The Benefits of Toxicity: M. smegmatis VapBC TA Module Is Induced by Tetracycline Exposure and Promotes Survival.

Toxin-antitoxin (TA) systems are widely present in bacterial genomes. Mycolicibacterium smegmatis, a common model organism for studying Mycobacterium tuberculosis physiology, has eight TA loci, including mazEF and vapBC. This study aims to investigate the physiological significance of these TA systems. Proteomic profiling was conducted on a culture overexpressing the VapC toxin, and the involvement of VapC in M. smegmatis stress responses to heat shock and antibiotic treatment was examined. While deciphering the underlying mechanisms of the altered stress resistance, we assessed the antibiotic susceptibility of vapBC, mazEF, and double vapBC-mazEF deletion mutants. Additionally, the mRNA levels of vapC and mazF were measured following tetracycline supplementation. The results reveal changes in the abundance of metabolic enzymes and stress response proteins associated with VapC overexpression. This activation of the general stress response leads to reduced thermosensitivity in M. smegmatis, but does not affect susceptibility to ciprofloxacin and isoniazid. Under tetracycline treatment, both vapC and mazF expression levels are increased, and the fate of the cell depends on the interaction between the corresponding TA systems.

Generation of a Cell Line Selectively Producing Functionally Active OATP1B1 Transporter.

The solute carrier organic anion transporter family member, OATP1B1, is one of the most important transporter proteins, which mediate penetration of many endogenous substances and xenobiotics into hepatocytes. A model system providing expression of the functional protein is needed to assess interaction of OATP1B1 with various substances. Based on the HEK293 cells, we obtained the HEK293-OATP1B1 cell line, constitutively expressing the SLCO1B1 gene encoding the OATP1B1 transporter. Expression of the SLCO1B1 gene was confirmed by real-time PCR analysis and Western blotting. Functionality of the transporter was assessed by the transport of atorvastatin, which is a substrate of OATP1B1. Cells of the resulting cell line, which selectively express the functionally active recombinant OATP1B1 transporter, can be used to study functions of the protein and to test drugs for being substrates, inducers, and inhibitors of OATP1B1, and to assess the risks of drug interactions.

Development of Heterologous Expression System and Optimization of the Method of Cholera Toxin ß-Subunit Production in E. coli.

Cholera is a deadly infection disease, which is usually associated with low hygiene levels and limited access to high-quality drinking water. An effective way to prevent cholera is the use of vaccines. Among active vaccine components there is the CtxB protein (cholera toxin ß-subunit). In the current work, we have developed a genetic system for production of the recombinant CtxB in E. coli cells and studied conditions for synthesis and purification of the target product at the laboratory scale. It has been found that the optimal algorithm for isolation of the recombinant protein is to grow E. coli culture in the synthetic M9 medium with glycerol, followed by CtxB purification out of the spent culture medium using Ni2+-chelate affinity chromatography techniques. Forty-eight hours after induction of CtxB expression, concentration of the target product could be up to 50 mg/liter in the culture medium. The CtxB protein retains its pentameric structure during expression and through purification. The latter makes it possible to consider the developed system as a promising tool for the industrial-level production of recombinant CtxB for medical and research purposes.

VapC toxin switches M. smegmatis cells into dormancy through 23S rRNA cleavage.

Mycobacterium tuberculosis is an extremely successful pathogen known for its ability to cause latent infection. The latter is connected with the bacterium resting state development and is considered to be based on the activity of toxin-antitoxin (TA) systems at least in part. Here we studied the physiological and proteomic consequences of VapC toxin overexpression together with the features of the protein synthesis apparatus and compared them with the characteristics of dormant mycobacterial cells in an M. smegmatis model. The findings allow suggesting the mechanism mycobacteria enter dormancy, which is realized through VapC-caused cleavage of the 23S rRNA Sarcin-Ricin loop followed by conservation of stalled ribosomes in a membrane-associated manner. The found features of resting mycobacteria protein synthesis apparatus hypothesize the mechanisms of resuscitation from dormancy through the ribosomes de-association off the membrane accompanied by the 23S rRNA break curing, and could be of value for the development of principally new antituberculosis agents.

Mycolicibacterium smegmatis possesses operational agmatinase but contains no detectable polyamines.

Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.