown to be involved in AMR. In mycobacteria, however, the role of cAMP in antimicrobial tolerance has been difficult to probe due to its particular complexity. In order to address this difficulty, here, through unbiased biochemical approaches consisting in the fractionation of clear protein lysate from a mycobacterial strain deleted for the known cAMP phosphodiesterase (Rv0805c) combined with mass spectrometry techniques, we identified a novel cyclic nucleotide-degrading phosphodiesterase enzyme (Rv1339) and developed a system to significantly decrease intracellular cAMP levels through plasmid expression of Rv1339 using the constitutive expression system, pVV16. In Mycobacterium smegmatis mc2155, we demonstrate that recombinant expression of Rv1339 reduced cAMP levels threefold and resulted in altered gene expression, impaired bioenergetics, and a disruption in peptidoglycan biosynthesis leading to decreased tolerance to antimicrobials that target cell wall synthesis such as ethambutol, D-cycloserine, and vancomycin. This work increases our understanding of the role of cAMP in mycobacterial antimicrobial tolerance, and our observations suggest that nucleotide signaling may represent a new target for the development of antimicrobial therapies.

Production and Purification of Phosphatidylinositol Mannosides from Mycobacterium smegmatis Biomass.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is regarded as the most successful pathogen of humankind and a major threat to global health. The mycobacterial cell wall is vital for cell growth, virulence, and resistance to antibiotics, and thus constitutes a unique target for drug development. To characterize the enzymes catalyzing the synthesis of the cell wall components, considerable amounts of substrates are required. Since many mycobacterial cell wall lipids, particularly phosphatidylinositol mannosides (PIMs), are not commercially available, isolation from cell biomass is the most straightforward way to obtain these compounds. In this study, we optimized a protocol to extract and purify PIM species, in particular Ac1 PIM2 and Ac1 PIM4 , which can be further used for the identification and characterization of target enzymes. PIMs were extracted from Mycobacterium smegmatis mc2 155 ΔPimE using organic solvents, and purified through three consecutive chromatography steps. Thin-layer chromatography (TLC) was used in-between purification steps to evaluate the success of lipid separation, and nuclear magnetic resonance (NMR) was used for product quantification and to assess purity. Typically, from a 60 g batch of M. smegmatis biomass we were able to isolate approximately 9 mg of Ac1 PIM2 and 1.8 mg of Ac1 PIM4 . This is the first time the purification of phosphatidylinositol tetramannoside has been reported. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of M. smegmatis mc2 155 ∆PimE Basic Protocol 2: Extraction of lipids from M. smegmatis mc2 155 ∆PimE Basic Protocol 3: Treatment of the lipid extract for isolation of phospholipids Basic Protocol 4: Isolation of phosphatidylinositol mannosides Basic Protocol 5: Quantification of phosphatidylinositol mannosides.

In silico and in vitro study of Mycobacterium tuberculosisH37Rv uncharacterized protein (RipD): an insight on tuberculosis therapeutics.

Tuberculosis caused byMycobacterium tuberculosis(Mtb)is responsible forthe highest global health problem, with the deaths of millions of people. With prevalence of multiple drug resistance (MDR) strains and extended therapeutic times, it is important to discover small molecule inhibitors against novel hypothetical proteins of the pathogen. In this study, a virtual screening protocol was carried out against MtbH37Rv hypothetical protein RipD (Rv1566c) for the identification of potential small molecule inhibitors. The 3D model of the protein structure binding site was used for virtual screening (VS) of inhibitors from the Pathogen Box, followed by its validation through a molecular docking study. The stability of the protein-ligand complex was assessed using a 150ns molecular dynamics simulation. MM-PBSA and MM-GBSA are the two approaches that were used to perform the trajectory analysis and determine the binding free energies, respectively. The ligand binding was observed to be stable across the entire time frame with an approximate binding free energy of -22.9916kcal/mol. The drug-likeness of the inhibitors along with a potential anti-tuberculosis compound was validated by ADMET prediction software. Furthermore, a CFU inhibition assay was used to validate the best hit compound's in vitro inhibitory efficacy against a non-pathogenic Mycobacterium smegmatis MC2155 under low nutrient culture conditions. The study reported that the compound proposed in our study (Pathogen Box ID: MMV687700) will be useful for the identification of potential inhibitors against Mtb in future.

Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis

Rifampicin is a critical first-line antibiotic for treating mycobacterial infections such as tuberculosis, one of the most serious infectious diseases worldwide. Rifampicin resistance in mycobacteria is mainly caused by mutations in the rpoB gene; however, some rifampicin-resistant strains showed no rpoB mutations. Therefore, alternative mechanisms must explain this resistance in mycobacteria. In this work, a library of 11,000 Mycobacterium smegmatis mc2 155 insertion mutants was explored to search and characterize new rifampicin-resistance determinants. A transposon insertion in the MSMEG_1945 gene modified the growth rate, pH homeostasis and membrane potential in M. smegmatis, producing rifampicin resistance and collateral susceptibility to other antitubercular drugs such as isoniazid, ethionamide and aminoglycosides. Our data suggest that the M. smegmatis MSMEG_1945 protein is an ion channel, dubbed MchK, essential for maintaining the cellular ionic balance and membrane potential, modulating susceptibility to antimycobacterial agents. The functions of this new gene point once again to potassium homeostasis impairment as a proxy to resistance to rifampicin. This study increases the known repertoire of mycobacterial ion channels involved in drug susceptibility/resistance to antimycobacterial drugs and suggests novel intervention opportunities, highlighting ion channels as druggable pathways.

Mutation in Eth A protein of Mycobacterium tuberculosis conferred drug tolerance against enthinoamide in Mycobacterium smegmatis mc2155

EthA is an NADPH-specific flavin adenine dinucleotide (FAD) containing monooxygenase that activates the –second-line drug ethionamide (ETH). ETH gets converted to an active form after interaction with the EthA (monooxygenase) protein. Upon activation, ETH interacts with NAD+ to form an ETH-NAD adduct, which hampers the activity of InhA (Enoyl-[(acyl-carrier-protein) reductase (NADH)]. This, in turn, inhibits the cell wall synthesis, thus killing the Mycobacterium tuberculosis (Mtb). Mutations in the EthA gene can modulate ETH activation. The mutation at 202 position (Val202-Leu) of EthA protein has been reported frequently in ETH resistance. In this study, the effect of this mutation on the function of the EthA protein was examined through structural and functional analysis. Molecular docking of wild type and mutated EthA protein with ETH were compared to inspect the effect of mutation on molecular mechanism of drug resistant. Docking results corroborated that the lower docking score of the mutant protein, larger binding cavity, and lower affinity towards ETH resulted in a less compact and energetically less stable structure than the wild type protein. The computational outcome was authenticated by in-vitro experiments. The wild type and mutated genes were cloned and expressed in M. smegmatis, a surrogate host. Antibiotic susceptibility testing demonstrated that the mutant showed high growth and survival in the presence of the ETH drug. Overall, the results indicated that a mutation in the intergenic region of EthA protein could result in the altered conversion of ETH to the active form, resulting in differential ETH sensitivity for M. smegmatis carrying the wild type and mutant gene.

First conjugation directed traverse of gene cassettes harboring α1,3GT from fast-growing Mycobacterium smegmatis mc2 155 to slow-growing pathogen Mycobacterium tuberculosis H37Rv, presumably opening up new scopes in tuberculosis treatment

Mycobacterium smegmatis strain mc2 155 is a fast-growing and non-pathogenic mycobacteria and widely used in genetic studies of mycobacteria. It has been shown that this species of mycobacterium can transfer its genomic DNA fragments to other species of mycobacteria during the conjugation process. Galα1–3Galβ1–4GlcNAc-R (α-gal) glycan epitope is a highly immunogenic epitope exerted by the enzyme α1–3-galactosyltransferase (α1,3GT) in mammalian cells on the glycan skeleton. However, the enzyme is inactive in humans, primates and Old World monkeys as a result of evolutionary mutations. The robust immunogenicity induced by the epitope in human, has attracted much attention to apply the epitope in vaccine research. In this study we proved successful transfer of desired expression cassettes from fast-growing Mycobacterium smegmatis mc2 155, to the slow-growing pathogen Mycobacterium tuberculosis H37Rv. We designed gene cassettes encoding the α1,3GT enzyme under control the potent G13 promoter and the cassette containing hygromycin resistance gene under a Mtb specific promoter, Ptpa in the vector pMV306DIG13 +FflucRT (harboring attP site). The resulting construct was electroporated into mc2 155 strain in combination with pBS-int containing the gene encoding Mycobacteriophage L5 integrase to integrate pMV306DIG13 +FflucRT-cassettes into mc2 155 genome. Following the integration, the recombinant clones were placed in vicinity to the Mycobacterium tuberculosis H37Rv strain to establish conjugation. Conjugated recombinant clones were selected on the medium containing the hygromycin B and transfer of the desired cassettes to Mycobacterium tuberculosis was confirmed. The enzyme α1,3GT in transconjugants were also investigated.

A multifactorial assessment of the SRP pathway constituent FtsY as a vital mycobacterial constituent.

The signal recognition particle (SRP) system plays an imperative role in transporting the secretory protein to its intended location. The SRP pathway running in Mycobacterium tuberculosis constitutes FtsY (signal receptor), FfH (SRP), and 4.5S RNA in which signal receptor acts in the GTP-dependent manner. In this study, we are rendering the essential facts of FtsY with respect to mycobacterial growth. The growth study experiment showed that downexpressed FtsY slowed the growth of Mycobacterium smegmatis mc2 155 from the initial lag phase to stationary phase. Previously, we have showed that GTPase activity of FtsY is metal ion dependent and showed the maximum activity with 10mM magnesium. The effect of Mg2+ and Mn2+ on mycobacterial growth showed that Mg2+ did not affect the growth, whereas higher concentration of Mn2+ decreases the bacterial growth. After searching the inhibitor database, 14 GTPase and ATPase inhibitors, Mac0182344, ML141, ITX3, NAV_2729, Br-GTP, Rhosin_HCl, Mac0182099, CCG_50014, CID_1067700, Mac0174809, Nsc_23766, Berberine, Nexinhib20, and EHT1864, were found to interact with FtsY. Further, ML141 and NAV2729 found to decrease the enzymatic activity of FtsY as well as the mycobacterial growth. Therefore, the conclusive statement of the present study can be stated as that the FtsY plays major role in mycobacterial cell survival and ML141 and NAV2729 can be used to constrain the SRP pathway.

Biosynthesis of the redox cofactor mycofactocin is controlled by the transcriptional regulator MftR and induced by long-chain acyl-CoA species

Mycofactocin (MFT) is a ribosomally synthesized and post-translationally-modified redox cofactor found in pathogenic mycobacteria. While MFT biosynthetic proteins have been extensively characterized, the physiological conditions under which MFT biosynthesis is required are not well understood. To gain insights into the mechanisms of regulation of MFT expression in Mycobacterium smegmatis mc2155, we investigated the DNA-binding and ligand-binding activities of the putative TetR-like transcription regulator, MftR. In this study, we demonstrated that MftR binds to the mft promoter region. We used DNase I footprinting to identify the 27 bp palindromic operator located 5′ to mftA and found it to be highly conserved in Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, and Mycobacterium marinum. To determine under which conditions the mft biosynthetic gene cluster (BGC) is induced, we screened for effectors of MftR. As a result, we found that MftR binds to long-chain acyl-CoAs with low micromolar affinities. To demonstrate that oleoyl-CoA induces the mft BGC in vivo, we re-engineered a fluorescent protein reporter system to express an MftA–mCherry fusion protein. Using this mCherry fluorescent readout, we show that the mft BGC is upregulated in M. smegmatis mc2155 when oleic acid is supplemented to the media. These results suggest that MftR controls expression of the mft BGC and that MFT production is induced by long-chain acyl-CoAs. Since MFT-dependent dehydrogenases are known to colocalize with acyl carrier protein/CoA-modifying enzymes, these results suggest that MFT might be critical for fatty acid metabolism or cell wall reorganization.

Complete Genome Sequences of Mycobacterium Phages Tripl3t and Zeuska.

ABSTRACTTripl3t and Zeuska are siphoviral bacteriophages that were isolated from Mycobacterium smegmatis mc2 155 and contain double-stranded DNA genomes 53,565 bp and 53,598 bp in length, respectively. Tripl3t and Zeuska were annotated by students at Bluff Dale High School (Bluff Dale, TX) and Tolar High School (Tolar, TX) in community engagement with Tarleton State University.

Investigating Ghanaian Allium Species for Anti-Infective and Resistance-Reversal Natural Product Leads to Mitigate Multidrug-Resistance in Tuberculosis.

The bulbs of Allium species are a known source of antibacterial phytochemicals. Anti-infective, efflux pump and biofilm inhibitory activities of bulb extracts of selected Ghanaian shallots Allium cepa var aggregatum were evaluated using the HT-SPOTi assay and other whole-cell phenotypic screening techniques to determine their possible mechanisms of action. Ethanol and aqueous extracts of white A. cepa inhibited the growth of Mycobacterium smegmatis mc2 155 and Escherichia coli, respectively. The majority of the Allium extracts significantly (p < 0.05) exhibited efflux pump inhibitory activity against all the acid-fast, Gram-positive and Gram-negative strains used. Hexane and chloroform extract of the pink A. cepa and the aqueous extract of the white A. cepa significantly inhibited M. smegmatis biofilm formation. For Pseudomonas aeruginosa, the inhibition was observed at 250 µg/mL for the aqueous extract (~77.34%) and 125 µg/mL for the hexane extract (~76.51%). The results suggest that Ghanaian shallots could potentially be useful when further developed to tackle antimicrobial resistance, particularly in tuberculosis (TB).

Complete Genome Sequences of Mycobacteriophages Dallas and Jonghyun.

ABSTRACTTwo temperate mycobacteriophages, Dallas and Jonghyun, were isolated from soil in Washington, DC, using the bacterial host Mycobacterium smegmatis mc2155. Analysis of the genomes revealed that Dallas and Jonghyun belong to clusters J and G, respectively. The structures of the genomes are typical of their respective clusters.

Structural characterization and Kemp eliminase activity of the Mycobacterium smegmatis Ketosteroid Isomerase

The Kemp elimination reaction, involving the ring-opening of benzoxazole and its derivatives under the action of natural enzymes or chemical catalysts, has been of interest to researchers since its discovery. Because this reaction does not exist in all currently known metabolic pathways, the computational design of Kemp eliminases has provided valuable insights into principles of enzymatic catalysis. However, it was discovered that the naturally occurring promiscuous enzymes ydbC, xapA and ketosteroid isomerase also can catalyze Kemp elimination. Here, we report the crystal structure of ketosteroid isomerase (KSI) from Mycobacterium smegmatis MC2 155. MsKSI crystallizes in the P212121 space group with two molecules in an asymmetric unit, and ultracentrifugation data confirms that it forms a stable dimer in solution, consistent with the 1.9 Å-resolution structure. Our assays confirm that MsKSI accelerates the Kemp elimination of 5-nitrobenzoxazole (5NBI) with an optimal pH of 5.5. A 2.35 Å resolution crystal structure of the MsKSI-5NBI complex reveals that the substrate 5NBI is bound in the active pocket of the enzyme composed of hydrophobic residues. In addition, the Glu127 residue is proposed to play an important role as a general base in proton transfer and breaking weak O–N bonds to open the five-membered ring. This work provides a starting point for exploring the artificial modification of MsKSI using the natural enzyme as the backbone.

Complete Genome Sequences of Mycobacteriophages HarryOW and Peeb.

HarryOW and Peeb are Mycobacterium smegmatis mc2 155 Siphoviridae temperate phages with 52,935 and 41,876 base pairs in genome length, respectively. HarryOW belongs to the A1 subcluster and Peeb to the G1 subcluster. They were isolated and annotated by students from the SUNY Old Westbury Science and Technology Entry Program.