magistrsko delo
Tanja Zupan (Author), Jerica Sabotič (Mentor), Miha Pavšič (Thesis defence commission member), Vera Župunski (Thesis defence commission member), Gregor Gunčar (Co-mentor)

Abstract

Povzetek: Listeria monocytogenes je Gram pozitivna patogena bakterija, ki povzroča listeriozo. Pri delu smo uporabili njej sorodno nepatogeno vrsto Listeria innocua, ki se pogosto uporablja kot modelni organizem za L. monocytogenes. Bakterije rastejo v obliki biofilma, ki jim omogoča rast na različnih površinah in ima pomembno vlogo pri preživetju bakterij v okolju. Biofilmi listerije so običajno v literaturi opisani kot monoslojni, pri našem delu pa smo opazili, da tvori tudi dvosloj in večslojne strukture, podobne piramidam. Proteini iz gob predstavljajo razmeroma neraziskano skupino proteinov z edinstvenimi lastnostmi. Med njimi izstopajo inhibitorji peptidaz in lektini, med katerimi sta tudi kokaprin in makrocipin. Kokaprin (KKP1) je majhen neraziskan protein izoliran iz gobe Coprinopsis cinerea in deluje kot inhibitor cisteinskih in aspartatnih peptidaz ter lektin. Makrocipin (Mcp1) pa je inhibitor cisteinskih peptidaz iz gobe Macrolepiota procera. V bakterijskem ekspresijskem sistemu smo pripravili rekombinantne proteine Mcp1, KKP1 in štiri mutante KKP1 (KKP1-N22R, KKP1-D47R, KKP1-FH32EE, KKP1-G13E). Z merjenjem inhibitorne aktivnosti za encim papain smo potrdili inhibitorno aktivnost makrocipina, z encimoma papain in pepsin pa smo preverili inhibitorno aktivnost za KKP1 in njegove mutante. Med njimi je izstopal mutant KKP1-N22R, pri katerem smo uspešno spremenili reaktivno mesto za inhibicijo papaina. S tem smo nakazali, da ima kokaprin ločena mesta za inhibicijo cisteinskih in aspartatnih peptidaz. Z analizo nativne poliakrilamidne gelske elektroforeze smo pokazali, da se KKP1 in njegovi mutanti povezujejo v oligomere. Preverili smo vpliv izoliranih proteinov iz gob na biofilm bakterije L. innocua, za kar smo predhodno uspešno pripravili bakterije, ki so izražale različne reporterske proteine, od katerih se je najbolje izkazal DsRED Express. Poleg tega smo za detekcijo divjega tipa bakterij L. innocua preizkusili različne označevalce in barvila. Za najbolj uporabno se je izkazalo komercialno barvilo Mycolight Red, ki se veže na DNA bakterij. S preverjanjem, ali izbrani proteini iz gob vplivajo na razvoj biofilma bakterije L. innocua, smo ugotovili, da tako kokaprin kot makrocipin povzročita zmanjšano adhezijo bakterij na substrat. Opazili smo tudi viden učinek kokaprina na razbijanje statičnega biofilma in učinek makrocipina na razbijanje dinamičnega biofilma. Na adhezijo bakterij na substrat in na razbijanje zrelega statičnega biofilma imajo močnejši vpliv od KKP1 tri njegovi mutanti KKP1-N22R, KKP1-FH32EE in KKP1-G13E. Ti spremenjeni aminokislinski ostanki se nahajajo na isti strani KKP1, zato sklepamo, da se tam nahaja mesto, ki je pomembno za vezavo KKP1 na bakterije L. innocua. Tako kokaprin kot makrocipin torej vplivata na tvorbo biofilma bakterije L. innocua, zato bi bilo smiselno njun vpliv preveriti tudi na patogeno vrsto L. monocytogenes, ki predstavlja velik zdravstven problem. Če se vpliv izbranih proteinov iz gob na tvorbo biofilma patogene bakterije potrdi, bi jih lahko uporabili za nadaljnje študije in razvoj novih protimikrobnih snovi ali celo zdravil za zdravljenje okužb z listerijo.

Keywords

Listeria monocytogenes;Listeria innocua;listerija;proteini iz gob;kokaprin;makrocipin;biofilmi;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [T. Zupan]
UDC: 577.112:579.86(043.2)
COBISS: 87666691 Link will open in a new window
Views: 235
Downloads: 60
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Other data

Secondary language: English
Secondary title: Effect of cocaprin and macrocypin on Listeria biofilm
Secondary abstract: Abstract Listeria monocytogenes is a Gram-positive pathogenic bacterium that causes listeriosis. We used the related non-pathogenic species Listeria innocua, which is often used as a model organism for L. monocytogenes. Bacteria grow in the form of a biofilm and resist different types of stress, and plays an important role in the survival of bacteria in the environment. Listeria biofilms are usually described as monolayers in the literature. In our work, we observed that they also form bilayer and multilayer pyramid-like structures. Fungal proteins represent a relatively unexplored group of proteins with unique properties. These include peptidase inhibitors and lectins, including cocaprin and macrocypin. Cocaprin (KKP1) is a small unexplored protein isolated from Coprinopsis cinerea that acts as an inhibitor of cysteine and aspartic peptidases. Macrocypin (Mcp1) is an inhibitor of cysteine peptidases from Macrolepiota procera. The recombinant proteins Mcp1, KKP1 and four KKP1 mutants (KKP1-N22R, KKP1-D47R, KKP1-FH32EE, KKP1-G13E) were produced in a bacterial expression system. By measuring the inhibitory activity for the enzyme papain, we confirmed the activity for macrocypin, and using the enzymes papain and pepsin, we analysed the inhibitory activity of KKP1 and its mutants. Among them, the mutant KKP1-N22R stood out, in which we successfully changed the reactive site for papain inhibition. This suggests that cocaprin has separate sites for inhibition of cysteine and aspartic peptidases. Native polyacrylamide gel electrophoresis analysis showed that KKP1 and its mutants form oligomers. We investigated the effect of isolated fungal proteins on the biofilm of L. innocua, for which we had previously successfully produced bacteria expressing different reporter proteins, of which DsRED Express proved to be the best. We also tested various markers and dyes for the detection of wild-type L. innocua. The commercial dye Mycolight Red, which binds to bacterial DNA, proved most useful. When investigating whether selected fungal proteins affect L. innocua biofilm development, we found that both cocaprin and macrocypin caused decrased adhesion of bacteria to the substrate. However, we also observed a visible effect of cocaprin on the degradation of static biofilms and the effect of macrocypin on the degradation of dynamic biofilms. Bacterial adhesion to the substrate and degradation of the mature static biofilm were more affected by the three mutants KKP1-N22R, KKP1-FH32EE, and KKP1-G13E than by KKP1. These altered amino acid residues are located on the same side of KKP1, suggesting that there is a site important for binding to L. innocua bacteria. Both cocaprin and macrocypin affect the formation of L. innocua biofilm, so it is worth analysing their effect on the pathogenic species L. monocytogenes, which is a major health problem. If the effect of selected fungal proteins on biofilm formation of pathogenic bacteria were confirmed, they could be used for further studies and development of new antimicrobial agents targeting biofilms or even drugs to treat listeriosis infections.
Secondary keywords: cocaprin;macrocypin;biofilm;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 77 str.
ID: 13595026