Karakterizacija AggLb agregacionog faktora soja Lactobacillus paracasei subsp. paracasei BGNJ1-64

Abstract

Odgovor bakterija na uticaj razliĉitih faktora sredine koja ih okruţuje, kao i njihova meĊusobna komunikacija, zavisi od procesa koji se odvijaju na površini bakterijskih ćelija. S obzirom na brojne mogućnosti primene bakterija roda Lactobacillus, kako u prehrambenoj industriji, tako i u medicini (u prevenciji i/ili leĉenju bolesti), potrebno je što bolje okarakterisati komponente koje ĉine površinski sloj ovih bakterija. Agregacija bakterija, determinisana faktorima lociranim na površini ćelija moţe se smatrati poţeljnom osobinom pri izboru probiotika, s obzirom da ima vaţnu ulogu u meĊubakterijskoj interakciji, u interakciji sa epitelijalnim ćelijama gastrointenstinalnog trakta i doprinosi sposobnosti bakterija da se adsorbuju na razliĉite površine, odnosno sposobnost agregacije omogućava uspešnu kolonizaciju staništa. Kolekcija laktobacila Laboratorije za molekularnu mikrobiologiju je pretraţena na prisustvo sojeva koji poseduju sposobnost agregacije (Agg+), nakon ĉega je za dalji rad odabrano jedanaest sojeva: BGSJ2-8, BGGR2-68, BGGR2-82, BGDP1-84, BGDP9-38, BGNJ1-3, BGNJ1-61, BGNJ1-64, BGNJ1-70, BGZLS30-6 i BGAR75. Odabrani Agg+ sojevi se na osnovu stepena agregacije i tipa formiranih agregata mogu podeliti u tri grupe: oni koji brzo, srednje i sporo agregiraju ili koji formiraju krupne, srednje i sitne agregate, pri ĉemu postoji direktna korelacija izmeĊu brzine agregacije i tipa agregata. Analizom sposobnosti vezivanja odabranih Agg+ sojeva laktobacila za komponente ekstracelularnog matriksa (ECM) zapaţen je širok dijapazon sposobnosti vezivanja za kolagen - od intenzivnog afiniteta vezivanja do odsustva sposobnosti vezivanja za matriks. Pritom su pojedini sojevi, BGGR2- 68 i BGGR2-82, ispoljili sposobnost vezivanja za plastiku (formiranje biofilma). Analizom plazmidnog sastava Agg+ sojeva konstatovano je prisustvo velikih plazmida u svim sojevima, a ĉišćenjem plazmida iz sojeva BGNJ1-64 i BGSJ2-8 potvrĊena je plazmidna lokacija gena odgovornih za ekspresiju agregacionog fenotipa. Nakon konstrukcije i analize plazmidnih biblioteka sojeva BGNJ1-64 i BGSJ2-8, izolovan je konstrukt pALb35 (koji nosi SacI fragment veliĉine 11,4 kb poreklom iz plazmida pNJ1 soja BGNJ1-64) koji dovodi do ponovne pojave autoagregacionog fenotipa transformisanog heterologog domaćina BGKP1-20 (Agg-)...

Response of bacteria to different environmental factors as well as their communication primarily depends on the processes that occur on their cell surface. Due to the continuously increasing number of application possibilities of lactobacilli, both in the food industry and in medicine (in the prevention and/or treatment of the disease), there is a need for a more comprehensive characterization of the components that form the surface layer of these bacteria. Aggregation ability of bacteria, determined by factors located on the cell surface could be considered as a desirable trait in the selection of probiotics. Since it plays an important role in the interaction between bacteria, in interaction with the epithelial cells of the gastrointestinal tract plus contributing to the bacterial capability to adhere to different surfaces, which is an important feature enabling them to successfully colonize diverse habitats. Lactobacilli collection of Laboratory for molecular microbiology was screened for the presence of strains possessing the aggregation ability (Agg+) and for the further work eleven strains have been selected: BGSJ2-8, BGGR2-68, BGGR2-82, BGDP1-84, BGDP9-38, BGNJ1-3, BGNJ1-61, BGNJ1-64, BGNJ1-70, BGZLS30-6 and BGAR75 which, according to the level of aggregation and the appearance of the aggregates formed, could be clasified into three groups: the fast, the medium and the slow aggregating ones or into the groups of those forming large, medium or small aggregates wherein a direct correlation between the aggregation kinetics and the type of the formed aggregates was found. By analyzing the binding ability of the selected Agg+ strains of lactobacilli to the components of extracellular matrix (ECM) a wide range of binding capacity to collagen was observed, varying from the intensive to the complete lack of the binding capacity to the matrix. Two strains (BGGR2-68 and BGGR2-82) showed the ability to bind even to plastics (form biofilms). By analyzing the plasmid composition of Agg+ strains, the presence of large plasmids was detected in all strains. The plasmid curing experiments confirmed the plasmid location of the gene(s) responsible for the expression of aggregation phenotype in strains BGNJ1-64 and BGSJ2-8. After the construction and analysis of BGNJ1-64 and BGSJ2-8 strains plasmid libraries, selected construct pALb35 (which carries the SacI fragment of 11.4 kb. derived from plasmid pNJ1 from strain BGNJ1-64) was able to reconstitute the autoagreggation ability of the transformed heterologous host BGKP1-20 (Agg-)...