Conditions and compounds for analysis of efflux pump activity in bacteria

Bakterijų atsparumas antibiotikams gali būti nulemtas įvairių mechanizmų ir ląstelių savybių. Daugiavaistis atsparumas – tai būsena, kai mikroorganizmai geba priešintis įvairių cheminių medžiagų poveikiui, tarp jų ir skirtingos struktūros antibiotikams. Išmetimo siurbliai sugeba išstumti iš ląstelių skirtingus lipofilinius ir ampifilinius junginius. Šiame darbe palyginome išmetimo siurblių aktyvumo tyrimų rezultatus, gautus naudojant 4 skirtingas indikatorines medžiagas ir 2 skirtingus tyrimo metodus, įvertinome, kokią įtaką tokiomis pačiomis sąlygomis tiriamai siurblių veiklai turi 9 skirtingi išmetimo siurblių substratai. Pademonstravome, kad gauti rezultatai gali reikšmingai skirtis priklausomai nuo terpės pH ir bakterijų išorinės membranos laidumo. Pirmą kartą tyrimams panaudoti laboratorijoje sukurti etidžiui ir PABN atrankūs elektrodai. Pirmą kartą buvo kiekybiškai įvertintas siurblių efektyvumas, t.y. apskaičiuotas iš ląstelės išmetamas indikatorinių medžiagų kiekis per laiko vienetą. Naudojant kelias fluorescuojančias indikatorines medžiagas, buvo ištirta jų sąveiką su nefluorescuojančiais substratais ir siurblių slopikliais Darbo metu gautos žinios leidžia kompleksiškai įvertinti skirtingais tyrimų metodais gautus rezultatus bei pasiūlyti optimalias sąlygas eksperimentams.Antimicrobial resistance of bacteria is determined by different mechanisms as well as cell properties. Multiple drug resistance is insensitivity of microorganisms to antibiotics and treatment with different compounds. Efflux pumps (EPs) are able to extrude different lipophilic and amphiphilic compounds. In this work we compared the results obtained by two different methods using four indicator compounds. The impact of 9 different EP substrates on registered efflux activity was evaluated at the identical conditions. We showed that the obtained results could significantly differ depending on medium pH and bacterial outer membrane permeability. The novel ethidium- and phenylalanylarginyl-β-naphtylamide- selective electrodes were used for research. For the first time, the quantitative assessment of EPs efficiency was performed, i.e. we determined the number of indicator molecules extruded by a cell per unit of time. Several fluorescent substrates were used to register their interaction with nonfluorescent EP substrates and inhibitors. Results of this study bring additional knowledge that enables a complex evaluation of methodological aspects to design the optimal experimental conditions

Quantative analysis of adenine and pyridine nucleotides in stem cells

Daugialąstelinių organizmų audiniai geba regeneruoti. Ši savybė priklauso nuo to, kiek audinyje šalia defirencijuotų, funkciją atliekančių ląstelių yra nediferencijuotų ląstelių, kurios geba greitai dalytis. Yra intensyviai tiriama skeleto raumens miogeninių kamieninių ląstelių transplantacija į pažeistą širdies raumenį. Mioblastų transfekcija koneksinu (Cx43 – širdžiai specifine izoforma) yra įrankis, didinant sąveiką tarp ląstelių ir transplantuotų mioblastų pagerėjusio išgyvenamumo skiepytame audinyje. Šio darbo tikslas buvo palyginti normalių ir transfekuotų koneksinu Cx43 mioblastų proliferacijos greitį, hiperterminės inkubacijos sukeltus gyvybingumo pokyčius ir su diferenciacija susijusius morfologijos ir adenino bei piridino nukleotidų kiekio pokyčius. Gauti rezultatai rodo, kad mioblastų transfekcija koneksinu Cx43 patikimai neveikia ląstelių gebėjimo dalytis. Dėl labai mažo redukuotų piridino dinukleotidų kiekio mioblastuose, koreliacija tarp NAD(P)H fluorescencijos intensyvumo ir nustatyto tripano mėlio testu gyvybingumo yra vidutinio arba silpno stiprumo. Po 3 dienų nuo diferenciacijos sukėlimo NAD(P)H fluorescencijos intensyvumas nesikeičia, tačiau po 7 ir 12 dienų nuo diferenciacijos sukėlimo – patikimai didėja, lyginant su kontrolinėmis nediferencijuotomis ląstelėmis. Atlikus kiekybinę adenino ir piridino nukleotidų HPLC analizę nustatyta, kad adenino nukleotidų ir piridino dinukleotidų bendras kiekis transfekuotose Cx43 ląstelėse yra 2,5-3 kartus mažiau, lyginant su kontroliniais mioblastais. Adenino nukleotidų kiekis, ypač ATP, didėja diferenciacijos metu. Diferenciacija yra susijusi su didėjančiu ADPATP santykiu, tikėtina, kad dėl didėjančio energijos vartojimo. Tiek NAD(P)H fluorescencijos matavimų, tiek chromatografinės analizės duomenimis, bendras redukuotų piridinų nukleotidų NADH ir NADPH kiekis diferenciacijos metu didėja. Šiam didėjimui yra svarbesni NADPH kiekio pokyčiai. Bendras netransfekuotų miogeninių ląstelių piridino dinukleotidų kiekis yra didžiausias (1,7 karto didesnis lyginant su kontrole) praėjus 7 dienoms po diferenciacijos pradžios, Cx43 transfekuotų ląstelių bendras piridino dinukleotidų kiekis yra apie 2,5 karto didesnis diferencijuojančiose ląstelėse, lyginant su kontrole.The regenerative potential of different tissues depends on the availability and specific properties of stem cells. The use of skeletal muscle myogenic stem cells for the transplantation into injured heart muscle is presently under intensive investigation. The transfection of myoblasts with connexin (specific heart isoform Cx43) is a tool for increasing interaction between cells and improving survival of transplanted myoblasts in the grafted tissue. The aim of this study was to compare the rate of proliferation of normal and Cx43 transfected cell lines, to estimate their viability changes induced by hyperthermic incubation and to evaluate differentation induced changes in cell morphology and amount of pyridine and adenine nucleotides. The obtained results show that the transfection of myoblasts by Cx43 does not significantly affect their ability to proliferate. Due to very low amount of reduced forms of pyridine dinucleotides in myoblasts the correlation between intensity of NAD(P)H fluorescence and viability detected by Trypan blue test is modest or weak. The intensity of NAD(P)H fluorescence does not increase after 3 days following initiation of differentiation but substantially increases after 7 and 12 days of differentiation in comparison to nondifferentiated control cells. The quantitative HPLC analysis of pyridine and adenine nucleotides has revealed that the total amount of adenine nucleotides and pyridine dinucleotides is 2,5-3 times lower in Cx43 transfected cells in comparison to the control myoblasts. The amount of adenine nucleotides, in particular ATP, increases during differentiation. Differentiation is related to the increase in ADP/ATP ratio, possibly due to the increased energy consumption. Both NAD(P)H fluorescence measurements and HPLC analysis reveals substantial increase in the total amount of the reduced forms of pyridine dinucleotides during myoblast differentiation, mostly due to the continuous changes in NAD(P)H amount. The total amount of pyridine dinucleotides is maximal (1.7 times higher in comparison to the control) after 7 days following the onset of differentiation of normal myogenic cells. In Cx43 transfected cells undergoing differentiation it was 2,5 fold higher in comparison to the nondifferentiated control.Vytauto Didžiojo universiteta

Studies of multidrug efflux pump activity in Lactococcus lactis using ethidium cations

Abstract of special issue: 22nd IUBMB & 37th FEBS Congress, Seville, Spain, September 4-9, 2012Multidrug resistance (MDR) efflux pump is the main reason of bacteria resistance to antibiotics. Lactococcus lactis is very important bacterium in food industry. We used spectrofluorimetric measurments to assay the activity of MDR pumps and to investigate the response of the pumps to cell growth conditions. We used ethidium (Et) as as indicatory compound, which increases its fluorescence because of the binding to DNA. We also used another MDR pump substrate tetraphenylphosphonium (TPP+ ) as a competitive compound and antibiotic gramicidin D as the cell permeabilizer. In this study we demonstrated that the differences in MDR efflux pump activity depend on the growth conditions (with or without heme source) and the energetic state of cellsBiochemijos katedraVytauto Didžiojo universiteta

Effect of phenylalanine-arginine beta-naphthylamide on minimal inhibitory concentration of antibiotics in the case of salmonella enterica cells

Resistance to antibiotics is the very common problem in medicine. It is important to understand the mechanisms of multidrug resistance in bacteria. We focused our study on efflux pump system using method of microdilutions. We used Salmonella enterica SL1344 (WT), L664 and ΔacrB strains to determine influence of phenylalanine-arginine β-naphthylamide (PAβN) on MICs of ampicillin, chloramphenicol and tetracycline. PAβN is very popular inhibitor of RND-type efflux pumps. It is toxic for bacterial cells only at high concentrations. We showed that low inactive concentrations of antibiotics can inhibit bacterial growth in combination with PAβN. Our results indicate that inhibitory activity of PAβN depends on the initial bacterial cell concentration. Such results suggest intensive binding of PAβN by the cellsBiochemijos katedraVytauto Didžiojo universiteta

Studies of multidrug efflux pump activity in salmonella enterica using nile red stain and tetraphenylphosphonium and tetraphenylarsonium cations

Multidrug resistance (MDR) efflux pumps is the main reason of Salmonella enterica cell resistance to antibiotics. We used spectrofluorimetric method to assay the activity of MDR pumps. The entry of lipophilic stain, such as Nile red (NR), into the cells increases its fluorescence because of the staining on intracellular lipids. We present data on the fluorimetrical assay of accumulation of tetraphenylphosphonium (TPP+) and tetraphenylarsonium (TPAs+) ions in bacteria. Effects of RND-type MDR pump inhibitor phenylalanyl-arginyl-β-naphtylamide were also studied. In this study we demonstrated competition between NR stain and TPP+ or TPAs+ ionsBiochemijos katedraVytauto Didžiojo universiteta

Synthesis and evaluation of the efficiency of RND type efflux pump inhibitors in bacteria

Abstracts of the 22nd IUBMB and 37th FEBS congress, Seville, Spain, September 4-9, 2012Biochemijos katedraVytauto Didžiojo universiteta

Salmonella enterica cells accumulate RND-family efflux pump inhibitor Phenylalanylarginyl-beta-naphthylamide

Abstract of special issue: FEBS EMBO 2014 conference, Paris, France, 30 August - 4 September 2014Biochemijos katedraVytauto Didžiojo universiteta

Interaction of ethidium and tetraphenylphosphonium cations with Salmonella enterica cells

Background and objective: One of the main causes of bacterial resistance to antimicrobials is multidrug resistance induced by the increased efficiency of the efflux pumps. In this study we analyzed how the conditions of assay affect the efflux of indicator substrates ethidium (Et+) and tetraphenylphosphonium (TPP+) in Salmonella enterica ser. Typhimurium cells. Impact of the outer membrane permeability barrier, composition and temperature of the medium on accumulation of the indicator compounds also was analyzed. Materials and methods: The fluorescence of Et+ and Nile Red was measured using 96-well plates and a plate reader. In parallel to traditional studies of fluorescence we applied a constructed selective electrode to follow the accumulation of Et+ in S. enterica cells. Simul- taneously with monitoring of Et+ concentration in the cell incubation medium, electrochem- ical measurements of TPP+ accumulation were performed. Furthermore, Et+ and TPP+ were used within the same sample as agents competing for the interaction with the efflux pumps. An inhibitor phenylalanyl-arginyl-b-naphtylamide (PAbN) was applied to evaluate the input of RND-family pumps in the total efflux of these indicator compounds. Results: S. enterica cells with the intact outer membrane (OM) bound very low amounts of Et+ or TPP+. Cells with the permeabilized OM accumulate considerably higher amounts of the indicator compounds at pH 8.0, but only Et+ was considerably accumulated at pH 6.5. At conditions of electrochemical monitoring accumulation of Et+ by the permeabilized cells at 37 8C was considerably faster than at 23 8C, but at the higher temperature most of the cell-accumulated Et+ was extruded back to the medium. The fluorescence of Et+ in suspension of cells incubated in 400 mmol/L Tris buffer was about twice higher compared to 100 mmol/L one. [...]

Interaction of ethidium and tetraphenylphosphonium cations with Salmonella enterica cells

This work was supported by Research Q2 Council of Lithuania, funding grant No. MIP-040/2015Background and objective: One of the main causes of bacterial resistance to antimicrobials is multidrug resistance induced by the increased efficiency of the efflux pumps. In this study we analyzed how the conditions of assay affect the efflux of indicator substrates ethidium (Et+) and tetraphenylphosphonium (TPP+) in Salmonella enterica ser. Typhimurium cells. Impact of the outer membrane permeability barrier, composition and temperature of the medium on accumulation of the indicator compounds also was analyzed. Materials and methods: The fluorescence of Et+ and Nile Red was measured using 96-well plates and a plate reader. In parallel to traditional studies of fluorescence we applied a constructed selective electrode to follow the accumulation of Et+ in S. enterica cells. Simul- taneously with monitoring of Et+ concentration in the cell incubation medium, electrochem- ical measurements of TPP+ accumulation were performed. Furthermore, Et+ and TPP+ were used within the same sample as agents competing for the interaction with the efflux pumps. An inhibitor phenylalanyl-arginyl-b-naphtylamide (PAbN) was applied to evaluate the input of RND-family pumps in the total efflux of these indicator compounds. Results: S. enterica cells with the intact outer membrane (OM) bound very low amounts of Et+ or TPP+. Cells with the permeabilized OM accumulate considerably higher amounts of the indicator compounds at pH 8.0, but only Et+ was considerably accumulated at pH 6.5. At conditions of electrochemical monitoring accumulation of Et+ by the permeabilized cells at 37 8C was considerably faster than at 23 8C, but at the higher temperature most of the cell-accumulated Et+ was extruded back to the medium. The fluorescence of Et+ in suspension of cells incubated in 400 mmol/L Tris buffer was about twice higher compared to 100 mmol/L one. [...]Biochemijos katedraGamtos mokslų fakultetasLietuvos sveikatos mokslų universitetas. Medicinos akademija. Kardiologijos institutasVytauto Didžiojo universiteta

Studies of RND-type efflux pump inhibitor Phenylalanylarginyl-beta-naphtylamide interaction with Salmonella enterica cells

Abstract of special issue: 38th FEBS Congress, Saint Petersburg, Russia, July 6-11, 2013Biochemijos katedraVytauto Didžiojo universiteta