Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Antimicrobial peptides (AMPs) are currently the most researched group of compounds for new
antimicrobial drugs especially with the rise in resistance to almost all available drugs by public
health relevant pathogens. In this study we set out to characterise small cyclic AMPs in terms of
their activity towards human pathogens Listeria monocytogenes, a food-borne pathogen causing
listeriosis and Plasmodium falciparum, a parasite that causes malaria respectively, each a threat
to public health.
One of the small cyclic peptide libraries examined is the tyrocidines (Trcs) and analogues, which
are cyclic decapeptides [cyclo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val-
(Orn/Lys)-Leu] produced by the Gram-positive bacteria Bacillus aneurinolyticus as part of the
tyrothricin complex which is non-ribosomally synthesised during sporulation. Previous research
found that the six major Trcs were active against Listeria monocytogenes and Plasmodium
falciparum and it was found that the identity of the aromatic residues in the aromatic dipeptide
unit has an important role in activity. We set out to extend the qualitative structure to activity
relationship (QSAR) studies using more Trc analogues and small synthetic Arg- and Trp-rich
cyclic peptides (RW-peptides) in a bid to establish essential structural motifs and pre-requisites
for activity. Eight natural and three synthetic Trc analogues and fifteen RW-peptides were either
naturally or by chemical synthesis produced and characterised in terms of chemical character and
biological activity. The Trcs were significantly more active than RW peptides, although much
more haemolytic and thus toxic. Results indicated the relevance for hydrogen bonding with an
aromatic amino acid residue for selective activity towards the leucocin A resistant L.
monocytogenes B73-MR1. However, structural properties favouring a tighter membrane
interaction hindered the Trc mode of action (MOA). We determined that Gln6 and hydroxyl
group of Tyr7 may be involved in interaction with the putative target in L. monocytogenes. There
was also need for an amphipathic balance between hydrophobicity and size/steric parameters for
optimal activity. From our QSAR studies we predict as lead peptide for a future library of
antilisterial Trcs: cyclo(VOMe3LfPWfNQY). Furthermore, the antilisterial activity of the Trcs
was found to be predominantly lytic and salt tolerant while RW-peptides were non-lytic and
sensitive to Ca2+. We confirmed that Ca2+ enhanced Trc antilisterial activity with Ca2+ increasing
the Trc anti-metabolic activity, but conversely inducing a non-lytic mechanism of action. From model membrane studies, we propose that the calcium induced Trc non-lytic MOA could be due
to detrimental lipid demixing, presence of a Trc sensitive Ca2+-induced non-membrane target in
the prematurely calcium induced intracellular anaerobic form of Listeria monocytogenes, and/or
the Trc-Ca2+ complexes may inhibit key components such as membrane bound electron transport
system or bacterial dehydrogenases.
We confirmed, as previously found, that the Trcs have potent antimalarial activity that is
sequence specific and non-lytic. The RW-peptides had very weak activity, but our results again
indicated that more hydrophobic and haemolytic peptides tend to be more active, particularly the
RW-peptide containing the Trp analogue β-(benzothien-3-yl)-alanine (Bal). A novel finding was
that one of the more polar Trc C analogues, namely tryptocidine C (Tpc C), in contrast to Trc C
showed potent antimalarial activity indicating the specific sequence and the role of the Trp7 in
activity. From these results a proposed lead peptide for future research is
cyclo[VOLfP(Bal)fNQ(Bal)]. Furthermore, in our search for the Trc and Tpc C target(s) we
employed high resolution fluorescence microscopy. Results show that Trc led to disorganisation
of neutral lipid structures and chromatin halting growth in late trophozoite/early schizont stages.
This indicated that membrane structures containing neutral lipids, as well as chromatin may be
targeted by the Trcs. Another novel finding in our studies was that chloroquine (CQ) resistance
not only correlated with resistance to Trcs, but the Trcs and CQ were found to be antagonistic
towards each other’s activity. This indicated a shared target and we propose the food vacuole as
another of the Trc targets in P. falciparum. / AFRIKAANSE OPSOMMING: Antimikrobiese peptiede (AMPe) is tans die mees nagevorsde groep verbindings in die soeke na
nuwe antimikrobiese middels, veral weens 'n toenemende weerstandigheid van patogene in die
openbare gesondheidsektor teen alle beskikbare middels. Die doel van hierdie studie was om
klein, sikliese AMPe in terme van hul aktiwiteit teenoor twee menslike patogene wat 'n
bedreiging vir openbare gesondheid is, Listeria monocytogenes, 'n voedsel-oordraagbare
patogeen wat listeriose veroorsaak, asook Plasmodium falciparum, die parasiet verantwoordelik
vir malaria, te karakteriseer.
Een van die klein, sikliese peptiedbiblioteke wat ondersoek is, is die tyrocidines (Trcs) en analoë
(sikliese dekapeptiede [siklo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val-
(Orn/Lys)-Leu]). Hierdie peptiede deur die Gram-positiewe bakterie Bacillus aneurinolyticus
word wat nie-ribosomaal gesintetiseer as deel van die tirotrisien kompleks word tydens
sporulasie. Vorige navorsing het gewys dat die ses hoof Trcs teen Listeria monocytogenes en
Plasmodium falciparum aktief is en dat die identiteit van die aromatiese residue in die aromatiese
dipeptiedeenheid 'n belangrike rol speel in die Trc-aktiwiteit. Ons het gepoog om die
kwalitatiewe struktuur-aktiwiteit-verwantskap (QSAR) studies uit te brei deur meer Trc analoë
en klein sintetiese Arg- en Trp-ryke sikliese peptiede (RW-peptiede) te gebruik en sodoende
essensiële struktuur-motiewe en voorvereistes vir aktiwiteit vas te stel. Agt natuurlike en drie
sintetiese Trc analoë, asook vyftien RW-peptiede is of deur natuurlike of chemiese sintese
geproduseer en gekarakteriseer in terme van chemiese karakter en biologiese aktiwiteit. Die Trcs
het beduidend meer aktiwiteit as RW-peptiede getoon, maar is ook meer hemolities en dus meer
toksies. Die resultate dui op die belang van waterstofbinding met 'n aromatiese aminosuurresidu
vir die selektiewe aktiwiteit teenoor die leucocin A weerstandige L. monocytogenes B73-MR1.
Strukturele eienskappe wat tot 'n sterker membraan-interaksie lei, verhinder egter die
werkingsmeganisme. Ons het vasgestel dat Gln en die hidroksielgroep van Tyr betrokke kan
wees in die interaksie met die vermeende teenmiddelteiken in L. monocytogenes. 'n Balans tussen
amfipatiese/hidrofobiese en grootte/steriese parameters is ook noodsaaklik vir optimale
aktiwiteit. Vanuit ons QSAR studies word die peptied siklo-(VOMe3LfPWfNQY) as die
voorloper vir 'n toekomstige peptiedbiblioteek van antilisteriale Trcs voorgestel. Verder is daar
gevind dat die antilisteriese aktiwiteit van die Trcs oorwegend lities en sout-verdraagsaam is, terwyl die RW-peptiede nie-lities en Ca2+ sensitief is. Ons het bevestig dat Ca2+ die Trc
antilisteriese aktiwiteit verbeter, deur die Trc se antimetaboliese aktiwiteit verhoog, maar
terselfdertyd 'n nie-litiese werkingsmeganisme induseer. Vanuit model-membraan studies word
voorgestel dat Trc se nie-litiese werkingsmeganisme, soos teweeggebring deur Ca2+, die gevolg
kan wees van nadelige lipied vermenging, die teenwoordigheid van 'n kalsium geïnduseerde Trcsensitiewe
nie-membraan teiken in 'n vervroegde kalsium geïnduseerde intrasellulêre anaerobiese
vorm van Listeria monocytogenes, en/of dat die Trc-Ca2+ komplekse belangrike komponente
soos ’n membraan-gebonde elektron transport sisteem of bakteriële dehidrogenases inhibeer.
Daar is ook bevestig, soos voorheen gevind, dat die Trcs kragtige, antimalaria aktiwiteit besit wat
volgorde-spesifiek en nie-lities is. Die RW-peptiede het swak aktiwiteit getoon, maar ons
resultate het weereens bewys dat peptiede wat meer hidrofobies en hemolities is, meer aktief is,
veral die RW-peptiede wat die Trp analoog β-(bensoteïen-3-iel)-alanien (Bal) bevat. 'n Nuwe
bevinding is dat een van die meer polêre Trc C analoë, genaamd triptosidien C (Tpc C), in
teenstelling met Trc C, sterk antimalaria aktiwiteit het, wat 'n aanduiding is van die spesifieke
volgorde en die rol van die Trp7 in aktiwiteit. Vanuit hierdie bevindinge word die peptied siklo-
(VOLfP(Bal)fNQ(Bal)) as 'n voorloper vir toekomstige navorsing aangedui.
Vir ons soeke na die Trc en Tpc C teiken(s), het ons hoë resolusie fluoressensie mikroskopie
aangewend. Resultate toon dat Trc tot die ontwrigting van 'n neutrale lipied strukture en
chromatien lei en sodoende groei beperk in die laat trofosoïet/vroeë skisont fases. Dit het
aangedui dat die membraanstrukture wat neutrale lipiede bevat, sowel as chromatien, deur die
Trcs geteiken word. 'n Verdere nuwe bevinding in hierdie studie was dat chloroquine (CQ)
weerstandigheid nie net korreleer met weerstandigheid teen Trcs nie, maar dat die Trcs en CQ
antagonisties optree teenoor mekaar se aktiwiteite. Dit dui op 'n gemeenskaplike teiken en die
kosvakuool as 'n addisionele Trc teiken in P. falciparum word voorgestel.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/86161 |
| Date | 04 1900 |
| Creators | Leussa, Nyango-Nkeh Adrienne |
| Contributors | Rautenbach, Marina, Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 295 p. : ill. |
| Rights | Stellenbosch University |
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