Delovanje ortokaspaze MaOC1 na par toksin-antitoksin Ipf_1067/1065 cianobakterije Microcystis aeruginosa PCC 7806

Halužan Vasle, Ana (author), Dolinar, Marko (mentor)

Sistemi toksin-antitoksin so genetski moduli na kromosomih in/ali plazmidih številnih bakterij in arhej. Zapisujejo stabilen toksin in kratkoživ antitoksin. Sprva so veljali zgolj za dejavnike stabilizacije plazmidov s postsegregacijskim ubijanjem, z nadaljnjimi raziskavami pa se je izkazalo, da sodelujejo tudi v drugih celičnih procesih, kot sta preživetje neugodnih življenjskih razmer in tvorba persistentnih celic. Izraženi toksini vplivajo na celične procese in porušijo običajno delovanje biokemijskih poti, kar onemogoči nadaljnjo rast celice. Te učinke lahko prepreči antitoksin, ki nasprotuje delovanju toksina. Analiza genoma cianobakterije Microcystis aeruginosa PCC 7806 je razkrila prisotnost šestih zapisov za ortokaspaze. To so prokariontski homologi kaspaz, ki verjetno delujejo kot regulatorji programirane celične smrti. V okolici zapisa za ortokaspazo MaOC1 sta tudi zapisa za dva para toksin-antitoksin – Ipf_1067 in Ipf_1065 ter Ipf_1064 in Ipf_1063. Oba para sta proteinska, pri čemer par Ipf_1067/1065 uvrščamo v tip RelBE, par Ipf_1064/ 1063 pa v tip VapBC. V magistrski nalogi smo želeli raziskati vpliv ortokaspaze MaOC1 na par toksin-antitoksin Ipf_1067/1065 cianobakterije Microcytis aeruginosa PCC 7806 v bakteriji Escherichia coli. Z bioinformacijskimi orodji smo pripravili teoretična strukturna modela toksina Ipf_1065 in antitoksina Ipf_1067. Primerjali smo ju s strukturama toksinov RelE in ParE ter antitoksinov RelB in ParD. Z namenom določitve kristalne strukture kompleksa smo na podlagi izsledkov bioinformacijske analize predvideli mesta mutacij, ki bi zmanjšale toksičnost toksina Ipf_1065, a hkrati ohranile možnost povezovanja z antitoksinom Ipf_1067 v kompleks toksin-antitoksin. Pripravili smo plazmide, ki so nosili zapise za ortokaspazo MaOC1, toksin Ipf_1065, antitoksin Ipf_1067 in par Ipf_1067/1065 v različnih kombinacijah. Plazmidi so omogočali izražanje omenjenih komponent v bakteriji Escherichia coli. Da bi določili način delovanja MaOC1 na par Ipf_1067/1065, smo z merjenjem OD600 v različnih časovnih intervalih spremljali rast bakterijskih kultur Escherichia coli BL21(DE3), transformiranih z ustreznimi plazmidi. Ugotovili smo, da izražanje toksina Ipf_1065 upočasni rast celic Escherichia coli, izražanje para Ipf_1067/1065 pa na hitrost rasti ne vpliva. Rast celic, ki so konstitutivno izražale MaOC, se je po začetku izražanja para Ipf_1067/1065 močno upočasnila. Predvidevamo, da je ortokaspaza cepila nastajajoč antitoksin, ki nato ni mogel nevtralizirati nastajajočega toksina in tako preprečiti njegovih učinkov na celice. Rezultati magistrske naloge so osvetlili delovanje ortokaspaze MaOC1 na par toksin-antitoksin Ipf_1067/1065.

• Language: Slovenian
• Keywords: sistemi toksin-antitoksin, ortokaspaza, Microcystis aeruginosa
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-137462
• COBISS.SI-ID: 115499267
• Publication date in RUL: 17.06.2022

Secondary language

• Language: English
• Title: Orthocaspase MaOC1 activity on Ipf_1067/1065 toxin-antitoxin pair from the cyanobacterium Microcystis aeruginosa PCC 7806

Abstract

Toxin-antitoxin systems are genetic modules, located on chromosomes and/or plasmids in numerous bacteria and archaea. They consist of a stable toxin and labile antitoxin. At first, they were thought of as the main components in the process of post-segregational killing. However, further research has shown they participate in other cellular processes, such as the response to harsh environmental conditions and the formation of persistent cells. Expressed toxins impact cellular processes and disturb biochemical pathways, resulting in diminished cell growth. These effects can be counteracted by a putative antitoxin that binds the toxin and thus neutralizes it. The analysis of the Microcystis aeruginosa PCC 7806 genome has revealed the presence of 6 orthocaspase encoding sequences. Orthocaspases are prokaryotic caspase homologues that probably regulate programmed cell death. Two toxin-antitoxin operons are located in the vicinity of the orthocaspase MaOC1 sequence, denoted Ipf_1067/1065 and Ipf_1064/1063. They were classified as RelBE and VapBC types, respectively. This thesis aims to determine the orthocaspase MaOC1 activity on the Ipf_1067/1065 toxin-antitoxin pair from the cyanobacterium Microcystis aeruginosa PCC 7806 in Escherichia coli. We prepared theoretical structural models of the Ipf_1065 toxin and the Ipf_1067 antitoxin. We compared these to structures of RelE and ParE toxins and RelB and ParD antitoxins. Based on this bioinformatical analysis, we predicted mutation sites that would diminish the toxicity of Ipf_1065 but preserve the toxin's ability to form a complex with the antitoxin. This was done in order to determine the crystal structure of the complex. We prepared plasmids carrying sequences for MaOC1, toxin, antitoxin and the toxin-antitoxin pair in different combinations. These plasmids enabled us to express the components in Escherichia coli. To determine the MaOC1 activity on the Ipf_1067/1065 complex, we prepared Escherichia coli BL21(DE3) bacterial cultures carrying appropriate plasmids and measured OD600 in different time intervals to determine the growth rate. Our results show that the expression of the toxin Ipf_1065 decelerates the growth rate of the Escherichia coli cells, however, expressing the pair Ipf_1067/1065 does not affect the growth rate. The growth rate of the cells that have been constitutively expressing MaOC1 has decelerated once we induced the expression of the pair Ipf_1067/1065. We presume that the orthocaspase cleaved the expressed antitoxin which was unable to neutralize the emerging toxin and therefore prevent its effects on the cells. The results of this thesis have highlighted the orthocaspase MaOC1 activity on the pair Ipf_1067/1065.

• Keywords: toxin-antitoxin systems, orthocaspase, Microcystis aeruginosa