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

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 243-271). Also available on the World Wide Web.

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 107-119). Also available on the World Wide Web.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

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.

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.

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.

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.

No description available.

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.