The ability of antimicrobial peptides to migrate across the gastrointestinal epithelial and vascular endothelial barriers

Thesis (MSc)--Stellenbosch University, 2018.ENGLISH ABSTRACT: Antibiotic resistance has become a major threat to humankind, necessitating research and development of alternative antimicrobial compounds. Many bacteria, including lactic acid bacteria, produce small antimicrobial peptides, referred to as bacteriocins. These peptides are generally not toxic, are active at low concentrations and have a narrow spectrum of antimicrobial activity. However, they usually have low in vivo stability due to degradation by proteolytic enzymes. Drug delivery systems are thus required to transport bacteriocins to the site of infection. Probiotic bacteria are an attractive delivery system, since these bacteria normally colonise the gastrointestinal tract and produce bacteriocins. Numerous studies have been done on the probiotic Entiro™ which consists of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA. The bacteriocins produced by these strains, plantaricin 423 and bacST4SA, respectively, are active against a variety of pathogens and are potential alternatives to antibiotics. However, it is unknown whether these bacteriocins are able to migrate across the gastrointestinal epithelium and vascular endothelium in order to enter the bloodstream. The aim of this study was to evaluate the stability, cytotoxicity and permeability of plantaricin 423 and bacST4SA in vitro and evaluate the potential use of these peptides as an alternative to antibiotics. The well-known lantibiotic, Nisin A, produced by Lactococcus lactis subsp lactis, was used as control and Listeria monocytogenes EGDe as target (sensitive) organism. Migration of the lantibiotic, nisin A and class IIa bacteriocins, plantaricin 423 and bacST4SA across simulated models of the vascular endothelial and gastrointestinal epithelial barriers was studied by growing human umbilical vein endothelial cells (HUVEC)- and human colonic adenocarcinoma (Caco-2) cells on transmigration inserts and adding fluorescently labelled nisin A, plantaricin 423 and bacST4SA to the inserts. All three peptides diffused across HUVECs and Caco2 cells. Only 21% nisin A, 11% plantaricin 423 and 12% bacST4SA remained attached to Caco-2 cells and only 6% nisin A and 3% bacST4SA attached to the HUVECs, and plantaricin 423 did not attach. The viability of both cell types remained unchanged when exposed to 50 μM nisin A, 50 μM plantaricin 423 and 50 μM bacST4SA, respectively. Furthermore, little extracellular lactate dehydrogenase (LDH) activity was recorded when cells were exposed to 100 μM of each peptide, suggesting that the peptides are not cytotoxic. The three peptides retained 60% of their antimicrobial activity when 25 μM of each were exposed to 80% human plasma for 24 h. However, at higher concentrations (50 μM) 68% of the original antimicrobial activity was recorded and at 100 μM the peptides retained 79% of their activity. This is the first report of nisin A, plantaricin 423 and bacST4SA migrating across simulated gastrointestinal- and vascular barriers. In vivo studies are required to confirm these findings and determine the effect these peptides may have in the treatment of systemic infections.AFRIKAANSE OPSOMMING: Antibiotika weerstandigheid het 'n groot bedreiging vir die mensdom geword en daarom is daar ’n behoefte aan navorsing en ontwikkeling van alternatiewe antimikrobiese verbindings. Die meerderheid van bakterieë, insluitend melksuurbakterieë, produseer klein antimikrobiese peptiede, bekend as bakteriosiene. Hierdie peptiede is oor die algemeen nie toksies nie, is aktief teen lae konsentrasies en het 'n noue spektrum van antimikrobiese aktiwiteit. Hulle het egter gewoonlik ’n lae in vivo stabilteit as gevolg van afbraak deur proteolitiese ensieme. Effektiewe vervoersisteme vir geneesmiddels is dus nodig om bakteriosiene na die plek van infeksie te vervoer. Probiotiese bakterieë is ’n aantreklike voervoersisteem, aangesien hierdie bakterieë gewoonlik die spysverteringskanaal koloniseer en bakteriosiene produseer. Talle studies is gedoen op die probiotika, Entiro ™ wat bestaan uit Lactobacillus plantarum 423 en Enterococcus mundtii ST4SA. Die bakteriosiene, plantarisien 423 en bakteriosien ST4SA (bakST4SA), wat deur hierdie stamme geproduseer word, is aktief teen ’n verskeidenheid patogene en is potensiële alternatiewe vir antibiotika. Dit is egter onbekend of hierdie bakteriosiene oor die spysverteringskanaal en vaskulêre endoteel kan migreer om die bloedstroom te bereik. Die doel van hierdie studie was om die stabiliteit, sitotoksisiteit en deurlaatbaarheid van plantarisien 423 en bakST4SA in vitro te evalueer om vas te stel of hierdie peptiede as alternatief vir antibiotika kan dien. Die bekende lantibiotikum, nisin A, geproduseer deur Lactococcus lactis subsp lactis, is gebruik as die kontrole baktieriosien en Listeria monocytogenes EGDe as teiken (sensitiewe) organisme. Migrasie van die lantibiotika, nisin A- en klas IIa-bakteriosiene, plantarisien 423 en bakST4SA oor gesimuleerde modelle van die vaskulêre endoteel en spysverterings versperrings was bestudeer deur endoteelselle vanaf menslike naelstring are (HUVECs) en kolonale adenokarsinoom (Caco-2) selle op transmigrasie houers te groei en fluoresserende nisin A, plantarisien 423 en bakST4SA by die houers in te voeg. Al drie peptiede het gediffundeer oor HUVECs en Caco2-selle. Slegs 21% nisin A, 11% plantarisien 423 en 12% bakST4SA het gebonde gebly aan Caco-2 selle. Slegs 6% nisin A en 3% bakST4SA het gebonde gebly aan die HUVECs. Plantarisien 423 het nie geheg nie. Die lewensvatbaarheid van beide seltipes bly onveranderd wanneer dit blootgestel word aan 50 μM nisin A, 50 μM plantarisien 423 en 50 μM bakST4SA, onderskeidelik. Verder is baie min ekstrasellulêre laktaat dehidrogenase (LDH)-aktiwiteit aangeteken wanneer selle aan 100 μM van elke peptied blootgestel is, wat daarop dui dat die peptiede nie sitotoksies is nie. Die drie peptiede behou 60% van hul antimikrobiese aktiwiteit wanneer 25 μM van elk vir 24 uur aan 80% menslike plasma blootgestel is. Daar is egter by hoër konsentrasies (50 μM), 68% van die oorspronklike antimikrobiese aktiwiteit aangeteken en by 100 μM het die peptiede 79% van hul aktiwiteit behou. Dit is die eerste verslag wat raporteer op die vermoë van nisin A, plantarisien 423 en bakST4SA om oor gesimuleerde spysverteringskanale en vaskulêre grense te migreer. In vivo studies word vereis om hierdie bevindinge te bevestig en die effek te bepaal wat hierdie peptiede moontlik kan hê in die behandeling van sistemiese infeksie