Profiling of the secondary metabolites and the characterization two novel antilisterial peptides, xenopep and rhabdin, produced by xenorhabdus khoisanae

Abstract

ENGLISH ABSTRACT: In the early 1900’s the discovery of sulfonamides and penicillin paved the way for antibiotics and led to a boom in the discovery of other antibiotics. Unfortunately, this boom was short lived and soon the discovery and approval of novel antibiotics by the food and drug association and other similar associations dwindled. With the ever-increasing prevalence of antibiotic resistant pathogens this soon became a problem that was not foreseen. Most antibiotics currently on the market have been isolated from a select few genera. With nearly all the antibiotics from such few sources, bacteria were able to acquire resistance at an enhanced pace. This study focused on a relatively unexplored niche for novel antibiotics, from the genus Xenorhabdus. Species of this genus is mutually associated with Steinernema nematodes and have a unique life cycle. Xenorhabdus spp. are known to produce various secondary metabolites (SMs) that have antimicrobial, insecticidal, antiviral, immunosuppressant and proteolytic properties. Species from this genus use different synthesis machineries to produce these compounds, although the majority are produced via the non-ribosomal peptide synthesis. The ability of non-ribosomal peptides to incorporate non-proteogenic amino acids, D-amino acids, fatty chains, or polyketide chains result in unique resistance to proteinases and environmental stressors. Xenorhabdus khoisanae J194 is mutually associated with Steinernema jeffreyense J194, a nematode that was isolated from soil in the Eastern Cape. Culture conditions, especially oxygen, greatly affected SM production of X. khoisanae J194. PAX peptides, xenocoumacins and xenoamicins were identified in the cell-free crude extract of X. khoisanae J194 cultures. Two novel antilisterial peptides, xenopep and rhabdin, were also detected in the cell-free crude extract of. Xenopep has a narrow spectrum of activity and inhibited the growth of only, Listeria monocytogenes and Staphylococcus epidermidis, while rhabdin is active against both Gram-positive and Gram-negative bacteria. Xenopep and rhabdin share numerous characteristics and both contain a tetra-peptide in their structure including a tetra-peptide in their structure. Both peptides share the same amphipathic characteristic and behave similar suspension. Membrane potential and ATP release assays have shown that xenopep formed pores/lesions in the cell membrane of L. monocytogenes within minutes, followed by a rapid decrease in cell numbers over 3 hours. Scanning electron microscopy (SEM) images of L. monocytogenes treated with xenopep became elongated and formed filaments. This suggests that xenopep may inhibit penicillin binding protein three. This is the first study reporting on SMs produced by X. khoisanae when cultured under different conditions and is the first detailed description of antilisterial peptides produced by the species.

AFRIKAANSE OPSOMMING: In die vroeë 1900’s het die ontdekking van sulfonamiede en penisillien die weg gebaan vir antibiotika en gelei tot ‘n oplewing in die ontdekking van ander antibiotika. Ongelukkig was hierdie oplewing van kort duur en gou het die ondekking en goedkeuring van nuwe antibiotikums deur die kos en dwelm vereniging (KDV) en ander soortgelyke verenigings afgeneem. Met die toename in antibiotika-weerstandige patogene het dit vinnig ‘n probleem geword wat nie voorsien is nie. Die meeste antibiotika wat tans op die mark is, is uit ‘n paar uitegesoekte genera geïsoleer. Met byne al die antibiotika uit so min bronne kon bakterieë weerstand teen ‘n groter tempo ontwikkel. Hierdie studie het gefokus op ‘n relatiewe onontginde nis vir nuwe antibiotika, bakterieë van die genus Xenorhabdus. Spesies van hierdie genus is wedersyds geassosieer met Steinernema nematodes en het ‘n unieke lewens siklus. Xenorhabdus spp. produceer verskeie sekondêre metaboliete (SMe) met antimikrobiese, insekdodende, antivirale, immuunonderdrukkende en proteolitiese eienskappe. Spesies van hierdie genus gebruik ook verskeie sintese-masjinerie om hierdie verbindings te produseer, alhoewel die meederheid deur die nie-ribosomale peptiedsintese geproduseer word. Die vermoë van nie-ribosomale peptiede om nie-proteogeniese aminosure, D-aminosure, vetkettings of polieketiedkettings te inkorporeer, het hulle ‘n unieke weerstand teen proteïenase en omgewings stres faktore. Xenorhabdus khoisanae J194 is wedersyds met Steinernema jeffreyense J194 geassosieer wat uit grond in die Oos-kaap geïsoleer is. Kultuurtoestande, veral suurstof, het ‘n groot invloed op SM produksie van X. khoisanae J194. PAX-peptiede, xenocoumasiene en xenoamisiene is in sel-vry kru-ekstrak van X. khoisanae J194 kulture geïdentifiseer. Twee unieke antilisteriale peptiede, xenopep en rhabdin, is ook in die sel-vry kru-ekstrak van X. khoisanae J194 geïdentifiseer. Xenopep het ‘n nou spektrum van aktiwiteit en het slegs die groei van Listeria monocytogenes en Staphylococcus epidermidis geïnhibeer, terwyl rhabdin aktief is teen beide Gram-postiewe en Gram-negatiewe bacterieë. Xenopep en rhabdin dell verskeie eienskappe en beide besit ‘n tetra-peptied in hul struktuur. Beide peptiede deel dieselfde amfipatiese eienskappe en reageer soortgelyke gedrag in suspensie. Membraan potensiaal en ATP vrystellings toetse het getoon dat xenopep porieë of letsels in die selmembraan van behandelde L. monocytogenes binne minute na blootstelling vorm, gevolg deur ‘n vinnige afname in selgetalle binne drie ure. Skandeer elektronmikroskopie (SEM) beelde van L. Monocytogenes wat met xenopep behandel is, het getoon dat selle verleng en filamente vorm. Dit mag dus wees dat xenopep penisillien bindede proteïen drie inhibeer. Hierdie is die eerste bekendmaking van SMe wat deur X. Khoisanae, blootgestel aan verskillende kultuur toestande, geproduseer word en is die eerste gedetailleerde beskrywing van antilisteriale peptiede geprodusser deur die spesie.