RESUMEN
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.
RESUMEN
Persisters are rare phenotypic variants of regular bacterial cells that survive lethal antibiotics or stresses owing to slowing down of their metabolism. Recently, we have shown that polyamine putrescine can upregulate persister cell formation in Escherichia coli via the stimulation of rpoS expression, encoding a master regulator of general stress response. We hypothesized that rmf and yqjD, the stationary-phase genes responsible for ribosome inactivation, might be good candidates for the similar role owing to their involvement in translational arrest and the ability to be affected by polyamines. Using reporter gene fusions or single and multiple knockout mutations in rpoS, rmf and yqjD genes, we show in this work that (i) E. coli polyamines spermidine and cadaverine can upregulate persistence, like putrescine; (ii) polyamine effects on persister cell formation are mediated through stimulation of expression of rpoS, rmf and yqjD genes; (iii) these genes are involved in persister cell formation sequentially in a dynamic fashion as cells enter the stationary phase. The data obtained in this work can be used to develop novel tools relying on a suppression of polyamine metabolism in bacteria to combat persister cells as an important cause of infections refractory to antibiotics.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Netilmicina/farmacología , Poliaminas/metabolismo , Farmacorresistencia Bacteriana , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Viabilidad Microbiana/efectos de los fármacos , Mutación , Proteínas Ribosómicas/genética , Espermidina/metabolismo , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Persisters are suggested to be the products of a phenotypic variability that are quasi-dormant forms of regular bacterial cells highly tolerant to antibiotics. Our previous investigations revealed that a decrease in antibiotic tolerance of Escherichia coli cells could be reached through the inhibition of key enzymes of polyamine synthesis (putrescine, spermidine). We therefore assumed that polyamines could be involved in persister cell formation. Data obtained in our experiments with the polyamine-deficient E. coli strain demonstrate that the formation of persisters tolerant to netilmicin is highly upregulated by putrescine in a concentration-dependent manner when cells enter the stationary phase. This period is also accompanied by dissociation of initially homogenous subpopulation of persister cells to some fractions differing in their levels of tolerance to netilmicin. With three independent experimental approaches, we demonstrate that putrescine-dependent upregulation of persister cell formation is mediated by stimulation of rpoS expression. Complementary activity of putrescine and RpoS results in ~ 1000-fold positive effect on persister cell formation.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Netilmicina/farmacología , Putrescina/metabolismo , Tolerancia a Medicamentos , Escherichia coli/crecimiento & desarrollo , Viabilidad Microbiana/efectos de los fármacos , Putrescina/análisis , Espermidina/análisis , Espermidina/metabolismoRESUMEN
Bactericidal antibiotics (fluoroquinolones, aminoglycosides and cephalosporins) at their sublethal concentrations were able to produce hydroxyl radicals, hydrogen peroxide and superoxide anions (ROS) in Escherichia coli cells, which resulted in damage to proteins and DNA. The cells responded to oxidative stress by a 2-3-fold increase in cell polyamines (putrescine, spermidine) produced as a consequence of upregulation of ornithine decarboxylase (ODC). Relief of oxidative stress by cessation of culture aeration or addition of antioxidants substantially diminished or even completely abolished polyamine accumulation observed in response to antibiotics. Alternatively, inhibition of polyamine synthesis resulted in enhancement of oxidative stress in antibiotic-processed cells. When added to antibiotic-inhibited culture, polyamines reduced intracellular ROS production and thereby prevented damage to proteins and DNA. These effects eventually resulted in a substantial increase in cell viability, growth recovery and antibiotic resistance that were more strongly expressed in polyamine-deficient mutants.