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In English The thesis deals with research of novel antimicrobial peptides (AMP) Halictines (HAL-1, GMWSKILGHLIR-NH2 a HAL-2, GKWMSLLKHILK-NH2) and their structural analogs isolated from the venom of the wild bee Halictus sexcinctus. The structure and antimicrobial activity of these peptides had been described earlier [1]. The goal of this diploma thesis is to find peptide which is strongly toxic only for cancer cells and nontoxic for normal cells. Using of the fluorescent marked peptides we aimed to acquire the information about mechanism of action of the studied peptides on the cells. Using the MTT test (determination of valuation IC50), the toxicity of HAL-1 and HAL-2 and their analogs against 2 normal cell lines (Human umbilical vein endothelial cells, HUVEC, and normal rat intestinal cells, IEC) and against 2 cancer cell lines (cancer cells of suppository uterine, HeLa-S3 and cancer cells of human colorectal carcinoma, CRC SW 480) was determined. First we tested antimicrobial peptides with antimicrobial activity and low hemolytic activity. For verification the toxicity of less active analogs was also determined. We found out that the HeLa-S3 cells are the most sensitive to these peptides. The most toxic peptides (HAL-1/9, HAL-1/18, HAL-2/2) kill 50% of cells in the concentration 2,5 - 10 µM. To obtain...

In this thesis optical microscopy techniques are used to consider aspects of viral and bacterial infections. In part 1, the physical effects of cytomegalovirus on lipid droplet dynamics in live cells are studied; in part 2, the effects of an antimicrobial peptide on the fluidity of model bacterial membranes are studied. The optical microscopy techniques used to study the effects of murine-cytomegalovirus (mCMV) on lipid droplets in live NIH/3T3 fibroblast cells in real-time are coherent anti- Stokes Raman scattering (CARS), two-photon fluorescence (TPF) and differential interference contrast (DIC) microscopies. Using a multimodal CARS and TPF imaging system, the infection process was monitored by imaging the TPF signal caused by a green fluorescent protein (GFP)-expressing strain of mCMV, where the amount of TPF detected allowed distinct stages of infection to be identified. Meanwhile, changes to lipid droplet configuration were observed using CARS microscopy. Quantitative analysis of lipid droplet numbers and size distributions were obtained from live cells, which showed significant perturbations as the infection progressed. The CARS and TPF images were acquired simultaneously and the experimental design allowed incorporation of an environmental control chamber to maintain cell viability. Photodamage to the live cell population was also assessed, which indicated that alternative imaging methods must be adopted to study a single cell over longer periods of time. To this end, DIC microscopy was used to study the lipid droplet dynamics, allowing lipid droplet motion to be tracked during infection. In this way, the effects of viral infection on the mobility and arrangement of the lipid droplets were analysed and quantified. It was found that the diffusion coefficient of the lipid droplets undergoing diffusive motion increased, and the droplets undergoing directed motion tended to move at greater speeds as the infection progressed. In addition, the droplets were found to accumulate and cluster in infected cells. The second part of this thesis presents a study on the effects of an antimicrobial peptide on model bacterial membranes. Giant unilamellar vesicles (GUVs) were produced as a simple model of E. Coli membrane using a 3:1 mixture of DPPC and POPG lipids. Incorporating Laurdan fluorescent dye into the lipid membrane of the GUVs allowed the membrane fluidity to be probed and visualised using TPF microscopy, whereby the fluidity was quantified by determining the general polarization (GP) values. Studying GUVs comprising single lipid and mixed lipid compositions over a temperature range from 25 C to 55 C enabled the lipid phase bands to be identified on the basis of GP value as gel phase and liquid crystalline phase. As such, the changes in lipid phase as a result of interaction with AMP were quantified, and phase domains were identified. It was found that the amount of liquid crystalline phase domains increased significantly as a result of AMP interaction.

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The need for alternative or supplementary treatments due to the global problem of microbial resistance towards conventional antimicrobials may be met by the development of novel drugs based on antimicrobial peptides. The antimicrobial peptides of interest to this study were the tyrocidines, cyclic decapeptides produced by Bacillus aneurinolyticus. Although these antimicrobial peptides were the first natural antibiotic to be discovered though a systematic search for antibacterial compounds, information regarding their bioactivity, structure-activity relationships, determinants of bioactivity and mode of action is limited. The aim of this study was to investigate the antibacterial and antiplasmodial activity, as well as to identify determinants of bioactivity modulation, of the natural tyrocidine library. The study indicated that the tyrocidines exhibit significant activity toward Gram-positive bacteria, notably Listeria monocytogenes, and the intraerythocytic parasite, Plasmodium falciparum. Both the antilisterial and antiplasmodial activity was found to be highly dependent on peptide identity and self-assembly. The antilisterial activity of the tyrocidines was shown to be associated with increased self-assembly within a membrane-like environment, which suggested that formation of lytic complexes within the bacterial membrane may play a crucial role in tyrocidine activity. In contrast to the observations for antilisterial activity, the antiplasmodial activity of the tyrocidines was shown to be associated with reduced self-assembly within a membrane-like environment, which suggested that the antiplasmodial activity of the tyrocidines is mediated by a mechanism other than the formation of lytic complexes within the target cell membrane. In addition to the influence of peptide identity and self-assembly, the bioactivity of the tyrocidines was found to be highly sensitive to environmental conditions, notably the presence of calcium. The antilisterial activity, as well as the mode of action, of the tyrocidines was also found to be highly sensitive to tyrocidine-Ca2+ complexation and the concomitant induction of higher-order structures. Tyrocidine-Ca2+ complexation was shown to greatly enhance antilisterial activity and change the mechanism of action from a predominantly membranolytic to an alternative, non-lytic mode of action. The results of this investigation suggest that the alternative mode of tyrocidine activity may be related to complexation with Ca2+. It is hypothesised that such complexation may either (1) promote tyrocidine-DNA complexation, and thus inhibition of transcription and/or replication; or (2) interfere with Ca2+ homeostasis, and thus influence vital cell functions. Overall, it may be hypothesised that tyrocidine activity and mode of action is modulated by a critical play-off between self-assembly, cation-complexation and membrane-interaction. As these modulators of activity are highly dependent on tyrocidine sequence/structure, the wide variety of tyrocidines found in the natural complex may allow for optimal interaction with and activity toward a variety of microbes. / AFRIKAANSE OPSOMMING: Die universele probleem van mikrobiese weerstand teen konvensionele antimikrobiese middels en die wêreld-wye noodsaaklikheid vir alternatiewe of bykomende behandeling mag deur die ontwikkeling van nuwe middels, gebasseer op antimikrobiese peptiede, vervul word. Die antimikrobiese peptiede van belang tot hierdie studie is die tirosidiene, sikliese dekapeptiede wat deur Bacillus aneurinolyticus geproduseer word. Informasie ten opsigte van die tirosidiene se bioaktiwiteit, struktuur-funksieverwantskap, determinante van bio-aktiwiteit en meganisme van aksie was beperk, alhoewel hierdie peptiede die eerste antimikrobiese peptiede was wat ontdek is deur ‘n sistematiese soektog vir antimikrobiese middels. Die doelwit van hierdie studie was die ondersoek van antibakteriële and antiplasmodiese aktiwiteit, sowel as om die determinante van bio-aktiwiteit modulering van die natuurlike tirosidienbiblioteek te ondersoek. Hierdie studie het getoon dat die tirosidiene merkwaardige aktiwiteit teenoor Gram-positiewe bakterië, in besonder Listeria monocytogenes het, asook teenoor die intra-eritrositiese parasiet, Plasmodium falciparum. Daar is bevind dat beide die antilisteriese en antiplasmodiese aktiwiteite hoogs afhanklik is van peptiedidentiteit en self-verpakking. Daar is gewys dat die antilisteriese aktiwiteit van die tirosidiene geassosieer is met verhoogde self-verpakking in ’n membraanagtige omgewing, wat ’n aanduiding is dat die vorming van litiese komplekse in die bakteriële membraan ’n kritiese rol in tirosidienaktiwiteit speel. Kontrasterend tot die waarnemings van antilisteriese aktiwiteit, is getoon dat die antiplasmodiese aktiwiteit van die tirosidiene geassosieer is met verlaagde self-verpakking in ’n membraanagtige omgewing. Dis ’n aanduiding dat die antiplasmodiese aktiwiteit van die tirosidiene gemediëer word deur ‘n ander meganisme en nie die vorming van litiese komplekse in die teikenselmembraan nie. Bykomend tot die invloed van peptiedidentiteit en self-verpakking, is daar bevind dat die bioaktiwiteit van die tirosidiene hoogs sensitief is vir die omgewing, in besonder die teenwoordigheid van kalsium. Daar is ook bevind dat die antilisteriese aktiwiteit, sowel as die meganisme van aksie, van tirosidiene hoogs sensitief is vir tirosidien-Ca2+ kompleksvorming en die gevolglike induksie van of hoër-orde strukture. Daar is gewys dat tirosidien-Ca2+ kompleksvorming die antilisteriese aktiwiteit drasties verhoog en dat die meganisme van aksie verander van ’n oorwegende membranolitiese meganisme na ’n alternatiewe nie-litiese meganisme van aksie. Die resultate van hierdie ondersoek het aangedui dat die alternatiewe meganisme van aksie van tirosidienaktiwiteit moontlik verband kan hou met kompleksvorming met Ca2+. Die hipotese is dat sodanige kompleksvorming moontlik of (1) tirosidien-DNA komplekvorming aanmoedig, en dus transkripsie en/of replikasie inhibibeer of (2) met Ca2+ homeostase inmeng, en sodoende lewensnoodsaaklike selfunksies beïnvloed. Die algemene hipotese is dat tirosidienaktiwiteit en meganisme van aksie deur ’n kritiese spel tussen self-verpakking, katioonkompleksvorming en membraaninteraksie gemoduleer word. Die wye verskeidenheid van tirosidiene, wat in die natuurlike kompleks gevind word, kan moontlik toelaat vir die optimale interaksie met, en aktiwiteit teenoor ’n verskeidenheid van mikrobes, aangesien die aktiwiteitmoduleerders hoogs afhanklik is van tirosidien struktuur/volgorde.

<p>Insects rely solely on innate immune reactions for protection against infect-ing microbes in their environment. In <i>Drosophila</i>, one major defense mechanism is the production of a battery of antimicrobial peptides (AMPs). The expression of AMPs is primarily regulated at the level of transcription and constitutes both constitutive expression in a tissue-specific manner and inducible systemic expression in response to infection. The aim of my thesis has been to investigate the regulation of AMP gene expression at different levels. I have studied a novel cis-regulatory element, Region 1 (R1) found in the proximal promoter of all Cecropin genes in Drosophila melanogaster, as well as in other species of <i>Drosophila.</i> We found that the R1 element was important for the expression of CecropinA1 (CecA1) both in vitro and in vivo. A signaling-dependent R1-binding activity (RBA) was identified in nuclear extracts from <i>Drosophila</i> cells and flies. The molecular nature of the RBA, has despite considerable effort, not yet been identified. I also have studied the role of the JNK pathway in transcriptional regulation of AMP genes. The role of the JNK pathway in the regulation of AMP genes has long been elusive, however, in this study we showed that the pathway is directly involved in the expression of AMP genes. Analysis of cells mutant for JNK pathway components showed severely reduced AMP gene expression. Fur-thermore, over-expression of a JNK pathway-inhibitor also inhibited AMP gene expression. Lastly, I have studied transcription factors that have not previously been implicated in transcriptional regulation of AMP genes. In a yeast screen, three members of the POU family of transcription factors were identified as regulators of CecA1. Two of them, Drifter (Dfr) and POU do-main protein 1 (Pdm1) were further characterized. We showed that Dfr was able to promote AMP gene expression in the absence of infection, suggest-ing it to play a role in constitutive expression of AMP genes. Indeed, down-regulation of Dfr expression using RNAi severely reduced the constitutive expression of AMP genes in the male ejaculatory duct. We also identified an enhancer element important for Dfr-mediated expression of CecA1. Pdm1, on the other hand, was shown to be important for the systemic expression of AMP genes. In Pdm1 mutant flies, several AMP genes are systemically expressed even in the absence of infection, suggesting that Pdm1 works as a repressor of those genes. However, at least on AMP gene, AttacinA (AttA) requires Pdm1 for its expression, suggesting that Pdm1 works as an activator for this gene. Upon infection, Pdm1 was rapidly degraded, but, regenerated shortly after infection. We propose that the degradation of Pdm1 is important for the activation of the Pdm1-repressed genes and that regeneration sup-ports the expression of AttA.</p>

<p>The fruit fly <i>Drosophila melanogaster</i> has been a powerful model system in which to study the immune response. When microorganisms breach the mechanical barrier of the insect, phagocytosing cells and a battery of induced antimicrobial molecules rapidly attack them. These antimicrobial peptides can reach micromolar concentrations within a few hours. This immediate response is reminiscent of the mammalian innate immune response and utilizes transcription factors of the NF-κB family. </p><p>We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the <i>Drosophila</i> immune response to microbes. Relish mutant flies have a severely impaired immune response to Gram-negative (G^-) bacteria and some Gram-positive (G⁺) bacteria and fungi and succumb to an otherwise harmless infection. The main reason for the high susceptibility to infection is that these mutant flies fail to induce the antimicrobial peptide genes. The cellular responses appear to be normal. </p><p>Relish is retained in the cytoplasm in an inactive state. We designed a set of expression plasmids to investigate the requirements for activation of Relish in a hemocyte cell line after stimulation with bacterial lipopolysaccharide. Signal-induced phosphorylation of Relish followed by endoproteolytic processing at the caspase-like target motif in the linker region released the inhibitory ankyrin-repeat (ANK) domain from the DNA binding Rel homology domain (RHD). Separation from the ANK domain allowed the RHD to move into the nucleus and initiate transcription of target genes like those that encode the inducible antimicrobial peptides, likely by binding to κB-like sites in the promoter region. </p><p>By studying the immune response of the Relish mutant flies in combination with mutants for another NF-κB-like protein, Dorsal-related immunity factor (Dif), we found that the <i>Drosophila</i> immune system can distinguish between various microbes and generate a differential response by activating the Toll/Dif and Imd/Relish pathways. The recognition of foreign microorganisms is believed to occur through pattern recognition receptors (PRRs) that have affinity for selective pathogen-associated molecular patterns (PAMPs). We found that the <i>Drosophila</i> PRRs can recognize G^- bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G⁺ bacteria such as <i>Micrococcus luteus</i> and <i>Bacillus megaterium </i>and fungal PAMPs from <i>Beauveria bassiana</i> and <i>Geotrichum candidum</i>. </p><p>This thesis also investigates the expression of the antimicrobial peptide genes, <i>Diptericin B</i> and <i>Attacin C</i>, and the putative intracellular antimicrobial peptide gene <i>Attacin D</i>, and explores a potential evolutionary link between them.</p>

Higher eukaryotes produce a vast range of antimicrobial peptides (AMPs) that play important roles in their defence against microbial infection. Beta defensins are small (3-5 kDa), cationic peptides that display broad, potent antimicrobial activity against a range of microbes and also act as chemoattractants of important immunomodulatory cells. To generate highly pure peptides for structural and functional studies, we developed a method to prepare recombinant human beta defensin-2 (HBD2). The HBD2 gene was synthesised by recursive PCR with codons optimised for expression in Escherichia coli. HBD2 was expressed as an insoluble fusion to a His-tagged ketosteroid isomerase. After cleavage from the fusion with cyanogen bromide, 1H NMR spectroscopy and mass spectrometry confirmed that the oxidised HBD2 was folded and possessed the correct b-defensin disulfide bond topology. The recombinant HBD2 was active against E. coli, P. aeruginosa, S. aureus and C. albicans and was also a chemoattractant against HEK293 cells expressing the chemokine receptor CCR6. 15N-labelled HBD2 was also prepared and was highly suitable for future structural studies. Since defensins are thought to interact with bacterial membranes we also tested the recombinant HBD2 in biophysical studies (surface plasmon resonance, SPR, Biacore). We observed different binding to artificial model membranes containing either E. coli Kdo2-lipid A or phospholipids. Bacterial resistance to AMPs has been linked to the covalent modification of the outer membrane lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N). This neutralises the charge of the LPS, thereby decreasing the electrostatic attraction of cationic peptides to the bacterial membrane. The pathogen Burkholderia cenocepacia displays extremely high resistance to AMPs and other antibiotics and the Ara4N pathway appears to be essential. To explore this further we expressed recombinant forms of two enzymes (ArnB and ArnG) from the B. cenocepacia Ara4N pathway. Purified ArnB is a pyridoxal 5’-phosphate (PLP)-dependent transaminase and we tested its ability to bind amino acid substrates. We investigated the binding of inhibitors L- and D-cycloserine to ArnB and tested their antibiotic activity against Burkholderia strains. We also studied the B. cenocepacia ArnG – a proposed membrane protein undecaprenyl-L-Ara4N flippase – and showed that the protein behaved as a dimer by non-denaturing gel analysis. The B. cenocepacia ArnG failed to complement E. coli knock-out strains encoding the equivalent flippase proteins ArnE and ArnF, suggesting that ArnG is a Burkholderia-specific protein.

La multiplication des résistances aux antibiotiques constitue une grave menace qui nécessite de nouvelles stratégies antimicrobiennes. Le but de ce travail est d'étudier le potentiel des nanocapsules lipidiques (NCLs) pour l'administration de peptides antimicrobiens (PAMs). Les premiers travaux ont porté sur le développement et l'optimisation de NCLs chargées en PAMs. Différentes stratégies d’association ont été testé (adsorption à la surface ou encapsulation dans le coeur de NCLs). Les résultats ont démontré une efficacité d'association comprise entre 20 et 40%, lorsque les peptides sont adsorbés à la surface et plus de 80%,lorsqu’ils sont encapsulés. La deuxième partie s’est concentrée sur l'évaluation de l’activité des complexes PAMs et NCLs ainsi que leur stabilité vis-à-vis des protéases. Les résultats ont montré que l'adsorption entraîne une potentialisation de l'activité antimicrobienne des PAMs, associée à une protection partielle contre la dégradation enzymatique. Inversement, l’encapsulation des PAMs montre une meilleure stabilité aux enzymes, corrélée à une efficacité d'encapsulation supérieure sans amélioration de l'activité antimicrobienne in vitro. Dans une troisième partie, les mécanismes impliqués dans les interactions LNC/PAM ainsi que l'interaction du complexe avec un modèle de membrane bactérienne ont été étudiés. Il a été montré que la structure et la flexibilité des PAMs aux interfaces solide-liquide gouverneraient l'adsorption des peptides à la surface des NCLs, entrainant un changement de leur comportement avec les membranes bactériennes. L’ensemble de ces résultats démontre le potentiel prometteur des NCLs en tant que vecteur de PAMs. / The rapid increase in drug-resistant infections presents an acute problem in the healthcare sector, generating interest in novel antimicrobial strategies. The aim of this work is to explore the potential of lipid nanocapsules (LNCs) for Antimicrobial peptides (AMPs) delivery. Firstly, the experiments were focused on the development and optimization of AMP-loaded LNCs. Different strategies were investigated to deliver AA230,LL37 and DPK060 using LNCs (peptides adsorption atthe surface or encapsulated in the core of modified LNCs). The results demonstrated an association efficiency of 20 to 40%, when peptide is adsorbed, and over 80% encapsulation efficiency, when peptides are encapsulated. The second part concerned the study ofthe influence of peptides loading on their activity and stability against proteases. The results showed that peptides adsorption induced a potentiation of the antimicrobial activity of the native peptides, with a partial protection against proteolytic degradation. Conversely, peptides encapsulation allowed better peptide stability, correlated with higher encapsulation efficiencies and a preservation of the in vitro antimicrobial activity. In a third part, the mechanisms involved in LNC/AMP interactions and the complex interaction with model bacterial membrane have been evaluated. It has shown that structure and flexibility at solid-liquid interfaces govern peptide adsorption on the surface of the LNCs, which in turn is expected to change LNCs properties and interaction with bacterial membranes. Taken together, these results demonstrate the potential of LNCto deliver AMPs as an alternative anti-infective therapy.

Due to the escalating challenge of antibiotic resistance in bacteria over the past several decades, interest in the identification and development of antibiotic alternatives has intensified. Antimicrobial peptides (AMPs), which serve as part of the innate immune systems of most eukaryotic organisms, are being researched extensively as potential alternatives. However, the mechanism behind their bactericidal capabilities is not well understood. Previous studies have suggested that AMPs may first attach to the cell membranes, leading to pore formation caused by peptide insertion, lipid removal in the form of peptide-lipid aggregates, or a combination of both mechanisms. In addition to the lack of mechanistic knowledge, a significant hurdle in AMP-based drug development is their potential cytotoxicity to mammalian cells. Understanding AMP interactions with eukaryotic model membranes would allow therapeutics to be tailored for preferential action toward specific classes of bacterial membranes. In this study, we developed novel methods of quartz crystal microbalance with dissipation monitoring (QCM-D) data analysis to determine the fundamental mechanism of action between eukaryotic and bacterial membrane mimics and select membrane-active AMPs. A new technique for creating supported membranes composed entirely of anionic lipids was developed to model Gram-positive bacterial membranes. Atomic force microscopy (AFM) imaging was also used to capture the progression of AMP-induced changes in supported lipid membranes over time and to validate our method of QCM-D analysis. QCM-D and AFM were used to investigate the molecular-scale interactions of four peptides, alamethicin, chrysophsin-3, sheep myeloid antimicrobial peptide (SMAP-29) and indolicidin, with a supported zwitterionic membrane, which served as a model for eukaryotic cell membranes. Since established methods of QCM-D analysis were not sufficient to provide information about these interaction mechanisms, we developed a novel method of using QCM-D overtones to probe molecular events occurring within supported lipid membranes. Also, most previous studies that have used AFM imaging to investigate AMP-membrane interactions have been inconclusive due to AFM limitations and poor image quality. We were able to capture high-resolution AFM images that clearly show the progression of AMP-induced defects in the membrane. Each AMP produced a unique QCM-D signature that clearly distinguished their mechanism of action and provided information on peptide addition to and lipid removal from the membrane. Alamethicin, an alpha-helical peptide, predominantly demonstrated a pore formation mechanism. Chrysophsin-3 and SMAP-29, which are also alpha-helical peptides of varied lengths, inserted into the membrane and adsorbed to the membrane surface. Indolicidin, a shorter peptide that forms a folded, boat-shaped structure, was shown to adsorb and partially insert into the membrane. An investigation of rates at which the peptide actions were initiated revealed that the highest initial interaction rate was demonstrated by SMAP-29, the most cationic peptide in this study. The mechanistic variations in peptide action were related to their fundamental structural properties including length, net charge, hydrophobicity, hydrophobic moment, accessible surface area and the probability of alpha-helical secondary structures. Due to the charges associated with anionic lipids, previous studies have not been successful in forming consistent anionic supported lipid membranes, which were required to mimic Gram-positive bacterial membranes. We developed a new protocol for forming anionic supported lipid membranes and supported vesicle films using a vesicle fusion process. Chrysophsin-3 was shown to favor insertion into the anionic lipid bilayer and did not adsorb to the surface as it did with zwitterionic membranes. When introduced to supported anionic vesicle films, chrysophsin-3 caused some vesicles to rupture, likely through lipid membrane disruption. This study demonstrated that molecular-level interactions between antimicrobial peptides and model cell membranes are largely determined by peptide structure, peptide concentration, and membrane lipid composition. Novel techniques for analyzing QCM-D overtone data were also developed, which could enable the extraction of more molecular orientation and interaction dynamics information from other QCM-D studies. A new method of forming supported anionic membranes was also designed, which may be used to further investigate the behavior of bacterial membranes in future studies. Insight into AMP-membrane interactions and development of AMP structure-activity relationships will facilitate the selection and design of more efficient AMPs for use in therapeutics that could impact the lives of millions of people per year who are threatened by antibiotic-resistant organisms.

Antimicrobial peptides are active against bacteria, fungi and viruses as part of the innate immune system in animals and insects. Such peptides are currently produced by extracting them from the host organism or by solid phase peptide synthesis; both techniques are expensive and produce low yields. Recombinant DNA technology opens a window to produce these peptides inexpensively and in large quantities utilizing E. coli expression systems. Two antimicrobial peptides, indolicidin and PGQ, were the focus of this work. They are short amphipathic alpha helical antimicrobial peptides that target a broad range of microorganisms. Genes encoding multimers of indolicidin, PGQ and a hybrid of indolicidin:PGQ were placed into protein expression vectors pET32a+ and pET43.1a+, for peptide production in E. coli. A combination of multimerization and the use of a fusion protein were utilized to mask the toxicity of these peptides in E. coli. The multimerized peptide fusion construct was purified using Ni/NTA affinity chromatography. Methionine residues flanking each monomeric unit were utilized to enable cleavage of the multimerized protein and liberating a biologically active peptide. A Trx:indolicidin trimer fusion was produced in the greatest yield of all constructs investigated. Upon cyanogen bromide cleavage, a band corresponding to the theoretical molecular weight of an indolicidin monomer was observed with SDS-PAGE. Antimicrobial activity of monomeric recombinant indolicidin was tested resulting in zones of clearing. Overall the results indicate that multimerizing antimicrobial peptide genes can potentially produce a larger quantity of peptide per bacterial cell. These studies suggest that multimerization of antimicrobial peptide genes represents a means to control in vivo toxicity of the recombinant peptides and increase production relative to single gene fusions.