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The comparison of biological properties of L- and D-enantiomeric antimicrobial peptidesKwok, Hoi-shan, 郭凱珊 January 2014 (has links)
Antibiotics have been used widely for the treatment of bacterial infections for over half a century. However, the emergence of resistance to antibiotics has aroused public health concern, leading to the development of antimicrobial peptides (AMPs) as potential alternative therapeutic agents against bacterial infections. AMPs are naturally found in many species and have important roles in our innate immune defense systems. AMPs are usually cationic amphipathic peptides with membrane destabilizing property. They have a relatively broad spectrum of antimicrobial activity and pathogens are less likely to develop resistance against AMPs. The major challenge of using AMPs as therapeutic agents is their toxicity towards mammalian cells. The biological stability of AMPs to protease in human body is another concern.
To address the latter problem, instead of the naturally occur L-enantiomers, Denantiomeric AMPs were introduced to enhance their stability. This study aimed to test the hypothesis that the D-enantiomeric AMPs are more resistant than the Lenantiomeric AMPs against proteolytic degradation. Three pairs of synthetic D-/LAMPs (D-LAO160-P13/LAO160-P12; D-LAO160-H/LAO160-H; and D-LAK-120-HP13/LAK-120-HP13) were employed to test for their stability when treated with trypsin, serum and gastric fluid, and the samples were analyzed by high performance liquid chromatography (HPLC). Generally, all the D-enantiomeric AMPs were found to be resistant towards proteolysis. Besides, to compare the cytotoxicity of D-/LAMPs, MTT and LDH assays of the D/L-LAK120-HP13 pair were carried out on two different cell lines, A549 cells (human lung adenocarcinoma epithelial cells) and RAW264.7 cells (mouse macrophage cells). Significant difference in cytotoxicity of D-LAK120-HP13 and LAK120-HP13 on RAW264.7 cells were obtained from MTT assay, but not in LDH assays or on A549 cells. Further analysis has to be done to validate the findings obtained from this research. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences
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Advanced studies on cyclic amino acids in neurological signaling and peptide antibiotics /Blanchard, David, L. January 1900 (has links)
Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 243-271). Also available on the World Wide Web.
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Adaptive diversity and divergence at frog antimicrobial peptide loci /Tennessen, Jacob A. January 1900 (has links)
Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 107-119). Also available on the World Wide Web.
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Nisin adsorption and function at hydrophobic surfaces coated with the poly[ethylene oxide]-poly[propylene oxide]-poly[ethylene oxide] triblock surfactant Pluronic® F108 /Tai, Yuan-Ching. January 1900 (has links)
Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.
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Cyclic lipodepsipeptides as lead structures for the discovery of new antiobioticsUnknown Date (has links)
With antimicrobial resistance to current drugs steadily rising, the development of new antibiotics with novel mechanisms of action has become an imperative. The majority of life-threatening infections worldwide are caused by "ESKAPE" pathogens which are encountered in more than 40% of hospital-acquired infections, and are resistant to the majority of commonly used antibiotics. Naturally occurring cyclic depsipeptides, microbial secondary metabolites that contain one or more ester bonds in addition to amide bonds, have emerged as an important source of pharmacologically active compounds or lead structures for the development of novel antibiotics. Some of those peptides are either already marketed (daptomycin) or in advanced stages of clinical development (ramoplanin). Structurally simple, yet potent, fusaricidin/LI-F and lysobactin families of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents capable of overco ming infections caused by multidrug-resistant bacteria. These natural products exhibit potent antimicrobial activity against a variety of clinically relevant fungi and Gram-positive bacteria. Therefore, access to these classes of natural products and their synthetic analogs, combined with elucidation of their mode of action represent important initial steps toward full exploitation of their antmicrobial potential. This dissertation describes a general approach toward the solid-phase synthesis of fusaricidin/LI-F and lysobactin analogs and an extensive structure-activity relationship (SAR) study. We have devised a simple and robust preparation strategy based on standard Fmoc solid-phase peptide synthesis protocols. / The SAR study revealed key structural requirements for fusaricidin/LI-F and related cyclic lipopeptides antibacterial activity, including the presence of the guanidino moietly at the end of the lipidic tail, hydrophobic amino acid residues, and peptide conformation Moreover, substitution of the ester bond with an amide bond significantly improved stability under physiologically relevant conditions and reduced toxicity. In addition, we have shown that these antibacterial peptides exert their mode of action via a novel mechanism, which invloves bacterial membrane interactions, followed by peptide internalization. Altogether, the research described in this dissertation demonstrates that new antibiotics derived from fusaricidin/LI-F natural products, have the potential to meet the challenge of antibiotic resistance in Gram-positive bacteria. / by Nina Bionda. / Thesis (Ph.D.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.
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Role of surfactin from Bacillus subtilis in protection against antimicrobial peptides produced by Bacillus speciesEyeghe-Bickong, Hans Andre 03 1900 (has links)
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.
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Tyrocidines, cyclic decapeptides produced by soil bacilli, as potent inhibitors of fungal pathogensTroskie, Anscha Mari 04 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The global rise in microbial resistance, ranging from the agricultural industry to the medical
sector, has created the urgent need for novel or supplementary antibiotics. Antimicrobial
peptides or “nature’s antibiotics” may be the answer to this major problem. In this study a group
of antimicrobial peptides, cyclic decapeptides named tyrocidines, produced by the soil bacterium
Bacillus aneurinolyticus, was investigated for their antifungal activity, possible mode of
antifungal action and potential applications.
The study illustrated that the tyrocidines have significant antifungal activity against a range of
phytopathogens, including Fusarium solani and Botrytis cinerea, as well as the human pathogen
Candida albicans. The activity of the tyrocidines is influenced by the identity of both the target
organism and the media environment. Further evidence was obtained in support of the
hypothesis that the tyrocidines are extremely sensitive to their environmental conditions and that
they tend to self-assemble to form oligomers. The assessment of a small tyrocidine library and
analogues, comprised of eight peptides, revealed no overt structure-activity relationships against
fungal pathogens, except for the importance of a tyrosine residue. This indicated an important
role for the conserved sequence of the tyrocidines, NQYVOLfP, together with the tendency of
the tyrocidines to oligomerise into higher-order active structures in their antifungal activity.
The tyrocidines were found to be membrane active toward the fungal pathogens. However,
supporting evidence was also obtained for additional mode(s) of antifungal action for the
tyrocidines which inter alia induces morphological abnormalities in filamentous fungal target
cells. Furthermore, the results also indicated that the membrane activity of the tyrocidines may
be influenced by additional factors to that of the composition of the target cell membrane, for
instance components of the fungal cell wall. This investigation also indicated the significant potential of the tyrocidines to be developed for
the commercial sector. The potent activity of the tyrocidines against agronomically important
phytopathogens (significantly higher than the commercial fungicide bifonazole) together with
their relative salt stability bodes well for their development as bio-fungicides for the agricultural
sector. The tyrocidines also exhibited an overt sinergistic effect on the in vitro candidacidal
activity of two key antifungal drugs, caspofungin and amphotericin B. Furthermore, tyrocidine A
and caspofungin exhibited synergistic activity in vivo which had a significant positive effect on
the survival of C. albicans infected Caenorhabditis elegans. Latter results highlighted their
potential to serve as candidates for combinatorial treatment in the medical industry. / AFRIKAANSE OPSOMMING: Die globale verskynsel van mikrobiese weerstand, wat strek vanaf die landbou sektor tot in die
mediese bedryf, het ’n dringende behoefte vir die ontwikkeling van nuwe antmikrobiese middels
geskep. Antimikrobiese peptiede of “die natuur se antibiotika”, kan moontlik die antwoord op
hierdie ernstige problem wees. Tydens hierdie studie is ‘n groep sikliese antimikrobiese
peptiede, naamlik die tirosidiene wat deur die grondbakterium Bacillus aneurinolyticus
geproduseer word, vir hulle antifungiese aktiwiteit, hulle moontlike meganisme(s) van
antifungiese werking en hulle potensiёle aanwendings bestudeer.
Hierdie studie het getoon dat die tirosidiene uitsonderlike antifungiese aktiwiteit teen ‘n reeks
fitopatogene, insluitend Fusarium solani en Botrytis cinerea, asook teen die mens patogeen
Candida albicans het. Die aktiwiteit van die tirosidiene is deur beide die identiteit van die
teikenorganisme sowel as die mediumomgewing beїnvloed. Daar is ook verdere bewyse verkry
wat die hipotese dat tirosidiene uiters sensitief is tot hulle omgewing en dat hulle neig om te
oligomeriseer, ondersteun. Die studie van die klein tirosidien-biblioteek, saamgestel uit agt
tirosidiene en analoё, het geen ooglopende struktuur-aktiwiteit verwantskappe opgelewer nie,
behalwe vir die oёnskynlike invloed van die tirosien-residu. Laasgenoemde het die belangrikheid
van die gekonserveerde aminosuurvolgorde van die tirosidiene, NQYVOLfP, asook die neiging
van tirosidiene om hoё-orde aktiewe strukture te vorm deur self-verpakking, beklemtoon.
Tydens die studie is daar gevind dat die tirosidiene membraan-aktiewiteit toon teenoor fungiese
patogene. Daar is egter ook goeie bewyse vir alternatiewe meganisme(s) van antifungiese
werking, wat ondermeer tot morfologiese abnormaliteite in filamentagtige fungi-teikenselle lei,
vir die tirosidiene verkry. Die resultate het verder ook daarop gewys dat die membraan-aktiwiteit van die tirosidiene ook deur ander faktore, soos deur komponente van die fungiese selwand, en
nie net deur die samestelling van die fungiese membraan beїnvloed word nie.
Hierdie ondersoek het ook die aansienlike potensiaal van die tirosidiene vir kommersiёle
ontwikkeling en gebruik uitgelig. Die merkwaardige aktiwiteit van die tirosidiene teen
fitopatogene van agronomiese belang (wat selfs beter as diè van die kommersiёle swamdoder
bifonazole was) tesame met die relatiewe sout stabiliteit van die tirosidiene, is belowende tekens
om die tirosidiene as bio-swamdoders vir die landbou sektor te ontwikkel. Die tirosidiene het
ook ‘n uitgesproke sinergistiese effek op die in vitro candidasidiese aktiwiteit van twee sleutel
antifungiese middels, caspofungin en amphotericin B, getoon. Verder is daar in vivo sinergistiese
aktiwiteit gewys deur die kombinasie van tirosidien A en caspofungin wat ’n beduidende
positiewe effek op die oorlewing van C. albicans geïnfekteerde Caenorhabditis elegans gehad
het. Laasgenoemde dui op die potensiaal van die tirosidiene om in die mediese bedryf as
kandidate vir kombinasie-behandeling te dien.
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Effects of cationic antimicrobial peptides on Candida and Saccharomyces speciesHarris, Mark R. January 2010 (has links)
Antimicrobial peptides (AMPs) are found throughout the animal kingdom and act as a natural defence against a broad spectrum of pathogens. These peptides are toxic to invading organisms without acting on host cells, so are of interest for their potential to act as potent new drugs against pathogenic organisms. AMPs traverse the cell wall and predominantly target the plasma membrane, resulting in destabilisation, leakage of intracellular components and cell death. In this thesis the mode of action of several AMPs was investigated. The role of the cell wall was studied and found to mediate peptide binding, the inhibition of certain cell wall components also increased peptide action, subsequent internalisation events were observed with varying localisation patterns and the effect of several genes that alter cell susceptibility to AMP were examined. Several Candida albicans mutants, each deficient in cell wall protein mannosylation, were tested in relation to their susceptibility to AMPs. It was discovered that cells lacking or deficient in the phosphomannan fraction, with a concomitant reduction in surface negative charge, correlated with reduced susceptibility to AMP action. To ascertain whether peptide binds to negatively charged phosphate, the effect of exogenous glucosamine 6-phosphate (but not glucosamine hydrochloride) was studied demonstrating that peptide efficacy was reduced due to the presence of exogenous phosphate. More specifically, sequestration of the truncated cationic AMP dermaseptin S3 (DsS3(1-16)) was reduced in these phosphomannan deficient mutants. Microscopy analysis of fluorescein tagged DsS3(1-16) also revealed the differential localisation patterns of this AMP: transiently binding to the plasma membrane, localisation to the vacuole or diffuse distribution throughout the cytoplasm. It is proposed that for these cationic AMPs to exert their full antifungal action they must first bind to the negatively charged phosphate. The echinocandins are a relatively new class of antifungal that function by inhibiting 1,3-β glucan synthase resulting in reduced 1,3-β glucan in the cell wall. As AMPs have to traverse the cell wall it was postulated that cells lacking this fraction would display increased AMP binding to the membrane. Clinical isolate strains of Candida and Cryptococcus spp. were acquired to test their susceptibility to AMP and echinocandin combinations. Comparing the fractional inhibitory concentration index (FICI) (supported by viable cell counts and on a solid surface using disc diffusion assays) synergy was observed between caspofungin, anidulafungin and several AMPs in vitro. In vitro toxicity assays revealed no increase in haemolytic or cytotoxic action on combination. These synergistic combinations could provide a novel treatment against fungal pathogens. The final area of study was based upon work that identified genes whose expression altered cell susceptibility to AMPs. Three genes were selected for investigation that upon deletion increased the action of DsS3(1-16) or magainin 2 on S. cerevisiae. Results from growth analysis, peptide sequestration and cell viability counts confirmed that deletion of HAL5, LDB7 or IMP2’ did increase susceptibility. Additionally, deletion of HAL5 increased the probability of cell depolarisation upon peptide exposure. Expression of GFP-tagged Imp2’ also increased when cells were exposed to DsS3(1-16). It was concluded that deletion of HAL5 increases depolarisation due to insufficient potassium efflux, leading to ion leakage and cell death facilitated by AMP action. Double strand break repair and DNA protection are probably compromised upon deletion of LDB7 and IMP2’, increasing the inhibitory action of DsS3(1-16) that has previously been shown to bind to DNA.
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Novel cationic peptides and polymers in the treatment of methicillin-resistant Staphylococcus aureus and multi-drug resistant Acinetobacter spp. skin infection isolatesKatvars, Laura K. January 2015 (has links)
No description available.
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Investigating Bacterial Lipopolysaccharides and Interactions with Antimicrobial PeptidesStrauss, Joshua 20 January 2009 (has links)
The goal of this research was to develop a novel biosensor for detecting and eliminating pathogenic E. coli. Traditionally, identifying pathogenic E. coli and distinguishing it from harmless environmental strains includes serotyping and DNA sequencing, which can take days or weeks. Our biosensor platform makes use of a material that is part of the immune system from single- multi- cellular organisms that target viruses, fungi, and bacteria called antimicrobial peptides (AMPs). Using the quartz crystal microbalance with dissipation monitoring (QCM-D), we characterized non-specific binding between CP1 to silicon nitride and gold, and covalent binding of cysteine-terminated CP1 (CP1-cys) to gold. QCM-D monitors frequency and dissipative changes resulting from adsorbed mass, and peptide film thickness and density can be calculated using Voigt Viscoelastic modeling. Viability of the E. coli was monitored using a live/dead kit consisting of nucleic acid stains Syto 9 and Propidium Iodide. Successfully immobilizing peptide to a substrate is particularly important if CP1 would be applied on a food processing surface. By immobilizing CP1 to silicon nitride, we measured the binding forces between bacteria and peptides with the atomic force microscope (AFM), and explored important bacterial features such as LPS composition and length that influence binding affinity with CP1. The structure of the LPS is comprised of 3 sections: lipid A, core group, and O-antigen. We are mostly interested in the initial binding between AMP and LPS since our goal is to develop a novel biosensor that can detect pathogenic bacteria within seconds of exposure. Considering the short exposure period, the AMP would only be exposed to the O-antigen and outer core groups, which are repeating sugar chains that are essential for bacterial pathogenicity and adhesion to substrates. Although geared for use as a novel biosensor, results of this study can also be applied to the use of AMPs for replacing or enhancing the activity of antibiotics. Our work suggests that CP1 may not be serotype-specific, but targets the O-antigen before interfering with phospholipid groups of the bacterial membrane. Other factors that assist in pathogenicity, such as LPS length, may also be important for the consideration of CP1 potency.
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