Thesis (PhD (Biochemistry))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Antagonism of antimicrobial action represents an alternative survival strategy for cohabiting
soil organisms. Under competitive conditions, our group previously showed that surfactin
(Srf) produced by Bacillus subtilis acts antagonistically toward gramicidin S (GS) from a
cohabiting bacillus, Aneurinibacillus migulanus, causing the loss the antimicrobial activity
of GS. This antagonism appeared to be caused by inactive complex formation. This study
aimed to elucidate whether the previously observed antagonism of GS activity by Srf is a
general resistance mechanism that also extends to related peptides such as the tyrocidines
(Trcs) and linear gramicidins (Grcs) from Bacillus aneurinolyticus. Molecular interaction
between the antagonistic peptide pairs was investigated using biophysical analytical methods
such as electrospray mass spectrometry (ESMS), circular dichroism (CD), fluorescence
spectroscopy (FS) and nuclear magnetic resonance (NMR).
Results from this study corroborated the previous findings, namely that Srf antagonised the
activity of GS towards Gram positive bacteria. However, for Micrococcus luteus synergism
of GS action was observed at low Srf concentrations, while antagonism only occurred at Srf
concentrations above the critical micelle concentration (CMC) of Srf when the bacteria were
pre-incubated with Srf. This result and an ultra-performance liquid chromatography massspectrometry
(UPLC-MS) study indicated that Srf pre-absorbed to cells, as well as Srf
micelles interacted with GS, preventing GS from reaching the membrane target. Antagonism
of GS action by Srf was also observed towards the Srf producer B. subtilis ATCC21332 and
B. subtilis OKB120, a non-producer. The Srf producer was less sensitive than the nonproducer
towards GS, possibly due to Srf production. Pre-incubation of Srf at different
concentrations caused a dose-dependent antagonism, from as low as 0.9 μM Srf of GS
activity towards B. subtilis OKB120. This antagonism at the low Srf concentration may be
related to the induction of more resistant biofilms by Srf in B. subtilis. It was also found that
Srf significantly improved the survival of B. subtilis OKB120 above that of M luteus in a
mixed culture. In addition, the Srf producer B. subtilis ATCC21332 grew in the inhibition
zone of the GS producer A. migulanus ATCC9999 during co-culturing, while B. subtilis
OKB120 growth was inhibited.
Srf induced biofilm formation in B. subtilis may be important in protecting the bacteria in
solution, but not on solid phase such as on or in agar plates. Also, the protection of various
cell types (previous studies by our group) by Srf from GS indicated a directed antagonistic
Srf mode of action. Srf formed complexes that are visible and stable under ESMS conditions
with GS, with the peptide bonds in the Val-Orn-Leu-D-Phe moiety of GS and the Val-Asp-
D-Leu-Leu moiety of Srf protected from fragmentation. 1H-NMR titration studies strongly
indicated that the molecular interaction of Srf and GS involved the re-orientation of the DPhe4,9
and Orn2,7 residues in GS. From CD spectra it was observed that Srf induced a
concentration dependent decrease in the β-turn component and increase in β-sheet structures
of the GS-Srf mixture. Diffusion orientated NMR (DOSY) indicated that Srf and GS formed
homo-oligomers with the Srf-GS mixture having a slightly higher diffusion coefficient
indicating the formation of smaller homo-oligomers or more compact hetero-oligomers.
These hetero-oligomers involve intermolecular interaction at <5Å between the Orn2,7 residue
of GS with Asp residue of Srf, as observed with ROESY-NMR. These results strongly
indicate that inactive complex formation between Srf and GS is part of the antagonistic
mechanism of action of Srf towards GS.
Two high performance liquid chromatography (HPLC) methods was developed to purify
peptides from the tyrothricin complex, namely the Trcs (contains one GS Val-Orn-Leu-DPhe-
Pro moiety) and Grcs. These peptides were used to assess if Srf has an antagonistic
activity beyond that of GS. Srf indeed showed antagonistic action against the antimicrobial
activity of Trcs towards B. subtilis ATCC21332 and OKB120, with the tyrocidine C (TrcC)
being more sensitive to antagonism than tyrocidine B (TrcB). Srf had an ambiguous effect
on the linear gramicidin A (GA) that is co-produced with Trcs in tyrothricin. GA acted
synergistically with Srf at low GA concentrations, but slight antagonism was observed at
high GA concentrations. In contrast, GA showed pronounced synergism with TrcB towards
the M. luteus. However, Srf at 30 μM, antagonised the synergistic action of a lethal mixture
of 25 μM GA and TrcB. The Srf producer was also able to withstand and grow in the
presence of the tyrothricin producer B. aneurinolyticus ATCC10068, indicating that
antagonism of peptide action may allow different organisms to cohabit. Basic NMR and
ESMS studies failed to show complex formation between Srf and the Trcs. However, CD
presented clear evidence of Srf induced changes in secondary structures and/or higher order
self-assembled structures of the Trcs-Srf mixture. FS also provided evidence of the
reorientation/exposure of the Trp6 residue of the Trcs in the presence of Srf. These results
corroborated the previous findings that complexation between Srf and GS or peptides
analogous to GS may be part of the mechanism of Srf antagonistic action.
In conclusion, this study showed that the antagonism of GS activity by Srf, conferred in part
by inactive complex formation, is a putative resistance mechanism that also extends to other
peptides containing the Val-Orn-Leu-D-Phe-Pro moiety such as the Trcs from B.
aneurinolyticus. / AFRIKAANSE OPSOMMING: Antagonisme van antimikrobiese aksie verteenwoordig ʼn alternatiewe oorlewingstrategie vir
grondorganismes wat in dieselfde habitat gevestig is. Ons groep het gewys dat surfaktien
(Srf), geproduseer deur Bacillus subtilis, antagonistiese werking teenoor gramisidien S (GS)
vanaf die bacillus Aneurinibacillus migulanus, onder kompeterende kondisies, toon. Die
antagonistiese werking, wat moontlik veroorsaak word deur vorming van onaktiewe
komplekse, lei tot die verlies van die antimikrobiese aktiwiteit van GS. Hierdie studie se doel
was die ontrafeling van die moontlikheid dat die antagonisme van GS aktiwiteit deur Srf,
soos deur vorige studies uitgewys, ʼn algemene weerstandsmeganisme is wat moontlik ook
verwante peptiede soos die tirosidiene (Trcs) en lineêre gramisidiene (Grcs), afkomstig vanaf
Bacillus aneurinolyticus, insluit. In hierdie studie is die molekulêre interaksie tussen
antagonistiese peptiedpare ondersoek met biofisiese analitiese metodes wat elektrosproeimassaspektroskopie
(ESMS), sirkulêre dichroïsme (SD), fluoressensie-spektroskopie (FS) en
kernmagnetiese resonansspektroskopie (KMR) insluit.
Die resultate wat tydens hierdie studie verkry is, het gewys dat Srf die werking van GS
teenoor Gram-positiewe bakterie teenwerk, en het die vorige waarnemings ondersteun. Daar
is egter sinergisme tussen Srf en GS werking by lae Srf-konsentrasies teenoor Micrococcus
luteus waargeneem, terwyl antagonisme slegs waargeneem is by Srf-konsentrasies hoër as
die kritiese miselêre Srf konsentrasie wanneer bakterieë vooraf met Srf met inkubeer is.
Hierdie resultaat, tesame met ʼn ultra-hoë verrigting vloeistofchromatografie gekoppelde
massaspektroskopie (UPLC-MS) studie, het daarop gedui dat Srf wat voorheen op selle
geabsorbeer het, sowel as Srf-miselle in die media, met GS interaksie het en sodanig kan
voorkom dat GS die membraanteiken bereik. Antagonisme deur Srf op die GS aktiwiteit is
ook waargeneem teenoor die Srf-produseerder B. subtilis ATCC21332 en B. subtilis
OKB120, ʼn nie-produseerder. Hierdie tipe antagonisme by ʼn lae konsentrasie van Srf mag
verwant wees aan die induksie van meer weerstandige biofilms deur Srf in B. subtilis. Dit is
ook gevind dat Srf die oorlewing van B. subtilis OKB120 aansienlik verhoog teenoor dié van
M luteus in ʼn gemengde kultuur. Daar is verder bevind dat die Srf-produseerder, B. subtilis
ATCC21332, in die inhibisiesone van die GS-produseerder, A. migulanus ATCC9999,
gegroei het tydens kokultivering, terwyl die groei van B. subtilis OKB120 geïnhibeer is.
Srf induseer biofilm-vorming in B. subtilis wat moontlik belangrik kan wees om die
bakterieë in suspensie te beskerm, maar nie op soliede fase soos byvoorbeeld agar plate nie.
Verder dui die beskerming van ʼn verskeidenheid sel-tipes (vorige studies deur ons groep)
deur Srf teen GS, ʼn direkte antagonistiese aksie van Srf. Sigbare en stabiele komplekse
tussen Srf en GS is waargeneem onder ESMS kondisies, waar die peptiedbindings in die
Val-Orn-Leu-D-Phe-Pro eenheid van GS en die Val-Asp-Leu-D-Leu eenheid van Srf
beskerm is teen fragmentering in die komplese. 1H-KMR titrasiestudies het duidelik
aangetoon dat die molekulêre interaksie van Srf en GS die D-Phe4,9 en Om2, 7 residue in GS
heroriënteer. SD-spektra van GS-Srf mengsels het daarop gedui dat Srf ʼn konsentrasieafhanklike
vermindering in die β-draai komponente van die mengsel veroorsaak, maar dat β-
plaat komponent van die mengsel vermeerder. Diffusie-georiënteerde KMR spektrometrie
(DOSY) toon dat Srf en GS homo-oligomere vorm, maar ʼn hoër diffusie koeffisiënt vir die
mengsel het aangedui dat die Srf-GS mengsel kleiner of meer kompakte hetero-oligomere.
ROESY-KMR toon dat hierdie oligomere intermolekulêre interaksie(s) van <5Å tussen die
Om2, 7 residue van GS en die Asp residu van Srf het. Die resultate gee ʼn sterk aanduiding dat
die onaktiewe kompleks-vorming tussen Srf en GS deelneem in die antagonistiese werking
van Srf teenoor GS.
Twee hoë verrigting vloeistofchromatografie metodes is ontwikkel om peptiede uit die
tirotrisienkompleks, naamlik die Trcs (bevat een GS Val-Om-Leu-D-Phe-Pro eenheid) en die
gramisidiene (Grcs), te suiwer. Hierdie peptiede is gebruik om te bepaal of Srf antagonistiese
aktiwiteit het wat verder strek as net dié van GS. Dit was inderdaad die geval en daar is
gevind dat Srf antagonisties is teenoor die antimikrobiese aktiwiteit van Trcs met B. subtilis
ATCC21332 en OKB120 as teikens, met tirosidien C (TrcC) wat meer sensitief vir
antagonistiese werking van Srf was as tyrosidien B (TrcB). Srf het ʼn gemengde effek getoon
teenoor lineêre gramisidien A (GA) wat saam met die Trcs in tirotrisien gekoproduseer
word. GA het sinergisties met Srf gewerk by lae GA konsentrasies, maar milde
antagonistiese werking getoon by hoë GA konsentrasies. Daarteenoor het GA en TrcB
uitgesproke sinergisme getoon teenoor M. luteus. In teenstelling het Srf by 30 μM die
sinergistiese aksie van die dodelike mengsel van 25 μM GA en TrcB elk geantagoniseer. Die
Srf produseerder was ook bestand en kon in die teenwoordigheid van die tirotrisien
produseerder B. aneurinolyticus ATCC10068 groei wat aangedui het dat die antagonisme
van antibiotiese peptiedaktiwiteit die kohabitasie van organismes toelaat. Basiese KMR en
ESMS studies kon nie kompleksvorming tussen Srf en die Trcs aantoon nie, terwyl SD
duidelike bewyse gelewer het dat Srf verandering geïnduseer het in die sekondêre strukture
en/of hoër orde/self-geassosieerde strukture van die Trc-Srf mengsel. FS het ook bewyse
gelewer van die reoriëntasie/blootstelling van die Trp6 residu in die Trcs in die
teenwoordigheid van Srf. Hierdie resultate ondersteun die vorige bevindinge dat
kompleksvorming tussen Srf en GS of GS-peptiedanaloë deel van die meganisme van Srf se
antagonistiese aksie uitmaak.
Samevattend het hierdie studie getoon dat die antagonisme van GS aktiwiteit deur Srf deels
toegeken kan word aan onaktiewe kompleksvorming tussen die twee peptiede en dat die
voorgestelde weerstandsmeganisme ook ander peptiede wat die Val-Orn-Leu-D-Phe-Pro
eenheid, soos die Trcs van B. aneurinolyticus, insluit.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/6773 |
| Date | 03 1900 |
| Creators | Eyeghe-Bickong, Hans Andre |
| Contributors | Rautenbach, Marina, University of Stellenbosch. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Stellenbosch |
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