ELEKTROPORACIJA KOT NARAVNI MEHANIZEM HORIZONTALNEGA PRENOSA GENOV PRI PROKARIOTIH

• Main Author: MARJANOVIČ, IGOR
• Other Authors: Kotnik, Tadej
• Format: Doctoral or Postdoctoral Thesis
• Language: Slovenian
• Published: 2015
• Subjects: Elektroporacija; horizontalni prenos genov; strele; evolucija; prokarioti; electroporation; horizontal gene transfer; lightning; evolution; prokaryotes; sami
• Online Access: https://repozitorij.uni-lj.si/IzpisGradiva.php?id=30673; https://repozitorij.uni-lj.si/Dokument.php?id=30664&dn=

Več vej znanosti si še vedno prizadeva ugotoviti, kako je življenje nastalo. Čeprav so si teorije nastanka življenja na Zemlji v podrobnostih lahko močno različne, so si v grobem enotne, da se živa bitja skozi naravno selekcijo prilagajajo okolju, v katerem se nahajajo. Temu procesu prilagajanja skozi čas pa pravimo evolucija. Vemo, da so bila enocelična bitja prisotna pred večceličnimi, celice brez jedra (prokarioti) pa pred celicami z jedrom (evkarioti). Do devetdesetih let prejšnjega stoletja je veljalo tudi prepričanje, da so bile najpomembnejši vir inovacij v evoluciji mutacije, ki so se z delitvijo celice (vertikalnim prenosom genov) prenašale na njene potomke. Ta teorija se je porušila, ko so znanstveniki pričeli sorodnost organizmov vrednotiti glede na sorodnost njihovih genomov (temu postopku pravimo filogenetska analiza) in pri tem ugotovili, da pri sledenju sorodnosti različnih genov pridejo do različnih sorodnostnih struktur (filogenetskih dreves). Študije genomov so pokazale tudi, da nekateri organizmi vsebujejo gen, ki ga njihovi bližji sorodniki nimajo, najdemo pa ga (enakega ali zelo podobnega) pri nekaterih evolucijsko zelo oddaljenih organizmih. Iz teh ugotovitev izhaja, da organizmi v splošnem prevzemajo gene ne le od celice, iz katere izvirajo, temveč tudi iz okolice oziroma od drugih organizmov. Ta proces imenujemo horizontalni prenos genov (HGT). Rezultati filogenetskih študij kažejo, da je bil HGT skozi evolucijo in je še danes pomemben vir inovacij, ki so omogočile hitrejši in bolj raznolik razvoj zgodnjega življenja. V znanstveni literaturi zasledimo tri mehanizme HGT: naravno kompetenco, konjugacijo in transdukcijo. Vsi našteti mehanizmi so biološki in temeljijo na proteinih z ozko specifičnim delovanjem, iz česar sledi, da so tudi sami nastali šele v določeni fazi evolucije, zato se zastavlja vprašanje, ali za HGT obstaja tudi kak preprostejši, denimo povsem fizikalen mehanizem, ki je lahko deloval že vse od nastanka življenja. Eden najobetavnejših tovrstnih mehanizmov je elektroporacija. Elektroporacija je pojav, ki omogoča vnos tujega materiala tako v prokariotske kot v evkariotske celice. Kot laboratorijska metoda je bila razvita pred štirimi desetletji, temelji pa na kratkotrajni izpostavitvi celice električnemu polju dovolj visoke jakosti, ki ga običajno ustvarimo z dovajanjem napetostnih pulzov na par elektrod, med katerima se nahajajo celice. Posledica izpostavitve celice takšnim pulzom je povečanje prepustnosti celične membrane, ki omogoči vnos najrazličnejših snovi, tudi DNA, iz okolice v celico, lahko pa tudi iztekanje snovi iz celice. Če iztekanje ni premočno in celica po končani izpostavitvi pulzom preživi, govorimo o reverzibilni, sicer pa o ireverzibilni elektroporaciji. V naravi se ob udaru nevihtne strele v habitat prokariotskih organizmov v bližini točke udara ustvari električno polje, zadostno za povzročitev elektroporacije – zelo blizu točke udara so prisotni pogoji za ireverzibilno elektroporacijo in s tem iztekanje DNA, nekoliko dlje od te točke pa pogoji za reverzibilno elektroporacijo in s tem vnos DNA. Za preučevanje elektroporacije kot naravnega mehanizma HGT je potrebno opraviti biološke poskuse, kjer se v kontroliranih laboratorijskih pogojih čim bolj približamo naravnim razmeram ob udaru strele. V ta namen je bilo potrebno razviti napravo, ki nam take poskuse omogoča. Po razčlenitvi zgoraj opisanih spoznanj in motivacije te disertacije sledi opis načrtovanja, izdelave in testiranja modularnega sistema za izpostavitev emulacijam električne strele (sistem »Znanstveni Emulator eVolucijskih Strel« – ZEVS) in pripadajočega visokonapetostnega generatorja (generator ZEVS). Sistem ZEVS nam omogoča, da biološke vzorce (celice ali tkiva) v kontroliranem okolju (točno določena dolžina obloka razelektritve, merjenje časovnega poteka električnega toka, ki teče skozi vzorec, snemanje poteka poskusov s hitro kamero) izpostavimo elektrostatični razelektritvi z nastavljivo amplitudo električnega toka (do nekaj sto amperov). To predstavlja ponovljivo emulacijo elektrostatične razelektritve, kakršna poteka pri naravni streli. Ta sistem raziskovalcem omogoča, da uporabijo poljubni generator elektrostatičnih razelektritev z ustrezno ozemljitveno elektrodo in prilagodljivo dolžino obloka. Modularna zasnova sistema omogoča hitro montažo in demontažo, kot tudi preprosto in temeljito čiščenje. Pri razvoju sistema smo si pomagali z računalniškim modeliranjem, kjer smo pred izdelavo prvega prototipa načrtovali vse komponente, virtualno sestavili sistem in njegove dimenzije iterativno določili z numeričnimi izračuni porazdelitve električnega toka in jakosti električnega polja, temelječimi na metodi končnih elementov. Sistem smo zasnovali tako, da se ga da hitro sestaviti in razstaviti, kar omogoča preprosto transportiranje in izvajanje poskusov v različnih laboratorijih. Pozorni smo bili tudi na to, da je mogoče sistem enostavno in temeljito očistiti, kar bistveno zmanjša tveganje kontaminacije, hkrati pa omogoča ponovljivost poskusov. Pri izbiri materiala smo za dele, ki zahtevajo električno neprevodnost, uporabili polietilen, kjer je bila poleg neprevodnosti sestavnega dela potrebna tudi njegova prozornost, pa pleksi steklo. Elektrode smo prvotno izdelali iz bakra, a se je izkazalo, da razelektritve povzročajo njihovo korozijo, zato smo baker v nadaljevanju razvoja nadomestili z nerjavečim jeklom, ki se je izkazalo za ustrezno odporno proti razelektritveni koroziji. Pri ozemljitveni elektrodi, ki je v neposrednim stikom z biološkim vzorcem, pa je nerjaveče jeklo ustreznejše tudi zato, ker se v primerjavi z bakrom precej manj elektrolitsko raztaplja in tako manj kontaminira vzorec. V prvih serijah preizkusov sistema ZEVS smo kot generator razelektritev uporabili komercialni električni paralizator (taser), ki je generiral razelektritveni tok s trajanjem nekaj sto nanosekund, nato pa smo načrtovali in zgradili visokonapetostni električni generator, ki temelji na krmiljeni 5 kV razelektritvi kondenzatorja s kapacitivnostjo 1 μF (generator ZEVS) in je po časovnem poteku generiranega razelektritvenega toka precej bolj podoben dejanskim nevihtnim strelam (dvig toka z ničle do maksimuma v približno 5 μs, nato pa eksponentno upadanje s časovno konstanto približno 75 μs). Prve biološke poskuse smo opravili na bakterijah Escherichia coli, nasajenih na agarju v petrijevkah z notranjim premerom 86 mm, razelektritve pa smo generirali z električnim paralizatorjem. Petrijevke z agarjem in nasajenimi bakterijami smo vstavili v sistem ZEVS in preko konične elektrode v središče petrijevke dovedli 10 zaporednih razelektritev. Tok vsake razelektritve je imel največjo vrednost pri ~100 A, dvižni čas od nič do največje vrednosti ~0.1 μs in čas upada do polovične vrednosti ~0.3 μs. Dolžina obloka pri vsaki razelektritvi je bila ~15 mm. Pri poskusih smo v krožnem področju do radija 5 mm od središča petrijevke dobili območje skoraj popolnoma brez kolonij E. coli. Izračunana jakost električnega polja na tej radialni razdalji je bila ~8 kV/cm. Opisani eksperimentalni rezultati in izračuni skupaj povedo, da je bilo osrednje območje brez živih bakterij zaradi njihove ireverzibilne elektroporacije. Drug sklop bioloških poskusov smo opravili na ovarijskih celicah kitajskega hrčka (celicah CHO), ki so evkariotske. Tudi pri teh poskusih smo za generator razelektritev še uporabljali električni paralizator. Celice CHO smo nasadili v petrijevke z notranjim premerom 52 mm. Preden smo petrijevke izpostavili razelektritvam, smo iz njih odstranili gojišče in nato dodali 1.5 ml svežega gojišča, ki je vsebovalo 4 μg/ml plazmidne DNA pEGFP‐N1, z izražanjem katere nastaja zeleno fluorescirajoči protein (GFP). Nato smo petrijevke zaporedoma vstavljali v sistem ZEVS in vsaki dovedli 10 elektrostatičnih razelektritev. Električni tok vsake razelektritve je imel največjo vrednost pri ~14 A, dvižni čas od nič do največje vrednosti ~0.5 μs in čas upada do polovične vrednosti ~1.5 μs. Dolžina obloka pri vsaki razelektritvi je bila ~7 mm. V krožnem pasu na razdalji od 3 do 15 mm od središča petrijevke smo zaznali fluorescenco GFP, ki je odražala vnos in izražanje pEGFP‐N1, torej je bilo to območje reverzibilne elektroporacije. Z izračunom smo ocenili jakost električnega polja 15 mm od središča petrijevke na 1.11 kV/cm, 3 mm od središča pa na 5.54 kV/cm, kar nakazuje, da so bile celice v osrednjem območju, kjer nismo zaznali fluorescence GFP, mrtve zaradi ireverzibilne elektroporacije, v zunanjem območju, kjer prav tako ni bilo zaznavne fluorescence GFP, pa niso bile elektroporirane, zato ni prišlo do vnosa DNA. Tretji sklop poskusov pa je bila ireverzibilna elektroporacija spor bakterije Bacillus pumilus, nasajenih na agarju v petrijevkah, pri teh poskusih pa smo kot napetostni generator uporabili tako električni paralizator kot generator ZEVS, ki smo ga takrat že razvili. Z obema generatorjema smo dosegli ponovljivo inaktivacijo spor. Pri poskusih s paralizatorjem smo z 20 razelektritvami dobili inaktivacijo na 0.65% celotne petrijevke, medtem ko je območje inakativacije pri uporabi generatorja ZEVS pokrivalo 7% celotne petrijevke pri eni razelektritvi, 27% pri desetih in 55% pri petdesetih razelektritvah. Opravljeni poskusi so pokazali, da je sistem ZEVS primeren za preučevanje vplivov elektrostatičnih razelektritev na prokariotske in evkariotske celice ter da z njim lahko povzročimo tako ireverzibilno elektroporacijo, katere posledica je lahko tudi iztekanje DNA, kot reverzibilno elektroporacijo, ki privede do vnosa DNA in njeno izražanje. Poskusa ireverzibilne elektroporacije na celicah E. coli in genske transfekcije na celicah CHO nakazujeta, da bi bila elektroporacija dejansko lahko četrti mehanizem prenosa HGT v naravi, vendar pa bo za zanesljivejši in kvantitativno relevanten odgovor potrebno s sistemom ZEVS opraviti dodatne poskuse na organizmih, katerih naravno okolje je dosegljivo nevihtnim strelam (denimo na bakterijah, ki naseljujejo površinske morske in sladke vode). Poleg tega pa bo potrebno namesto modelnih laboratorijskih molekul DNA, kot so tiste z genom za GFP ali odpornostjo na antibiotike, uporabiti naravno DNA brez modifikacij, s katerimi umetno povečamo njihovo stabilnost ter verjetnost vnosa in izražanja. Multiple scientific disciplines are still trying to determine how life began. Although competing theories on the origins of life on Earth differ in many aspects, they all agree that the genetic makeup of organisms is adapted to the environment in which they live by the forces of natural selection this process is known as evolution. We know that single‐cell organisms existed before multi‐cell organisms and that cells without a nucleus (prokaryotes) existed before cells with a nucleus (eukaryotes). Up until the 1990s, it was widely assumed that the prevailing source of innovations in evolution are mutations occurring during cell division and thus transferred to daughter cells (vertical gene transfer). This theory collapsed when scientists began to analyze the relatedness of organisms by looking at the similarities of their genomes (a process called phylogenetic analysis). They discovered that tracking the similarities of different genes can lead to different branching diagrams of relatedness (phylogenetic trees). Genome studies have also shown that some organisms contain a gene that is absent in their close relatives, but present in identical or only slightly altered form in some evolutionarily very distant organisms. These findings implied that the genetic material is not only inherited from the parent cells, but can also originate from the surroundings and from other organisms. This process is known as horizontal gene transfer (HGT). The results of phylogenetic studies show that HGT has been an important source of innovation for evolution that enabled a faster and more diverse development of early life. The scientific literature recognizes three mechanisms of HGT: natural competence, conjugation and transduction. All of the stated mechanisms are biological and are based on proteins, each with a highly specific function, which implies that these mechanisms are themselves products of evolution and had thus only occurred during a certain stage of the evolutionary history. Consequently, we are left with the question whether there exists a mechanism, perhaps based on simpler physical principles, that could have acted ever since the dawn of life. One of the most promising such mechanisms is electroporation. Electroporation is a phenomenon that enables the entry of exogenous matter into prokaryotic as well as eukaryotic cells. As a laboratory method it was developed four decades ago and is based on short‐term exposure of cells to a sufficiently strong electric field. The field is usually created by delivering voltage pulses to a pair of electrodes between which the cells are positioned. The result of exposure to such pulses is increased permeability of the cell plasma membrane, which enables the entry of a wide range of molecules, including DNA, from the environment to the cell, as well as release of such molecules from the cell into the environment. If the outflow from the cell is not too strong and the cell survives the exposure to the pulses, this phenomenon is termed reversible electroporation, otherwise it is known as irreversible electroporation. In natural habitats hit by a lightning stroke, the electric field in the ground near the lightning’s point of entry is sufficient for electroporation very close to that point the conditions are those for irreversible electroporation and hence release of DNA, while in the adjacent region in the downward and outward direction the conditions for reversible electroporation are met, and hence for uptake of DNA. To assess electroporation as a natural mechanism of HGT, it is necessary to conduct biological experiments, where in controlled laboratory conditions we strive to come as close as possible to emulating natural conditions of lightning striking the ground. For this purpose, we needed to develop a setup allowing such experiments. The analysis of the abovementioned findings and motivations for this dissertation are followed by the description of design, construction and testing of a modular system for lightning exposures (Scientific Emulator of Evolutionary Lightning, with the acronym ZEVS in Slovene) and the corresponding high‐voltage generator. The ZEVS system allows to expose biological samples (cells or tissues) in a controlled environment (precisely determined length of the discharge arc, monitoring the time course and amplitude of the electric current flowing through the sample, filming the experiments with a high‐speed camera) to electrostatic discharges with adjustable amplitude of electric current (up to several hundred amperes). This provides a reproducible emulation of electrostatic discharges that occur in natural lightning strokes. The system allows the researchers to use an arbitrary generator of electrostatic discharges with an adequate receiving (ground) electrode and an adjustable arc length. The modular design of the system enables quick assembly and disassembly, as well as simple and thorough cleaning. For the development of the system, we used computer modeling, where we designed and analyzed the entire system virtually before buildng the first actual prototype. The dimensions of the system were determined iteratively using numerical calculations of the distribution of electric current and field based on the finite elements method. The system was designed such that it was easy to assemble and disassemble, facilitating transport and thus allowing to conduct experiments in different laboratories. We also paid attention to allow for the system to be cleaned simply and thoroughly, which substantially decreases the risk of contamination, while allowing for the reproducibility of experiments. As a material for components that are required to be nonconductive, we chose polyethylene. For components where non‐conductivity as well as transparency was required, we used Plexiglas. Electrodes were initially made of copper, but we discovered that the electric discharges caused substantial corrosion of such electrodes, so we later replaced copper with stainless steel, which turned out to be sufficiently resistant to corrosion caused by electric discharges. For the ground electrode, which is in direct contact with the biological sample, the choice of stainless steel proved additionally advantageous as it is less susceptible to electrolytic dissolution and thus results in a much weaker contamination of the biological sample by the metal ions. In the first experimental trials of the ZEVS system, we modified a commercial electric Taser and used it as the electric discharge generator, yielding a discharge current that lasted several hundred nanoseconds. Later, we designed and constructed a high‐voltage electric generator that delivers arcs by a controlled 5 kV discharge of a 1 μF capacitor (the ZEVS generator). Compared to the Taser, the ZEVS generator discharge current was much closer in its time course to an actual lightning stroke (zero‐to‐peak time of ~5 μs followed by exponential decay from the peak with a time constant of ~75 μs, corresponding to a peakto‐ half time of ~100 μs). The first biological experiments were conducted on Escherichia coli bacteria planted on agar in petri dishes having inner diameter of 86 mm, with discharges generated by the Taser. Petri dishes with agar and the plated bacteria were inserted into the ZEVS system, the discharge was delivered from the conical electrode, entering vertically downwards into the center of the petri dish, and we supplied 10 consecutive such discharges. The current of each discharge had the peak value of ~100A, zero‐to‐peak time of ~0.1 μs, and peak‐to‐half time of ~0.3 μs. The length of the arc of each discharge was ~15 mm. The experiments produced a circular region of radius of 4 mm from the center of the petri dish in which there were almost no detectable colonies E. coli. The calculated electric field strength at that radial distance was ~8 kV/cm. The acquired results together with these calculations imply that the region devoid of viable bacteria was due to their irreversible electroporation. The second set of biological expeiments was conducted on Chinese Hamster Ovary (CHO) cells, which are eukaryotic, again using the Taser to generate the discharges. CHO cells were plated in petri dishes having inner diameter of 52 mm. Before exposing the petri dishes to the discharges, we removed the original culture medium and then added 1.5 ml of a fresh culture medium containing 4 μg/ml plasmid DNA pEGFP‐N1 that contains a gene encoding the green fluorescent protein (GFP). We then placed the petri dishes into the ZEVS system and exposed each dish to 10 electrostatic discharges. The electric current of each discharge had a peak value of ~14 A, zero‐to‐peak time of ~0.5 μs and peak‐to‐half time of ~1.5 μs. The length of the electric arc in each discharge was ~7mm. On the area spanning radially from 3 to 15 mm from the center of the petri dish, we detected GFP fluorescence, reflecting uptake of pEGFP‐N1 and its expression, and thus corresponding to the area of reversible electroporation. By calculattion, we estimated the electric field strength at 15 mm from the center of the petri dish as 1.11 kV/cm, and at 3 mm as 5.54 kV/cm. This suggests that the central region with no gene expression was subject to irreversible electroporation and thus cell death, while in the outer region in which there was also no detectable expression the cells were not electroporated, and thus there was no DNA uptake. The third set of experiments was irreversible electroporation on bacterial spores of Bacillus pumilus planted on agar in petri dishes. For these experiments, we used the Taser generator, as well as the ZEVS generator that we had already developed at that stage. With both discharge generators we achieved reproducible inactivation of the spores. With experiments utilizing the Taser, we achieved inactivation in 0.65% of the entire petri dish after delivering 20 electric discharges. Using the ZEVS generator, the area of inactivation was 7% using one discharge, 27% after 10 discharges, and 55% after 50 discharges. The conducted experiments have shown that the ZEVS system is suitable for studying the effects of discharges on both prokaryotic and eukaryotic cells, and that with it we can achieve irreversible electroporation that causes leakage of DNA, as well as reversible electroporation that results in uptake and expression of DNA. Experiments of irreversible electroporation in E. coli and of gene uptake in CHO cells suggest that electroporation could act as the fourth natural mechanism of HGT. To arrive at a reliable and quantitatively relevant answer, however, it is necessary to conduct further experiments on organisms whose natural environment is accessible to lightning strokes (e.g. bacteria populating the top layers of seawater and freshwater habitats). Furthermore, for reliable conclusions it is important to use natural DNA, devoid of artificial modifications often present in commercially available DNA with the aim to increase its stability and/or the efficiency of uptake and expression.