Search

There are numerous resources in Nature that can be utilised, and frogs' intricate living circumstances enables them to serve a purpose by secreting substances on their skin that shield them from the outside environment. Some of the complex problems of today may have been aided by their evolution. As many bacteria have evolved resistance to current antibiotics in use, antimicrobial peptides from frog skin secretions are among the most promising novel antimicrobial agents due to their wide-ranging antimicrobial activities and different mechanisms of action. This project aims to identify two novel antimicrobial peptides and to evaluate their biological activities and to find peptides with drug potential through modification. And their structure-activity relationships were studied. In Chapter 3, the full-length cDNA sequences of brevinin-1HL and palustrin-2LTb, two novel antimicrobial peptides, were cloned from skin secretion of Hylarana latouchii through "shotgun" cloning. In Chapter 4, brevinin-1HL identified in the skin secretion of Hylarana latouchii was evaluated for its biological activity. Five analogues were designed through D-type amino acid substitution and changing the amino acids in the hydrophobic face. It was found that the secondary structure, especially the helicity between these peptides, plays a significant role in their actions. And the designed analogue, brevinin-1HL-D-9L, was considered as a potential drug candidate with potent antibacterial activity with low haemolysis at its effective values. In Chapter 5, a novel peptide, palustrin-2LTb, identified in the skin secretion of Hylarana latouchii was shown to have good antibacterial activity against selected pathogens. To reduce the manufacturing cost and decrease toxicity, trypsin-cleavage mimicking modification strategy was ultilised to produce predicted fragments. Seven such truncated derivatives of palustrin-2LTb were synthesised and tested for their biological activities. Functional screening data indicated that truncated fragment 3 not only maintained and optimised antimicrobial efficacy of the parent peptide, but also showed great in vivo therapeutic potential in an MRSA-infected insect larvae model. Due to its strong antibacterial activity, minimal haemolytic activity, and ability to kill MRSA in an in vivo model, fragment 3 showed outstanding effects and had obvious potential to become a new antimicrobial agent.

With the increasing emergency of drug-resistance, the AMPs from the amphibians have raised much interest due to their broad-spectrum antimicrobial activity and promising immunological competence. Among the many species of amphibians, Odorrana grahami was found to be one of the most abundant sources of skin AMPs. In this thesis, three experimental chapters describe two peptide templates, the design of respective analogues and structure-activity studies conducted on different aspects and at different depths. In Chapter 3, a novel peptide was obtained from the skin secretion of a frog. This peptide has broad-spectrum antimicrobial activity and potent anti-biofilm activity. As an important motif, the "Rana box" was hitherto considered as important in affecting the antimicrobial activity of parent peptide, while the role of the cationic residues in the "Rana box" has not been discussed before. This study revealed the significant role of the specific residues in the "Rana box" and reduced the significance of the "Rana box" in the antimicrobial activity of parent peptide family, which was accepted widely in previous studies. In Chapter 4, the study here examines the imperfect/perfect amphipathic mode of a peptide. The parent peptide has a broad-spectrum antimicrobial activity against all the pathogens tested herein. Analogues of it exhibited advantages and disadvantages in many ways, mainly in their antimicrobial activity effects. This study has revealed the rationality of the imperfect amphipathic model and possibility of further modification targeting the residues of the hydrophobic face. Additionally, the disulfide bond also has been shown to have a special influence on anti-biofilm activity. Further structure-function relationship studies could be performed based on the bio-functionality data of these analogues presented here. In Chapter 5, another peptide isolated from the same frog above, which was studied before and showed no antimicrobial activity, was revisited. This peptide, possessing lectin-like activity and specific immune system bio-functions, underwent structural modifications in an attempt to investigate structure-function relationships for analogue design as potential drug leads. However, these attempts proved unsuccessful.

Antimicrobial peptides (AMPs) obtained from amphibian skin, have made significant strides toward becoming possible substitutes for traditional antibiotics. In this study, two new AMPs were initially identified from frog skin secretion. In order to assess their bioactivities, a number of bioassays were carried out, and the natural AMPs were utilized as models to investigate the structure-function correlations and enhance their bioactivities. The first novel peptide, namely Kas-4, was identified in the skin secretion of the African running frog, Kassina senegalensis, and possessed both intense anti-bacterial and anti-cancer activities. In Chapter 3, several modifications such as net charge enhancement, hydrophobicity decrease and Proline removal, were employed to explore its optimised structure for bioactivities. Then, one antimicrobial-oriented peptide Des-Pro-Kas-4 and one anticancer peptide, W7W10-Kas-4, were designed. Based on Chapter 3, Chapter 4 induced a unique motif "FFY" to further increase the bioactivities of Kas-4. Here, F-Kas-4 was found to possess similar antimicrobial activities but better antibiofilm activity towards MRSA 12493. These discoveries provided both novel and useful new understanding of how natural medicines are discovered. The second novel peptide, Kas-2, was identified from the skin secretion of the African hyperoliid frog, Kassina maculata. Studies showed that Kas-2 had potent bactericidal effects on Gram-positive bacteria and anti-proliferation activity on tested cancer cells. After the modifications were performed in Chapter 5 and Chapter 6, the results showed that charge enhancement was effective in Kas-2 design. Besides, the first helix was ineffective in the sequence when interacting with bacterial cell membranes. Four designed cell-penetrating peptides with a special motif with tryptophan and arginine residues, displayed potent antimicrobial and anticancer activities but high haemolysis. These data may provide some ideas to improve the bioactivities of AMPs from Kassina species in further studies. Under the studies on the identification and bioactivity evaluation of this two novel peptides Kas-4 and Kas-2, from the skin secretion of two different African frogs, Kassina senegalensis and Kassina maculate, respectively, we found that the positions of two prolines had a great influence on the bioactivities of peptides from Kassina species rather than cationicity and hydrophobicity. Also, through rational design, activities and haemolysis of Kas-4 and Kas-2 were well improved. This study provided a fresh perspective on the creation of antimicrobial derivatives.

The continuing evolution of bacterial resistance against commercially available antibiotics is recognised as a global health threat, and the discovery of novel antimicrobials is urgently required. Antimicrobial peptides (AMPs) are emerging as important players in the fight against antibiotic resistance. In parallel, the field of microfluidics is maturing, and its benefits are being exploited in applications related to biomimicry and standardised testing. Developing a fundamental understanding of the modes of action of AMPs against membrane models is critical for developing these compounds into novel therapeutics. Throughout my studies, my efforts have been focused on developing microfluidic platforms that can streamline the evaluation of membrane active peptides in a systematic and standardised manner. As a result, I present the "GUV Studio", a bespoke multilayer microfluidic platform to quantify membranolytic efficacy and characterise the mode of action of AMPs. The platform is a biomimetic vesicle-based screening assay, which integrates an element for the high-throughput generation of Giant Unilamellar Vesicles (GUVs) in physiological salt concentrations on demand. Thousands of GUVs are individually immobilised downstream in hydrodynamic traps connected to separate perfusion inlets that facilitate the total fluid exchange of the solutions surrounding the vesicles, and enable the controlled, continuous administration of peptides for 8 different experiments in parallel. Membranolytic activity is expressed as a function of the time needed for an encapsulated dye to leak out of individual GUVs as a result of membrane permeabilisation or lysis. The platform has been used to study 20 native and de novo synthesised peptides at different concentrations. The results generated provide novel insights into the activity of a range of membrane active peptides, and demonstrate the capability of the lab-on-a-chip system to differentiate various modes of action defined by the response of the vesicle population to the peptides. My platform provides a quantitative, high-resolution tool to investigate the activity of any membrane-active compounds in a controlled, highly parallelised and high-throughput manner.

The transport of ions and small molecules across membranes is a ubiquitous process in biological systems. Effective studies of membrane transport phenomena require suitable model membrane systems, efficient methods to quantify transport kinetics and a robust theoretical framework to derive constitutive transport parameters. This thesis advances all of these aspects of transport measurements through a combination of experimentation and modelling. First, Octanol-assisted Liposome Assembly (OLA) - an existing microfluidic Giant Unilamellar lipid Vesicle (GUV) formation method - is refined by the integration with a novel Pinched Flow Fractionation (PFF) purification module. PFF removes formationassociated residues at a range of length-scales, from surfactant molecules to oil droplets, with high efficiency, leading to more homogeneous model systems for transport studies. The application of PFF to soft systems like GUVs reveals that PFF separates particles based on deformability and not merely on size as previously assumed. Integrating OLA and PFF allows for controlled fusion between oppositely charged vesicles on a single microfluidic device, enabling the sequential assembly of advanced membrane models directly on-chip. Next, we develop computational image processing algorithms to quantify transport in fluorescence experiments with single-vesicle resolution. We apply these techniques to the analysis of GUVs whose membranes are disrupted by membrano-lytic peptides, determining distributions of peptide-induced membrane disruption rates. By modelling the shapes of the extracted distributions, different modes of action by different peptides are revealed, providing insight into how these peptides function as membrane-permeabilizing agents. Finally, we combine microfluidic manipulation and DNA nanotechnology to develop a novel technique for the quantification of potassium ion transport at the single vesicle level. By theoretically modelling ion transfer across model cell membranes, we can quantify ion transport parameters, including potassium ion permeability and ionic selectivities. We apply this technique to the study of different membrane models, including lipid vesicles formed using different methodologies and membranes with embedded prototypical ion channels, building further complexity in our biomimetic system. These experiments demonstrate the versatility of our method for the quantification of transport in membranes with different compositions and physical properties.

The aim of this research was to identify a novel bioactive peptide from the skin secretion of Rana pipiens, evaluate the bioactivities of the its synthetic replicate and explore the action mechanism following the identification of its full length of the prepropeptide-encoding cDNA by molecular cloning. Afterwards, novel peptide QUB-2607 was identified and then a series of modified peptides were designed to improve its bioactivity. Furthermore, the relevance between the Rana box motif of the brevinin-1 subfamily peptides and their bioactivity were also explored. With the worldwide emergency of super-resistant bacteria, antimicrobial peptides (AMPs) have been considered antibiotic candidates because of their broad-spectrum antimicrobial activity, low risk of causing drug-resistance and strong antibacterial activity. Furthermore, skin and soft tissue infections (SSTIs) have been growing continuously, and Methicillin-resistant Staphylococcus aureus-infected (MRSA) SSTIs occupied over half of the cases. In Chapter 3, a novel peptide, QUB-2607, was isolated from the skin secretion of Rana pipiens. The sequence of QUB-2607 was translated by molecular cloning. After alignment of QUB-2607 with other peptides recorded in the antimicrobial peptide database (APD), QUB-2607 was identified as belonging to the brevinin-1 family. Solid phase peptide synthesis (SPPS) was used to synthesise QUB-2607, and the synthesised peptide was identified and purified by Matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry (MS) and reverse phase-high performance liquid chromatography (RP-HPLC). A series of assays were performed to evaluate the bioactivity and mechanism of QUB-2607 and the results showed that QUB-2607 exhibited broad-spectrum antimicrobial activity, being especially potent against Gram-positive bacteria. However, the haemolytic activity of QUB-2607 was also strong. Furthermore, QUB-2607 could penetrate the cell membrane of bacteria according to the action mechanism assay results. Therefore, the aim of Chapter 4 was modification of QUB-2607 to optimise its antimicrobial and haemolytic activity. A series of peptide analogues were designed by substitution of amino acids and hybridisation with another peptide, GG3. The bioactivity assay results showed that QUB-3456 exhibited broad-spectrum antimicrobial activity and weak haemolytic activity, whereas it also had a synergistic effect with conventional antibiotics and anti-infection activity in vivo. It seems that QUB-3456 combined the advantages of QUB-2607 and GG3. In the course of searching the APD database, brevinin-1Pk was discovered, and its sequence was quite similar to that of QUB-2607 and its antibacterial activity was also broad-spectrum and especially strong against Gram-positive bacteria. Apart from this, the haemolytic activity of brevinin-1Pk was weaker than that of QUB-2607. Therefore, a series of modified peptides were designed by hybridisation of brevinin-1Pk with other chosen Rana boxes. Subsequently, the antimicrobial activities of these modified peptides were all enhanced, particularly against MRSA and the anti-biofilm actions of these peptides were dramatically increased. However, their haemolytic activities were also stronger than brevinin-1Pk, except for b-1Pk2. Among all the peptides, b-1Pk4, b-1Pk6 and QUB-G exhibited excellent antimicrobial activity, and then these peptides were evaluated for their drug potential further. These three peptides had synergistic effects with conventional antibiotics and were stable in a physiological salt environment. However, the in vivo anti-infection assay identified the cytotoxicity of these peptides which would negatively influence their clinical research. The next research step would be the optimisation of the haemolytic activity of these peptides.

Antimicrobial peptides (AMPs), vital components of innate immune systems in many organisms against invasion by pathogenic microorganisms, could be considered as promising antimicrobial agents. Palustrin-2 peptides were found to exhibit broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria and have low haemolytic activity. Therefore, GL-29, a truncated form of palustrine-2ISb, was used as a template in this study and in an attempt to investigate its structure-activity relationships and try to find a peptide with higher antimicrobial potency and good selectivity through modification. In the third chapter, GL-29 as well as the truncated analogue GL-22, were studied to identify the function of the "Rana box", which was confirmed to have no impact on antimicrobial activity, but the deletion of this motif notably decreased the haemolytic activity and antiproliferative activity both against human cancer cells and normal cells. In chapter 4, five short truncates of GL-22, including the N-terminal short analogue GL-9 and C-terminal short derivatives LF-10, FV-9, VN-8, FV-8 were designed and they all exhibited nearly no antimicrobial activities. In the fifth chapter, the structure-activity relationships were studied by designing eight short analogues of GL-9, named from GL-9-a to GL-9-h with different characteristics. And the result showed that for the short N-terminal derivatives, higher hydrophobicity was crucial for the antimicrobial activity and the position of the hydrophobic residue is of great importance, however, the improvement of charge was useless for these short compounds. In the sixth Chapter, 9 analogues of GL-9-h were synthesized and the results exhibited that the enhancement of Trp could further increase the antimicrobial activities but led to serious haemolysis further increase the antimicrobial potency and the introduction of the motif "RRR" at the C-terminal end could further enhance the antimicrobial activity against Gram-negative bacteria and biofilm, accompanying with an attenuation of haemolysis and cytotoxicity against HaCAT. Overall, the structure-activity study illustrated that the deletion of "Rana box" in the Palustrine peptide GL-29 could decrease the haemolysis and maintain the antimicrobial activities at the same time, and the study of structure-activity relationship elucidated that hydrophobicity was the most important characteristic for short peptide to maintain the antimicrobial potency and the introduction of the motif "RRR" with Trp together could expand the antimicrobial potency against Gram-negative bacteria of short analogues.

Antimicrobial peptides (AMPs) have been considered potential alternatives to traditional antibiotics. To discover more AMPs that can be developed into clinical drugs, Brevinin-2GUb, Temporin-SHf and QUB-1841, extracted from the skin secretion of Hylarana guentheri, Pelophylax saharica and Pelophylax kl. esculentus, respectively, were selected as the main subjects of the project. Some rational designs were made on these peptides based on diverse principles and the assays of identification, structure characterisation and bioactivities were conducted on them and their derivatives. The Rana box has little influence on the antimicrobial activity of Brevinin-2GUb and its core structure is the 1st to 19th residues at the N-terminal region. The introduction of Arginine at the N-terminal end of Temporin-SHf can facilitate the peptide to penetrate the bacterial membrane, while the branched analogues cannot enhance its bioactivities possibly due to the overlapping of the high percentage of hydrophobic residues. The introduction of D-amino acids can reduce the haemolysis activity of QUB-1841, but the analogues still have no effect on gram-negative bacteria. The replacements with Lysine can provide it with more potent and broad-spectrum antimicrobial activity and the haemolysis can be undermined as well. The optimal number of charges for QUB-1841 is three. The positions of the substitution are also critical and it is better to make modifications on the polar face. The three analogues, tB2U-6K, 3R-T-SHf and QUB-1841-3K, display potent and broad-spectrum antimicrobial activities in vitro and in vivo, high selectivity, low possibility to induce resistance and excellent stability, which makes them potential for clinical use.

Human demand for novel antimicrobials has increased due to the rapid development of antibiotic-resistant bacteria. Because of their remarkable antimicrobial capacity, antimicrobial peptides are at the forefront of new antibacterial drugs. In our research, we are working with the isolation and identification of small molecule peptides with potent bioactivity from frog skin secretions and performing functional experiments on them. In chapter 3, DRP-AC4, a novel dermaseptin family peptide was isolated and identified. DRP-AC4 possesses broad-spectrum antimicrobial activity and inhibits the proliferation of a wide range of cancer cells. In chapter 4, another novel peptide named Temporin-LTd was identified. Temporin-LTd is a linear cationic short alpha-helical peptide with strong antimicrobial activity against Gram-positive bacteria but weak activity against Gram-negative bacteria. In chapter 5, three analogues of DRP-AC4 were designed to employ the influence of cationicity, hydrophobicity and C-terminal amidation on bioactivity. Extended hydrophobic face exhibited higher haemolytic activity and anti-proliferative activity. cationicity-enhanced analogue exhibited higer antimicrobial activity without increasing haemolytic activity.finally, C-terminal amidation removed analogue lost most of its activity. In chapter 6, eight derivatives of Temporin-LTd template were designed and compared their helical content regarding their biological activity. The helical content of Temporin-LTd analogues is associated with their haemolytic and anti-cancer cell proliferative activity, but not associated with their antibacterial activity. Furthermore, we found that the cationic residue type could affect the activity and helical content of Temporin-LTd. Surprisingly, our addition of leucine over glycine at position 9 resulted in the loss of the antimicrobial activity of Temporin-LTd. Finally, we found that disruption of the C-terminal helical structure provides initial thoughts for the development of novel anti-fungal drugs.

Antimicrobial resistance is a growing threat that will have profound effects on global health and the economy. In order to avoid a worst-case scenario, there is a need to develop structurally distinct classes of antibiotics. Herein, the synthesis of cyclic lipopeptides based on the tridecaptins is reported. In particular, analogues where residues D-Trp5 and Phe9 were substituted for D-Cys and Cys respectively were synthesized and cross-linked with various benzylic linkers of various sizes. Ortho-xylyl, meta-xylyl and para-xylyl cross-linked Oct-TriA1 maintained strong activity against Escherichia coli and other ESKAPE pathogens (including carbapenem-resistant Acinetobacter baumannii). Interestingly, these analogues were resistant to enzymatic degradation in the presence of recently reported D-stereoselective peptidases. Secondly, brevicidine represents a promising candidate for further research and development and this ultimately relies on a synthetic route to acquire sufficient material. The first reported synthesis of brevicidine is reported within this thesis. Briefly, an on-resin approach was used to pre-form the sensitive macrocyclic ring with a modified Yamaguchi esterification. Subsequent Fmoc-SPPS and global cleavage yielded the desired peptide in impressively high yields. With a synthetic route in hand, early structure-activity relationship studies were commenced. Subtle alterations did not ablate the antimicrobial activity while theoretically stabilizing the peptide towards in vivo hydrolysis. Finally, attempts were made to synthesize various lipid tails to install on the depsipeptide globomycin. The natural and non-allo versions of linear globomycin were synthesized and tested using a recently developed fluorescence resonance energy transfer (FRET) assay developed by the Caffrey group.

Respiratory infections are of significant concern for individuals with chronic inflammatory lung diseases and there is an unmet need for novel antibiotics. Antimicrobial peptides possess antibiotic potential due to their antimicrobial and/or immunomodulatory properties. The project aim was to assess the therapeutic potential of several snake-derived AMPs and to evaluate the suitability of peptide delivery by nebulisation. In in vitro assays the peptides exhibited antimicrobial activity and/or anti-inflammatory effects with minimal cytotoxicity. We considered the impact of lung proteases on the peptides. Peptide incubation with CF sputum and protease inhibitors revealed the peptides were susceptible to proteolytic cleavage. Cleavage predictions highlighted neutrophil elastase (NE) as most likely responsible. All peptides were susceptible to cleavage by NE, and several peptides retained function following NE-incubation. Mass spectrometric analysis and cleavage predictions informed truncated derivative development, aiming to reduce production cost and potential immunogenicity. SnE1 and derivatives were tested as therapeutics for acute lung infection using a murine model of chronic lung disease. SnE1N treatment reduced lung bacterial load, whereas, SnE1 did not. SnE1 increased protein and IL6 concentration in the BAL fluid and reduced the number of immune cells, possibly due to cytotoxic effects, indicating increased inflammation; whereas, SnE1N did not. In nebulised delivery suitability testing, nebulisation did not alter peptide function. Sn1b and C2 were shown to be respirable in an adult spontaneously breathing model and all test peptides were found to be respirable in a mechanically ventilated breathing model. Assessment of nebulised droplet distributions suggested all test peptides would likely reach the lower airways. To conclude, AMPs remain an attractive candidate for antibiotic development. In this study, we found that SnE1N reduced the bacterial burden during acute lung infection in mice with chronic lung inflammation, warranting further investigation. Additionally, peptide delivery via nebulisation is likely feasible.

During pregnancy, the maternal immune system must adapt to allow for the growth of a semiallogenic fetus whilst maintaining the ability to respond to pathogens. Maternal immune system adaptions are imperfect. Pregnant women are more susceptible to infections and have a higher morbidity from infections than non-pregnant women. An inappropriate inflammatory response to the developing fetus can result in miscarriage or preterm birth. The maternal immune system is exposed to circulating cell-free fetal DNA (cff-DNA) in pregnancy, produced by the cell death of placental syncytiotrophoblasts. Circulating DNA is pro-inflammatory through various cellular pathways, including through intracellular toll-like receptor 9 (TLR9). Fetal DNA can elicit inflammation both in vivo, resulting in fetal resorption in mouse models, and in vitro in peripheral blood mononuclear cells (PBMCs) from pregnant women. As such, cff-DNA has potential to be involved in the pathogenesis of preterm birth. When compared to microbial DNA, it is more difficult for mammalian DNA (such as cff-DNA) to reach intracellular TLR9. Cathelidicin, a circulating host defence peptide, facilitates DNA entry to cells, and can enhance TLR9-mediated inflammation. It is not known whether cathelicidin can mediate inflammation in pregnancy. Indeed, little is known about microbial and cff-DNA sensing mechanisms in pregnancy. This thesis was based on the following hypotheses: (1) cff-DNA is pro-inflammatory in pregnancy; (2) cathelicidin modulates cff-DNA induced inflammatory response in pregnancy; and (3) the pathways of DNA-induced inflammation are altered in pregnancy. To investigate these hypotheses, I aimed to: (1) assess the pro-inflammatory potential of cff-DNA (2) explore the role of cathelicidin in cff-DNA-mediated inflammation in pregnancy, (3) to determine if PBMC responses to DNA-stimuli differ between pregnant and non-pregnant women. To assess the pro-inflammatory potential of cff-DNA, cff-DNA was generated using human placental explants. cff-DNA was not pro-inflammatory to PBMCs from pregnant women and in an in vivo model of preterm birth, injection of mouse placental DNA did not decrease time to delivery. These data suggested that cff-DNA and placental DNA alone are not proinflammatory, possibly due to the inability of cff-DNA to reach intra-cellular TLR9. When cathelicidin was added, cff-DNA successfully induced inflammation in the form of chemokine interferon-γ inducible protein ϭϬ ;CXCLϭϬͿ production from PBMCs from pregnant women. In a lipopolysaccharide (LPS) ʹ induced inflammatory preterm birth model, cathelicidin deficient mice had decreased rates of preterm birth compared to wildtype mice. Cathelicidin deficient mice also had significantly less circulating interleukin 6, suggesting that cathelicidin contributes to inflammation in LPS-induced inflammatory preterm birth. To examine whether pregnancy results in an altered inflammatory response to DNA-stimuli, the inflammatory responses of PBMCs from pregnant and non-pregnant women to CpG-ODN (a synthetic DNA TLR9 agonist) were compared. PBMCs from pregnant women produced a significantly smaller type 1 interferon-driven response (at protein level) and an overall lesser inflammatory response (at transcriptional level) compared to PBMCs from non-pregnant women. PBMCs from pregnant women also had a lower proportion of TLR9-positive plasmacytoid dendritic cells (pDCs), providing a possible mechanism for the reduced inflammatory response to DNA-stimuli in pregnancy. The placenta produces large amounts of progesterone in pregnancy, and progesterone can affect TLR9 responsiveness. Therefore, the ability for progesterone to alter TLR9 expression and function on PBMCs from non-pregnant women was explored. Progesterone pre-treatment significantly decreased the CXCL10 response of PBMCs from non-pregnant women to CpG-ODN, and decreased TLR9 expression in pDCs from non-pregnant women. Together, this suggested that DNA-mediated inflammation from PBMCs is altered during pregnancy, potentially due to progesterone. The altered inflammatory response to DNA-stimuli in pregnancy described in this thesis is a novel finding. It represents a mechanism that contributes to the altered immune response in pregnancy. This mechanism may be a protective physiological response of the maternal immune system against increasing cff-DNA levels during pregnancy. It may, however, also contribute to the known vulnerability of pregnant women to infections. Together, the findings of this thesis provide an avenue for the future investigation of the altered maternal immune system and of the ability of cathelicidin to modulate its response to DNA-stimuli.

Antimicrobial peptides (AMPs) and host defence peptides (HDPs) have been extensively investigated for their antimicrobial activity and evidence suggests that they can also modulate the host immune system. However, little is known about their effects on macrophages, a key cell population in inflammatory responses that mediates communication between innate and adaptive immunity. Macrophages are a pleiotropic cell population composed of different subsets that orchestrate diverse stages of inflammation with roles ranging from pro- to anti-inflammatory and thus, targets for immunomodulation in different diseases. Here, THP-1-derived M1- and M2-like cells (pro- and anti-inflammatory respectively) under stimulation of TLR4 by LPS were used as a model to investigate potential immunomodulatory effects of these peptides on macrophages. Human β-defensin 2 in the presence of the TLR4 agonist K12 LPS, up-regulated the release of the pro-inflammatory cytokines TNFα and IL-1β from M1- and M2-like macrophages. Conversely, epidermicin NI01 strongly up-regulated release of a number of pro-inflammatory cytokines in the presence and absence of K12 LPS in M1-like cells but only a subset of those in M2-like cells. Various inhibitors of pro-inflammatory cytokine release pathways allowed us to partially elucidate the mechanisms via which this happens. Regulation of macrophage function by these and other peptides may have clinical implications in a wide range of immune-related disorders, but there is much we do not yet understand about what role, if any, epidermicin may have in these processes. Human β-defensin 2-induced upregulation of TNFα and IL-1β from M2-like cells might be important in conditions driven by sustained M2 anti-inflammatory factors such as fibrosis.

Antimicrobial peptides (AMPs) are defined as any peptides and proteins that kill or inhibit the growth of microorganisms. They form integral parts of the innate immune systems of almost all living organisms, and are considered to be a potential new class of antibiotics, whose use could abate the current antimicrobial resistance crisis. Their sequential composition also suggests the possibility of tailoring activity, and as such a great interest has grown around the possibility of synthetically designed AMPs. Studies have indicated that nonpolar residues, such as tryptophan, and basic residues, such as arginine, are common in AMPs and important for their activity. However beyond this, little is known about the exact sequence-feature relationships of AMPs. In this thesis I explore two different datasets of AMPs to determine exact sequence-activity relationships, one a pre-existing set and the other a complete set of peptides. Examination of the pre-existing set indicated that sequence-activity relationships are different for different microorganisms, with the positioning of basic residues being shown to be important for activity against Gram-negative bacteria, and tryptophan content as important for activity against Grampositive bacteria, eukaryotic microorganisms and erythrocytes. To uncover more specific relationships, a complete set of peptides composed of all combinations of arginine and tryptophan for sequences, ranging in length from 1 to 7 residues, was screened for antimicrobial activity against S. aureus, P. aeruginosa and C. albicans, as well as haemolytic activity. Analysis of the resulting activity landscapes revealed an optimal ratio of 3:2 tryptophan to arginine residues for antimicrobial activity against all organisms, and an optimal ratio of 4:1 for haemolytic activity. Tryptophan clustering was also shown to be important for antimicrobial activity, and an even distribution of arginine residues throughout the sequence. Activity landscape analysis suggested that landscape roughness increased dramatically from single length subsets to the complete set. Aggregation analysis showed that whilst aggregation was common at high concentrations, it was much less prevalent at lower concentrations, suggesting aggregation was reversible. Membranebinding analysis highlighted significant correlations between antimicrobial activity and mass binding to anionic membranes, suggesting that the peptides in the complete set were membrane active. Scanning electron and atomic force imaging of bacteria treated with a subset of efficacious peptides exhibited an increase in morphological deformities, further indicated this. The findings outlined in this thesis will be of use in future work to direct synthetic AMP design.

Bacterial capsules are a virulence factor, especially in uropathogenic Escherichia coli (UPEC) that cause urinary tract infections (UTIs). The study of K1 capsule morphology and biosynthesis provide valuable information for the development of alternative treatments to problematic antibiotics. To resolve sub-diffraction limited details in the morphology of K1 bacterial capsule, direct stochastic optical reconstruction microscopy (dSTORM) with 50 nm lateral resolution was used to visualise the inhomogeneous thickness of the K1 bacterial capsules. The variation of capsule thicknesses (~49%) from polar and equatorial regions will affect bacterial adhesion and the infection of host cells. The investigation concluded the bimodal thickness was due to internal capsular biosynthesis in agreement with HPLC results. The heterogeneity of capsule thickness is independent of the bacterial curvature due to the dense grafting and strong polyelectrolyte properties of fully stretched K1capsular brushes. The steric stabilization due to the osmotic pressure of bacterial capsular brushes was confirmed using atomic force microscopy (AFM). The K1 biosynthesis of capsular polysaccharide was investigated via polysialyltransferase (NeuS) dynamics using a novel Halotag self-labelling method. The rapid dynamics of NeuS in capsular biosynthesis compared with a CPS mutant revealed a sub-diffusive regime with a 0.2-0.5 ms characteristic time for interaction using fluorescence correlation spectroscopy (FCS). The variation of NeuS interaction influences the polydispersity of capsular chains. Although densely grafted K1 capsular brushes fully cover the bacterial surface, positively charged amphiphilic peptides, G(IIKK)3I-NH2, could penetrate the thick layer of the bacterial capsule. This is similar to permeability in the nutrient consumption pathway. The inhomogeneous landscape of the bacterial surface mixture, which comprised proteins, lipid islets and other elements, is one of the factors. Thus, the K1 bacterial capsule is a permeable shield with a bimodal thickness that protects the bacterial surface from antimicrobial peptide treatment by introducing aggregation on its surface, through partial interaction with the bacterial membranes.

Antimicrobial resistance (AMR) is one of the greatest world-wide threats to human health and urgent action is needed to reduce the potentially devastating social and economic impacts that may result from continued spread of drug resistant infections. These actions include discovery of new antibiotics and community engagement in AMR. Natural product screening methods are arguably the most efficient way to identify novel antibiotics, but can be frustrated by the repeated discovery of known compounds, wasting valuable resources, effort and time. Thus, exploiting obscure, hard to reach environments and implementing combined bioinformatic and proteomic screening approaches may short-cut the process of discovery of unique agents. In addition, educating and engaging the public will raise their awareness about AMR, which could reduce misuse of antibiotics. In this project, a total of 633 bacterial isolates were obtained from various clinical and environmental samples, including those provided by members of the general public. These isolates were screened for antimicrobial production using various antagonism assays. Draft genomes of 26 selected antagonistic strains were determined and mined in-silico using BAGEL-3 and antiSMASH-4.0 tools. Based on the findings, six isolates were further analysed using various proteomic strategies. The spectrum of activity, physicochemical properties and/or toxicity of purified compounds were investigated. This has led to the discovery of multiple novel compounds, including nisin-P, EU4-B synthetic peptide and EU4-natural product, which displayed diverse activity spectra towards pathogenic microorganisms. Aside from these results, the engagement of the general public, many of whom had limited prior awareness of AMR, is another important outcome. The optimized proteogenomic analysis has provided a means to shortcut some of the more laborious dereplication methods and has facilitated rapid identification of novel antimicrobial agents. In addition, this work has provided further evidence indicating that bacteria from the deep sea could be a rich source of novel compounds. Further investigation of all of the compounds discovered here may provide evidence justifying pre-clinical progression for future use against clinically relevant, drug resistant bacteria.

In recent years, advances in computer architecture and lipid force field parameters have made Molecular Dynamics (MD) a powerful tool for gaining atomistic resolution of biological membranes on timescales that other tools simply cannot explore. With many key biological processes involving membranes occurring on the nanosecond timescale, MD allows us to probe the dynamics and energetics of these interactions in molecular detail. Specifically, we can observe the interactions taking place as a peptide or protein comes into contact with a lipid bilayer, and how this may shape or alter the bilayer either locally (changes in headgroup orientation, lipid fluidity) or in bulk (lipid demixing, membrane curvature). The resolution achieved through atomistic MD can be directly compared with other tools such as NMR and EPR to gain a full perspective of how these biological systems behave over different timescales. As my background is in computational physics, this thesis not only looks into broadening our understanding of various interactions with biological membranes, but also into the development of construction and analytical software to assist in my research and benefit others in the field. One aspect of biological membranes that could vastly benefit from MD simulations is that of antimicrobial peptides (AMPs). These peptides primarily target and destroy microbes by permeabilising the cell membrane through a variety of proposed mechanisms, where each mechanism relies on the AMP to adopt specific conformations upon contact with bacterial membranes. In this thesis, I present an investigation into the interactions between a synthetic AMP and an inhibitor peptide designed to regulate antimicrobial activity through the formation of a coiled coil structure, which restricts the AMP from adopting new conformations. Simulations captured the spontaneous formation of coiled coils between these peptides, and specific residues in their sequences were identified that promote unfolding. This knowledge may lead to better design of coiled coil forming peptides. Another aspect of biological membranes that can be explored with MD is the interactions between model bacterial membranes and amphipathic helices, such as the MinD membrane targeting sequence (MinD-MTS). This 11-residue helix is responsible for anchoring the MinD protein to the inner membrane of Bacillus subtilis and plays a crucial role in bacterial cell division. MinD is known to exhibit sensitivity to transmembrane potentials (TMVs), whereby its localisation and binding affinity to bacterial membranes are disrupted upon removal of the TMV. Simulations revealed rapid insertions of MinD-MTS peptides into the headgroup region of a model bacterial membrane. Analytical software was constructed to measure the membrane properties of the lipids surrounding inserted MinDMTS peptides, which revealed splayed lipid tails and suggests the MinD-MTS may be capable of inducing membrane curvature. Additional simulations were conducted to investigate the influence of a TMV on model bacterial membranes, where software was constructed to measure changes in membrane properties. An analysis of these simulations suggests that a TMV is capable of lowering the transition temperature of a model bacterial membrane by a few degrees, yielding increased fluidity in the lipids and increased perturbations on the membrane surface. Finally, another aspect of biological membranes that can be explored through MD is that of electroporation. This induction of transient water pores in cell membrane provides an exciting aspect for drug delivery applications into cells, whereby electric fields are applied to cells to increase the uptake of therapeutic drugs. Simulations of membranes with high voltage TMVs were conducted that sought to investigate the implications of electroporation across a variety of bilayer compositions at different temperatures. Software was constructed to measure changes in membrane and system properties, which revealed that pore formation occurred at the same threshold voltage for different bilayer compositions in the fluid phase (~1.9 V) and a higher voltage for DPPC bilayers in the gel phase (~2.4 V). The TMV was found to be highly dependent on the area per lipid (APL), implying that bilayers with bulkier lipids or those transitioning from gel to fluid will experience smaller TMVs and fewer pore formations. These simulations also revealed lipid flip-flopping through pores, where charged lipids tended to translocate in the direction of the electric field to produce an asymmetrically charged bilayer. Finally, simulations utilising charged peptides with membranes yielded electroporation effects, whereby the charged peptides generate an identical TMV to those produced by an ion imbalance of equal magnitude. This suggests that charged peptides, such as AMPs, may be capable of permeabilising cell membranes through electroporation mechanisms.

Poultry are vital to food security, with 60 billion chickens reared worldwide per annum and demand fast accelerating. For many years antibiotic growth promoters have been used to promote energy retention from the diet and control intestinal bacterial growth. Antibiotic use for prophylaxis or growth-promotion in farmed animals is prohibited under EU Directives due to human health concerns, but a pressing need exists to maintain the efficiency of animal production by finding alternatives. Antimicrobial peptides (AMPs), part of the innate immune system exist naturally in most species and could provide a vast array of potential therapeutics. Microbial resistance to AMPs is unlikely due to their relatively unspecific mode of action, their ability to target multiple sites within a cell and diverse immune-modulatory activities. The avian egg provides antimicrobial protection through many mechanisms including AMPs which are incorporated into the egg white by the hen. The ovodefensin family and ‘transiently expressed in neural precursors’ (TENP) have been identified as potential novel antimicrobials in egg white and therefore formed the basis of the peptide portfolio of this study. TENP was first identified as having a role in neurological development but has since been shown to be an important egg component constituting ~0.1-0.5% of the total protein. TENP is conserved across avian species being found in chicken, turkey, duck and zebra finch. Its homology with the bacterial permeability-increasing family of innate immune genes suggests it may contribute to antimicrobial function in the egg. This study confirmed that expression of TENP is confined to the albumen forming region of the oviduct in adult hens and is under gonadal steroid control, typical of an oviduct and egg specific gene. The ovodefensin family are β defensin related antimicrobial peptides thought to be restricted to the albumen producing region of the avian oviduct. This study identified twenty five novel ovodefensin members through genome analysis, expanding the ovodefensin family to include reptiles for the first time. Phylogenetic analysis showed a unique example of the evolution of a cysteine spacing motif alongside traditional sequence evolution. The expression of eight ovodefensins was shown to be oviduct specific supporting the hypothesis that ovodefensins evolved to protect the egg. Antimicrobial activity for three ovodefensins from chicken and duck was investigated against gram negative organisms E. coli and Salmonella including pathogenic strains as well as a gram positive organism, S. aureus, for the first time. The spectrum of activity varied greatly between peptides suggesting a link between structure and function. Inclusion of recombinant ovodefensin peptides in the feed of chickens showed beneficial effects on the gut microbiome, metabolite profile and most crucially an increase in mean body weight. This demonstrates the potential of antimicrobial peptides as alternatives to antibiotic growth promoters in poultry.

Cathelicidins are antimicrobial peptides (AMPs) that were first discovered to have microbicidal properties but more recently to be multifunctional immunomodulators and thus important in influencing host defence against infectious disease. Whilst roles in various organs have been demonstrated, their expression patterns in health and disease in other organs are less clear and their key immunomodulatory functions remain undefined, particularly with regard to the balance of immunomodulatory properties and microbicidal activity in their ability to promote defence against infection. I therefore set out to describe LL-37 expression (human cathelicidin) in the female reproductive tract (across the menstrual cycle) and in the lung (during specific lung diseases), to define the effects on the function of airway epithelial cells during bacterial infection and to evaluate the key in vivo roles of endogenous cathelicidin (using a knockout mouse model) as well as the effect of therapeutic administration of LL-37 in a pulmonary Pseudomonas aeruginosa infection model. I demonstrated that cathelicidin protein and transcription shows a cyclical pattern of expression in female reproductive tissues which is maintained at high levels in decidua. LL- 37 protein was also detected in hTERT endometrial epithelial cells but despite the suggestion that cathelicidin may be regulated by steroid hormones there was no direct effect of progesterone on transcription. LL-37 is barely detected in healthy airways however is well known to increase during infection or inflammation. I observed that sputum from patients with bronchiectasis showed a correlation between the level of LL-37, TNF, MPO and chronic colonisation of Pseudomonas aeruginosa. Patients with lung cancer expressed much less LL- 37 than the bronchiectasis patients but there was a trend towards increased production postsurgery compared to pre-surgery. LL-37 was previously shown by our lab to selectively promote BAX and caspase-dependant death of infected epithelial cells. I went on to show that this appears to be a partially caspase- 1 dependent mechanism and that human bronchial epithelial (HBE) cells and A549 cell lines both express several of the components required to form inflammasomes, a caspase-1 dependant form of inflammatory cell death. Finally, I showed using murine models that cathelicidin enhances bacterial clearance during pulmonary infection in vivo, a response which is defective in mice lacking endogenous cathelicidin and that administration of exogenous, synthetic LL-37 at the time of infection can promote an early protective neutrophil influx in the absence of endogenous cathelicidin production.

Antimicrobial peptides (AMPs) are small proteins produced by epithelial surfaces, which have broad-spectrum antimicrobial and immunomodulatory activities. In the lung, skin and alimentary tract AMPs are known to be important in infectious and inflammatory conditions. Far less is known regarding the role of AMPs within the female reproductive tract, but as infection and inflammation are causes of preterm labour, AMPs may have a key function in maintain and protecting pregnancy. The major groups of human AMPs include the human beta defensins (HBDs), two antileukoproteinases (secretory leukocyte protease inhibitor (SLPI) and Trappin-2/Elafin), and the human cathelicidin hCAP18/LL-37, with several studies identifying their presence at sites throughout the reproductive tract. The cervix in pregnancy is positioned between the upper genital tract containing the developing fetus and the lower tract where infections usually arise. I hypothesise that AMPs are fundamental to mucosal immune defence of the cervix in pregnancy, preventing ascending infection and excessive inflammation that can cause preterm labour. This thesis focused on the human cathelicidin hCAP18/LL-37 and its role within the cervix and vagina. The aims of this thesis were to; investigate the inflammatory effects of LL-37 from cervical and vaginal derived epithelial cells and determine the pathways and receptors in which LL-37 may elicit its effects and how production may be regulated; investigate the role of CRAMP in a mouse model of preterm birth; and determine the production of AMPs by the pregnant cervix whilst investigating the relationship between AMP concentrations in cervicovaginal secretions and preterm labour. The inflammatory effect of LL-37 was investigated using cell lines derived from endocervical, ectocervical and vaginal epithelium. The study of these cell lines suggests divergent responses of cervical and vaginal epithelial cells. LL-37 mediated induction of IL-8 and IL-6 production from endocervical epithelial cells was observed in a dose-dependent and time-dependent manner, whilst ectocervical and vaginal cells also respond to treatment with LL-37 through IL-8 and IL-6 production. To determine a possible mechanism of action of LL-37 on IL-8 and IL-6 in the three cell lines, inhibitors against MAPK cascades, ERK, p38 MAPK and JNK, and known LL-37 receptors were investigated. In endocervical cells LL-37 mediated IL-8 occurs via activation of unidentified GPCRs, whilst in ectocervical cells this effect on IL‐8 and IL-6 is via the activation of ERK and p38 MAPK cascades. The mechanism by which LL-37 induces IL-8 secretion in vaginal epithelial cells remains unknown. Expression of LL-37 was shown to be mediated by vitamin D3 in vitro in cervical and vaginal epithelial cells. However when this relationship was investigated in vivo, using matched serum and cervicovaginal secretions from woman at early pregnancy, no correlation was observed between circulating vitamin D and cervicovaginal or circulating hCAP18/LL-37. However, the majority of women in this study reported with insufficient levels of vitamin D, which may effect the relationship observed with hCAP18/LL-37. Using a mouse model of LPS-induced preterm labour, to mimic the presence of intrauterine infection bacterial infection, I aimed to characterise the role of CRAMP, the mouse orthologue of hCAP18/LL-37, in the lower inflammatory and immune response that results in preterm labour. Wild type C57Bl/6J mice receiving an intrauterine injection of LPS deliver prematurely, within 24 hours of injection. However mice deficient in CRAMP (Camp -/-) receiving an intrauterine injection of LPS deliver significantly later and have a non-significant increase in pup survival compared to wild type C57Bl/6J mice. Cervical tissue collected post partum showed no difference in inflammatory markers between wild type C57Bl/6J and Camp -/- mice, however there was increased expression of the neutrophil chemoattractant marker, Cxcl5, and the neutrophil marker, Ngp in Camp -/- mice. In the lower genital tract, levels of antimicrobial peptides were determined in samples of cervicovaginal secretions collected from pregnant women. AMPs, hCAP18/LL-37, HBD-2 and SLPI were found in cervicovaginal secretions, and levels of hCAP18/LL-37 were increased in women with the common vaginal infection bacterial vaginosis. However no relationship was identified between the concentration of AMPs and preterm birth in this study. This work has shown that the lower genital tract, where infections that are associated with preterm labour originate, expresses the human cathelicidin hCAP18/LL-37. It may play an important role in modulating the immune response to invading infection associated with preterm labour. Further investigation of these responses may increase understanding of the physiology and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.

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.

Probiotics have been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The beneficial effects of probiotics in the gut are well described and roles including immunomodulation and colonisation resistance have been documented. Recent reports suggest that topical use of probiotic bacteria may be an effective strategy to promote skin health or inhibit disease. Therefore, in this thesis the potential of probiotics to protect skin from pathogenic bacteria was assessed using primary keratinocytes as a model system, and the skin pathogen, Staphylococcus aureus. The ability of three probiotics, L. reuteri ATCC 55730, L. rhamnosus AC413 and L. salivarius UCC118 to inhibit the growth of S. aureus was tested using well-diffusion assays and spot on the lawn assays. All three probiotics inhibited the growth of S. aureus in well-diffusion assays, though this property was dependent on growth medium. Inhibition of S. aureus growth was principally via the production of organic acids rather than bacteriocin production. Next, to determine whether probiotics could protect keratinocytes, confluent normal human epidermal keratinocytes (NHEK) were infected with S. aureus (106 CFU/ml) in the presence or absence of the probiotic (108 CFU/ml). NHEK viability was measured using trypan blue exclusion assays. L. reuteri had a significant protective effect on NHEK when applied 1h prior to (P=0.0003), or simultaneously with S. aureus (P=0.002). L. reuteri did not however protect NHEK when applied 1h after S. aureus addition. There was no change in the number of viable S. aureus in cell culture assays. To determine whether the protective effect was due to the inhibition of adhesion, NHEK were either pre-exposed to the probiotic for 1h, simultaneously exposed to the probiotic and S. aureus for 1h, or exposed to the probiotic 30 minutes after S. aureus addition for 1h. Pre-exposure of NHEK to L. reuteri (exclusion) and simultaneous exposure to L. reuteri and S. aureus (competition) resulted in significantly less staphylococci adhering to NHEK (P=0.03 and P=0.008 respectively). However when L. reuteri was added after S. aureus (displacement), the number of adherent staphylococci was not reduced. The necessity of S. aureus adherence for the inactivation of NHEK was demonstrated using a α5β1 integrin blocking antibody. Finally, to compare the innate response of NHEK to probiotics with S. capitis and S. aureus, TLR-2, antimicrobial peptide (AMP) expression and IL-8 production were measured. TLR-2 protein (but not mRNA) expression was reduced in the presence of S. aureus (P=0.018). NHEK pre-exposed to S. capitis prior to S. aureus infection however, exhibited elevated TLR-2 protein and mRNA expression (P<0.0001 and P=0.009 respectively). NHEK pre-exposed to L. reuteri prior to S. aureus had no significant change in TLR-2 expression compared to untreated controls. ELISAs demonstrated that IL-8 production was significantly increased in NHEK pre-exposed to L. reuteri prior to S. aureus infection (P=0.0001). In conclusion, L. reuteri protected NHEK from the toxic effects of S. aureus at least partly through competitive exclusion of binding sites on NHEK. Finally, NHEK innate responses to probiotic bacteria were akin to those to the skin commensal, S. capitis. L. reuteri induced expression of a neutrophil chemoattractant, suggesting it could be of importance in priming the innate immune response against S. aureus infections. Taken together, these results suggest that probiotic bacteria could be used prophylactically within skin creams and soaps to prevent S. aureus colonisation and infection in skin.

The emergence of multidrug resistant strains of bacteria has resulted in the need for novel therapeutic agents. The ApoEdp peptide, derived from the receptor-binding region of the human apolipoprotein E, had previously been shown to have activity against herpes simplex viruses, human immunodeficiency virus and certain bacterial species. However, its antibacterial mode of action was not elucidated, therefore the present study aimed to determine this mechanism. The susceptibility of several different strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium smegmatis, Staphylococcus epidermidis and Escherichia coli, to ApoEdp was investigated. No significant difference was observed between the minimal inhibitory concentrations (MICs) of ApoEdp against a range of Gram positive and Gram negative bacteria. The presence of E. coli K5 capsular polysaccharide in the growth medium led to a decrease in ApoEdp susceptibility of the non-capsulated E. coli MS101 DeltakfiC strain. Bacteria with non-functioning multidrug efflux pumps showed no difference in susceptibility. A mutation in the phoP gene of Salmonella enterica Serovar Typhimurium LT2, which regulates cell surface modifications led to an increase in ApoEdp susceptibility. Transmission electron microscopy (TEM) images showed changes in the membrane and internal structures of strains incubated with a minimal bactericidal concentration (MBC) of ApoEdp for 5 min. ApoEdp was able to depolarise the cytoplasmic membrane. The ability of ApoEdp to induce cell lysis was assessed by the release of β-galactosidase into the supernatant. There was no significant difference in the supernatant β-galactosidase levels of ApoEdp treated and unlysed cells. ApoEdp, however was able to form pores in artificial lipid bilayers and decrease intracellular ATP levels. The effect of ApoEdp on transcription and translation was determined using an in vitro transcription/translation system. Results showed that ApoEdp did not affect protein synthesis. ApoEdp also worked in synergy with rifampicin, chloramphenicol, ampicillin and ciprofloxacin against bacteria. Overall, the results showed that ApoEdp acts by targeting the cytoplasmic membrane, although it may also have intracellular targets. Its ability to work in combination with conventional antibiotics and antibacterial activity against a range of different bacteria species demonstrates its therapeutic potential.

A range of simulations have been conducted to investigate the behaviour of a diverse set of complex biological membrane systems. The processes of interest have required simulations over extended time and length scales, but without sacrifice of molecular detail. For this reason, the primary technique used has been coarse-grained molecular dynamics (CG MD) simulations, in which small groups of atoms are combined into lower-resolution CG particles. The increased computational efficiency of this technique has allowed simulations with time scales of microseconds, and length scales of hundreds of nm. The membrane-permeabilizing action of the antimicrobial peptide maculatin 1.1 was investigated. This short α-helical peptide is thought to kill bacteria by permeabilizing the plasma membrane, but the exact mechanism has not been confirmed. Multiscale (CG and atomistic) simulations show that maculatin can insert into membranes to form disordered, water-permeable aggregates, while CG simulations of large numbers of peptides resulted in substantial deformation of lipid vesicles. The simulations imply that both pore-forming and lytic mechanisms are available to maculatin 1.1, and that the predominance of either depends on conditions such as peptide concentration and membrane composition. A generalized study of membrane protein aggregation was conducted via CG simulations of lipid bilayers containing multiple copies of model transmembrane proteins: either α-helical bundles or β-barrels. By varying the lipid tail length and the membrane type (planar bilayer or spherical vesicle), the simulations display protein aggregation ranging from negligible to extensive; they show how this biologically important process is modulated by hydrophobic mismatch, membrane curvature, and the structural class or orientation of the protein. The association of influenza hemagglutinin (HA) with putative lipid rafts was investigated by simulating aggregates of HA in a domain-forming membrane. The CG MD study addressed an important limitation of model membrane experiments by investigating the influence of high local protein concentration on membrane phase behaviour. The simulations showed attenuated diffusion of unsaturated lipids within HA aggregates, leading to spontaneous accumulation of raft-type lipids (saturated lipids and cholesterol). A CG model of the entire influenza viral envelope was constructed in realistic dimensions, comprising the three types of viral envelope protein (HA, neuraminidase and M2) inserted into a large lipid vesicle. The study represents one of the largest near-atomistic simulations of a biological membrane to date. It shows how the high concentration of proteins found in the viral envelope can attenuate formation of lipid domains, which may help to explain why lipid rafts do not form on large scales in vivo.

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.

Deep learning has become widely used in biological sciences. More specifically, the development of protein deep learning models has leveraged the evergrowing collection of biological data to learn the patterns that govern protein biochemistry. Here, we focus on the assessment of different protein deep learning models to better understand each of their capabilities, benefits and drawbacks. Our work aims to provide insights for future protein engineering efforts and for the discovery of protein homologs. In Chapter 2 we assessed a structure-based protein ML model in its ability to make biochemically meaningful predictions and tested weather or not the model can predict specific allowed amino acids in a protein. We compared the performance of models trained on different input sizes and correlated model predictions with natural variation in order to better understand how these models learn protein structure and biochemistry. In Chapter 3, we compared the predictions of two structure models and two language models to determine if different protein representations affect what information each model type learns and their performance. Finally, in Chapter 4, we apply a sequence-based protein model to searching for antibacterial microcin peptides in bacterial genomes.

Antimicrobial resistance is a growing threat to global health. One pathway to confer antimicrobial resistance is through the overexpression of multidrug (MDR) efflux pumps, making them important to study. Antimicrobial peptides (AMPs) are ideal candidates to explore for synergistic effects with antibiotics to revive the efficacy of antibiotics which are subject to resistance. This dissertation work aims to understand the mechanism the MDR efflux pump, AcrAB, that confers antimicrobial resistance to E. coli, and create a method to express the AMPs, Moringa oleifera chitin-binding protein (MOCBP) and Moringa oleifera coagulant protein (MOCP), in E. coli for future study. In this dissertation, we determined the transport rates of protons and substrates through the proton driven pump, AcrAB. We used two optical spectroscopic techniques, stopped flow and fluorescence correlation spectroscopy, in concert to estimate average transport through AcrAB incorporated into lipid vesicles. This research not only addresses the urgent public health concern of antimicrobial resistance but also pioneers a photonically enabled in vitro assay, opening new possibilities for the study and development of proton-coupled transporters and their inhibitors. We also developed a novel expression system in E. coli for MOCP and MOCBP and optimized the purification methods. MOCP and MOCBP are antimicrobial peptides found in Moringa oliefera seed protein extract. Both platforms were shown to be scalable, allowing for future studies of and alterations to their properties and functions. This has the potential for future applications in food safety, water purification, and cosmetic products.

Urinary tract infections (UTIs), including cystitis and pyelonephritis, are one of the most common infections across species. Approximately 80% of these infections are caused by uropathogenic Escherichia coli (UPEC). The host’s innate immune response is paramount in the defense against UPEC and is initiated by urinary tract epithelial cells with subsequent recruitment of leukocytes. Despite a robust immune response, UPEC can persist in the urinary tract, can cause recurrent infections, and can be resistant to antibiotic therapy. There is a growing and imminent risk for antibiotic resistant UTIs, thus necessitating investigations into alternative therapeutic options. One possibility is utilizing mechanisms to enhance existing host innate immune defenses; however, our understanding of innate immunity against UPEC, especially in the kidney, is limited. In this work, we aimed to characterize the host immune response to UPEC in cell culture and in various important mouse models of UTI to identify potential targets for augmenting host defense against UPEC.The first portion of this thesis investigates the in vitro pathogenesis of UPEC in one of the host’s most important UTI renal defenders, the kidney collecting duct intercalated cell. Previous work from our lab and others have shown that intercalated cells are required for protection against UPEC due to direct binding to these cells, their role in immune cell recruitment, and secretion of bactericidal antimicrobial peptides. We found that upon UPEC infection, intercalated cells activate multiple innate immune pathways including those associated with pattern recognition receptor (PRR) signaling. We identified that nucleotide oligomerization domain (NOD) 2 and Toll-like receptor (TLR) 4 are two intercalated cell PRRs that, upon initial activation, can protect against subsequent UPEC infection. Additionally, NOD2 activation induces upregulation of multiple innate immune pathway genes along with genes associated with various antimicrobial peptides, cytokines and chemokines. This work expanded on UPEC pathogenesis in intercalated cells and discovered a novel intercalated cell PRR, NOD2, utilized in host defense against UPEC.The second portion of this research focuses on the in vivo pathogenesis of UPEC and histopathologic characterization of three commonly used mouse models of cystitis and pyelonephritis, the C57BL/6J, C3H/HeN, and C3H/HeOuJ strains. There have been previous studies acknowledging the bacterial burden differences between mouse strains but in-depth assessments correlating urinary tract organ pathology and host innate immune responses to bacterial burden are limited. Additionally, current UTI histopathologic scoring systems are subjective and may not adequately reflect true observable pathologic differences. We developed an updated scoring system, identified stark differences in strain susceptibility to UPEC UTI, and correlated bacterial burden to organ pathology and urinary biomarker expression. Upon UPEC infection, C57BL/6J mice had rapid clearance of urine bacterial burden and cystitis without pyelonephritis. In contrast, the C3H/HeN and C3H/HeOuJ had more persistent bacterial burden but have differential susceptibility to cystitis and pyelonephritis. The C3H/HeOuJ mouse had delayed but persistent cystitis and severe pyelonephritis while the C3H/HeN mouse did not demonstrate significant pyelonephritis. Finally, urine bacterial burden and histopathology differences between strains corresponded with varying expression of urinary pro-inflammatory proteins, such as interleukin (IL)-1β, IL-6, KC/GRO (CXC ligand 1), and tissue necrosis factor (TNF)-α expression. This research demonstrates the utility of a novel UTI scoring system and highlights key strain specific bacterial burden, pathologic, and immune response differences in UPEC pathogenesis, which can impact in vivo studies aimed at treating UTIs. Lastly, given the observable protective effects of NOD2 signaling in vitro, we assessed impact of NOD2 loss in vivo and in cell culture. Upon infection with UPEC, NOD2-/- C57BL/6J mice exhibited increased urinary burden and bladder pathology with no significant differences in pyelonephritis susceptibility but had rare formations of renal urothelial intracellular bacterial communities. Unexpectedly, loss of NOD2 or disruption of NOD2 signaling in vitro did not impact UPEC attachment and invasion in intercalated cells. This work demonstrates that although NOD2 is protective, it may not be required for intercalated cell defense. In addition, given the observed pathology differences in the lower urinary tract NOD2 may have a role elsewhere in the urinary tract. In summary, this thesis addresses, in part, the complex pathogenesis of UTIs and innate immune response in both in vitro and in vivo modeling systems. We have identified a novel intercalated cell innate immune signaling pathway, NOD2, that can be activated during UPEC UTIs. We have also highlighted the spectrum of mouse model pathology during UTI and developed a novel histopathologic scoring system which can be utilized for thorough assessment of pre-clinical therapeutic efficacy.

Although the exact cause of Alzheimer’s disease (AD) is still unknown, it is widely considered that the accumulation of amyloid plaques composed of the amyloid-β (Aβ) peptide in the brain is linked to neurodegeneration. Co-localization of viral DNA with Aβ plaques, the association of brain infection and AD, and research indicating the protective effect of Aβ against bacteria and fungi in mice and human cells have led to the hypothesis that Aβ expression and deposition may be central to its function as an antimicrobial peptide (AMP). In my thesis research, we seek to elucidate how Aβ functions as an AMP by investigating its toxicity toward bacteria, particularly the role aggregation plays in its antimicrobial activity. A computational study was also undertaken to acquire a better understanding of Aβ aggregates' interactions with neuronal and bacterial-mimicking lipid membranes. Later, a molecular dynamics (MD) simulation was performed to examine the effect of methionine (Met35) oxidation on the destabilization of Aβ aggregates, which can potentially lead to its clearance. This research also focused on evaluating and modulating conjugated polyelectrolytes' antibacterial efficacy in solution, as a disinfectant spray, and in hydrogel platforms. In summary, this study revealed the antimicrobial effect of conjugated polyelectrolytes and Aβ aggregates.

Antibiotic tolerance is the prolonged survival of a bacterial population in the presence of antimicrobials. Tolerance can impact infection outcome and promote the evolution of outright resistance. Opportunistic pathogens, like Enterobacter cloacae, present a danger to immunocompromised individuals and have been identified by the CDC as an urgent threat to human health. Here, using E. cloacae as a model, we study mechanisms of 1) tolerance to cell wall-acting antibiotics and 2) resistance to killing by an antimicrobial peptide (AMP). We observed that E. cloacae displays meropenem (a β-lactam antibiotic) tolerance by forming cell wall-deficient spheroplasts, which resume normal growth after removal of the antibiotic. Presumably, spheroplasts rely on their outer membrane (OM) for structural integrity maintenance. In Chapter 2, we identified the PhoPQ signaling system as a key driver of tolerance. PhoPQ is activated upon exposure to meropenem, promoting OM modifications to enhance OM strength. PhoPQ is essential for optimal meropenem tolerance development, as a deletion mutant exhibits drastically reduced survival in the presence of drug. Further, we identified an inhibitor of PhoPQ which significantly reduces E. cloacae tolerance to meropenem. Interestingly, the OM modifications that promote tolerance are the same that promote resistance against antimicrobial peptides (AMPs). These short, naturally occurring peptides display potent antimicrobial activity across a wide range of pathogens, often through interactions with bacterial membranes. It is vital to understand mechanisms by which bacteria resist the effects of AMPs as they may become a viable infection treatment strategy. In Chapter 3, we investigated E. cloacae resistance mechanisms against cecropin, an insect AMP of therapeutic interest. E. cloacae cecropin resistance is heterogeneous within a population and highly dependent on initial cell density. Surviving cells are susceptible to subsequent doses of cecropin, suggesting unstable, stochastic resistance (i.e., heteroresistance). Heteroresistance collectively relies on outer membrane modifications, potential capsule production, and membrane-bound protease activity.This work reveals mechanisms by which an opportunistic pathogen survives treatment with cell-envelope targeting antimicrobials. These insights lay the groundwork for the development of novel treatment strategies to combat the looming threat of multidrug resistant infections.

Metamorphosis is a radical morphological and environmental transition from a larval phase to an adult. This process is utilized by ~80% of all animal species and is a particularly common life history strategy among marine invertebrates. It has been well established that across species developmentally competent marine larvae utilize bacterial cues to indicate a suitable habitat conducive to adult survival and reproduction. Despite the high incidence of bacterially induced metamorphosis, the underlying mechanisms of how animals directly sense these signals remain under-studied, especially in Echinoderms (e.g., sea urchins). This study aims to a investigate component of the innate immune system, antimicrobial peptides (AMPs), as a potential candidate for connecting bacterial presence to metamorphosis. To explore AMPs as a player in bacterially induced metamorphosis, I utilized the rapid and synchronously developing sea urchin Lytechnius pictus. I first identified a homolog of the Echinoderm specific AMP Strongylocin 2 in L. pictus. I found that LpStrongylocin 2 (LpS2l) is only transcribed in early larvae in the event of bacterial infection. In nascent late-stage larvae, LpS2l transcription is upregulated and accumulates within cells in developing adult structures, likely a subtype of blastocoelar cells. The spatiotemporal regulation of LpS2l transcription in L. pictus larvae suggests a role for AMPs in the development of new structures and recognizing metamorphosis-inducing bacterial cues. Future experiments will be aimed at assessing functional role of LpS2l in metamorphosis by disrupting transcription using CRISPR/Cas9 sgRNA guides designed in this study.

Orthopaedic devices and biomaterials have significantly improved the livelihood of patients suffering from debilitating health conditions and have offered healthcare institutions and governments the tools to intervene for better health outcomes. However, like with many other life-saving technologies, there are challenges hindering the optimal effectiveness of orthopaedic biomaterials, key amongst which is bacterial infections. These infections often result in multiple surgeries and implant failure. Traditional therapeutic strategies that have relied on antibiotics are being challenged by the rapid evolution and prevalence of antibiotic resistant bacteria. Therefore, exploring alternative strategies to kill bacteria and reduce medical devices associated infections is the need of the hour. Traditional antibiotics are susceptible to bacterial resistance primarily because of their mechanisms of action which involve identifying and binding to specific targets within bacteria. Alternatives to traditional antibiotics such as antimicrobial peptides and their mimics, quaternary ammonium compounds, antibacterial hydrogels, metal ions, etc., have emerged as potent antibacterial agents, some of which are less susceptible to bacterial resistance due to their non-specific modes of action. In the context of biomedical devices and implants, the attachment strategies employed for tethering antimicrobial compounds to their surfaces play crucial roles in determining the magnitude of antibacterial activity observed on the device surfaces and in the surrounding region. Plasma Immersion Ion Implantation and Deposition (PIIID) has emerged as an exciting single-step process for attaching molecules to material surfaces. It is relatively easy to perform and successfully generates stable coatings for materials. In this, study, we have explored two small-molecule antimicrobial peptidomimetics, an antimicrobial polymer, two antimicrobial peptides, a quaternary ammonium polymer, and an vi antimicrobial hydrogel as coatings and fillings for hydroxyapatite (HA) and polyether ether ketone (PEEK) surfaces. We have explored two strategies for attachment of these compounds to the materials, physical attachment via weak interactions and covalent attachment via PIIID treatment. Chapters 2 and 3 describe the attachment and properties of the peptidomimetics-based coatings for HA surfaces while chapter 4 details the properties of the polymer-based coatings for the same. Chapter 5 deals with the two antimicrobial peptides and chapter 6 talks about the quaternary ammonium polymer-based coating and the antimicrobial hydrogel-based filling for HA discs. Finally, chapter 7 describes the properties of peptidomimetics and polymer-coated PEEK sheets. Overall, several non-traditional strategies for generating antibacterial coatings for materials of relevance in orthopaedics were investigated. All the compounds demonstrated excellent antibacterial activity and were successful in inhibiting the attachment of bacteria (S. aureus (peptidomimetics, peptides, quaternary ammonium polymer and hydrogel), E. coli (polymer, peptides, and hydrogels) and P. aeruginosa (peptides)) to the material surfaces and in the surrounding solution. The covalently coated surfaces could retain their activity over a prolonged period of time while the physically coated surfaces exhibited significant activity immediately after the coating. These compounds and attachment strategies represent exciting alternatives to traditional therapeutic strategies.

Healthcare-associated infections are the most common adverse event affecting hospital patients. With the emergence of antibiotic-resistant bacterial strains, there is an increasing number of untreatable infections. Therefore, there is a clear need for the development of novel antibacterial strategies to both prevent, and treat, healthcare-associated infections. This thesis explores three key approaches to treat and prevent the transmission of healthcare-associated infections. First of all, a library of novel antibacterial compounds were assessed in vitro for their clinical potential. The compounds with the greatest clinical potential were then tested in various biomaterial applications (biocompatibility in an animal model, and their activity when covalently attached to biomaterial surfaces). Finally, this thesis explores antibacterial strategies to prevent the transmission of bacteria from clinical surfaces. The lead antimicrobial candidates, melimine and RK758, are peptidomimetics that address the drawbacks of conventional antibiotic therapy. They showed broad-spectrum antibacterial activity (including clinical antibiotic-resistant isolates), anti-biofilm activity and thermal stability. When peptidomimetics were used in combination with traditional antibiotics, bacteria were unable to develop resistance towards antibiotics or peptidomimetics. This combination antibiotic-peptidomimetic therapy also showed synergistic activity, which further increases the potential for these compounds to treat multidrug-resistant infections. When assessed in two clinical applications, peptidomimetics showed promise for their continued clinical development. Both melimine and RK758 were biocompatible in animal models. When peptidomimetics were loaded into bone allograft, there was no inhibition of bone healing in a critical-sized distal femur defect model. Moreover, when these peptidomimetics were covalently attached using a new polydopamine coating method to biomaterial surfaces, they retained their broad-spectrum antibacterial activity and prevented biofilm formation in Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. This thesis also explores the transmission of pathogens from clinical surfaces, and how ultraviolet-C disinfection could be used in healthcare-facilities to reduce the bacterial bioburden on various clinical surfaces. Overall, adjunct ultraviolet disinfection significantly reduced the bioburden on surfaces in operating theatres and clinical rooms. Overall, these three strategies work together to prevent healthcare-associated infections. Preventing the transmission of pathogens from the environment to patients, is known to reduce healthcare-associated infection rates. Furthermore, the development and application of novel-acting antibiotics offers a solution to the antibiotic-resistance crisis and addresses the urgent need for antimicrobials with broad-spectrum antibacterial activity. If commercialised, the strategies proposed in this thesis have the potential to increase activity of ineffective antibiotics and prolong their use in the clinic.

Surgical site infections and chronic wounds, especially those caused by antibiotic-resistant microorganisms, result in hospitalization and fatalities each year. Methods to prevent these infections, such as cleaning and preparing medical tools, have had minimal success in preventing infections. Further, antibiotic treatments have become less successful in treating infections and wounds because of antibiotic-resistant bacteria. Antimicrobial peptides (AMP) are a possible treatment solution for wound infections. AMPs are oligopeptides that occur in nature or can be synthesized in vitro which possess a broad spectrum of antimicrobial activity against bacteria and other harmful microorganisms. AMPs operate by disrupting the packing arrangements of biomembranes in prokaryotes through their insertion into negatively charged phospholipid bilayers. However, many AMP products fail clinical trials because of their difficulty to be encapsulated and delivered at high concentrations in an active form. This project proposed the use of bicontinuous microemulsions (BMEs) as a possible system to encapsulate and deliver AMPs. BMEs are thermodynamically stable mixtures consisting of a surfactant, oil, aqueous mixture, and sometimes a cosurfactant. They are optically clear and the surfactant(s) in BMEs solubilize nearly equal amounts of oil and water creating elongated nanodomains. AMPs are typically cationic, and the following hypothesis was tested: BMEs created with anionic surfactants would induce a more highly folded, hence more biologically active, conformation for melittin. Several different BME systems composed of biocompatible oils were identified and evaluated for their ability to encapsulate melittin, a model AMP, and to test the system’s antimicrobial activity. Small-angle x-ray scattering showed melittin effected the BMEs quasi-periodic repeat distance and correlation lengths. Circular dichroism data showed a higher percentage of melittin was in its active form when encapsulated in a BME compared to an aqueous solution. Fluorescence measurements showed melittin resided within the surfactant monolayers of the BMEs. Antimicrobial diffusion assays proved that there was a larger zone of inhibition against bacteria commonly found in surgical site infections and chronic wounds than the BMEs without melittin. This research was successful in adding an AMP into BMEs created with biocompatible materials and may be a viable option in combating the rise in antibiotic-resistant organisms.

Infections and disease caused by the obligate human pathogen Bordetella pertussis (Bp)are resurging worldwide, despite widespread vaccinations. The current acellular pertussis vaccinesremain ineffective against nasopharyngeal colonization, carriage, and transmission. To developmore effective vaccines, it is important to understand how Bp resists killing actions of hostimmunity. In the current work, we tested the hypothesis that Bordetella polysaccharide (Bps), amember of the poly--1,6-N-acetyl-D-glucosamine (PNAG) family, promotes respiratory tractcolonization of Bp by resisting killing from two major innate immune factors: (1) antimicrobialpeptides (AMPs) and (2) neutrophils.First, we show that Bps enhanced Bp survival in the presence of AMPs by functioning bothas a surface shield and a decoy. While surface-bound Bps inhibited AMP binding to the bacterialsurface, soluble Bps neutralized AMP activity by direct binding interactions. Simultaneousinfection of mice with Bps-proficient and Bps-deficient strains resulted in greater survival of theBps-deficient strain in the mouse respiratory tract. Furthermore, production of Bps in a nonpathogenicE. coli strain increased AMP resistance in vitro, and increased bacterial survival andaugmented pathology in the mouse respiratory tract.Second, we show that Bps enhanced survival in the presence of hydrogen peroxide andhypochlorous acid in vitro. Consistent with these data, Bps enhanced resistance to killing byhuman neutrophils and suppressed oxidative burst and neutrophil extracellular trap (NET)formation. Together, the results obtained from studies involving AMPs and neutrophils serve as afoundation for studying other PNAG polysaccharides, as well as for the development of aneffective Bp vaccine that includes Bps.Traditionally, pertussis is described as an acute disease with coughing fits and other severesymptoms. However, in recent years, many individuals who contract pertussis are eitherasymptomatic or show very mild symptoms. We hypothesize that biofilms, a community mode ofexistence, prolong the human carriage of Bp, thereby contributing to continued bacterial survivaland transmission. While factors that drive biofilm formation on abiotic surfaces have been wellcharacterized,biofilm architecture and matrix composition in humans are ill-defined. Therefore,we utilized a well-differentiated primary human airway epithelial culture system grown at airliquidinterface. We show that these airway cells promoted robust growth of cilia-adherent,structured Bp biofilms. Bacterial cells that attached to cilia were encased in an extracellular matrix(ECM) consisting of the polysaccharide Bps and extracellular DNA (eDNA). Distinct complexes(mono-, bi-, and tripartite) between the ECM and bacterial cells were visualized. Mutant strainsthat fail to produce the polysaccharide Bps or the protein filamentous hemagglutinin (FHA) weredefective in attaching to cilia and in biofilm formation compared to the WT strain. Contrary toprevious reports of abiotic biofilms, adenylate cyclase toxin (ACT) was required for mature Bpbiofilm formation on human airway epithelial cells. This highlights the importance of utilizingsystems resembling the natural infection of a microbial pathogen to gain insights regarding theirpathogenesis. Altogether, the studies conducted in this thesis provide new insights into how Bphas evolved to survive in the mammalian respiratory tract.

Ticks acquire a bloodmeal from vertebrate hosts, ranging from mammals to reptiles, and are regularly interacting with a variety of microbes via the bloodmeal host or environment. I set out to characterize ways in which ticks interact with a diverse set of organisms, spanning biological kingdoms.One focus of my work was venomous saliva from the tick Ixodes scapularis and its influence on multiple processes that are required for successful blood feeding from a variety of vertebrate hosts. Some of these processes include immune evasion through interference of the host’s defense system and protecting the tick from harmful microbes. I examined the activity of defensins, a family of antimicrobial peptides, found in tick salivary venom. Studies of tick saliva revealed that defensins are secreted into mice and protect ticks against microbes commonly found on the skin of their host. However, several observations point to a role for defensins that is independent of tick–microbe interactions. Through a collaborative effort, I identified a second, intriguing function where these defensins function as mast cell activators through an interaction with a G protein-coupled receptor, MRGPRX2. When ticks feed on mice missing an MRGPRX2 ortholog, they have a decrease in weight, suggesting that activation of this receptor benefits ticks during the feeding process. Another area I explored is the complicated interactions between Ixodes ticks and Borrelia burgdorferi, the causative agent of Lyme disease. In order for ticks transmit B. burgdorferi, the bacteria must migrate from the tick’s midgut to the salivary glands. This migration process is poorly understood. My exploratory research summarized here suggests that this necessary migration of B. burgdorferi is a passive process facilitated by ticks expelling water during blood feeding. Another arm of exploratory research validated previous findings that serum from a common vertebrate host for I. pacificus, the western fence lizard Sceloporus occidentalis, is bactericidal for B. burgdorferi. Both of my exploratory projects are suitable starting points for new research projects which seeks to better characterize cross-kingdom interactions of Ixodes ticks. Together, my studies detailed in this dissertation provide a greater understanding of how ticks survive their varied interaction with a diverse group of organisms.

Within the mammalian intestine, various microbes utilize flagellar motility to traverse the mucus layer and reach the epithelium, challenging the spatial segregation established by the host. For many of these microbes, contacting the epithelium to adhere or invade can provide them an advantage over other members of the highly competitive resident microbiota. These actions threaten homeostasis and can result in pathological consequences, which presents a direct risk to the health of the host. In response, the host has a collection of innate and adaptive immune countermeasures that target and inhibit flagellar motility, thereby preserving the spatial segregation of microbes from epithelium. These methods include pattern recognition receptors capable of responding to bacterial flagella, secreted innate effector molecules that inhibit microbial swimming through a variety of mechanisms, and flagella-specific mucosal antibody that can regulate the movement and behavior of flagellated microbes. Together, these host factors possess a redundancy in function that provides the intestinal epithelium with an overlapping defense against motile microbes. This work elucidates that human α-defensin 6 (HD6), an antimicrobial peptide secreted by Paneth cells in the small intestine, is capable of inhibiting bacterial flagellar motility. HD6 was previously described to protect the host from infection with Salmonella enterica serovar Typhimurium (S. Typhimurium) by limiting its invasion and dissemination into peripheral tissues through non-lethal means. It was found that HD6 could bind to bacterial flagellin and self-assemble into large polymers, termed nanonets, which resulted in bacterial agglutination and inhibition of cellular invasion. However, it remained unknown if HD6 could also have an effect on individual bacteria through agglutination-independent mechanisms. Due to the ability of HD6 to bind flagellin, the major component of the flagellar filament, we hypothesized that HD6 could inhibit the motility of individual flagellated bacteria through a process of binding and self-assembly. Initially, we employed a semi-solid agar bacterial motility assay to demonstrate that the motility of S. Typhimurium was hindered after treatment with HD6. The degree of this inhibition was comparable to treatment with α-flagellin IgG, a known inhibitor of flagellar motility. Next, we explored how HD6 affected the motility of individual bacteria by developing a live-cell fluorescence microscopy assay, capable of resolving the movement of single motile bacteria. Through this method, we showed that the population of actively motile S. Typhimurium was significantly reduced after treatment with HD6 at bacterial densities where agglutination does not occur and α-flagellin IgG can no longer inhibit motility. We also found that the loss of bacterial motility caused by HD6 coincided with the appearance of immobilized bacteria, which were distinct from actively motile and diffusing bacteria due to an absence of perceptible movement. Using an advanced segmentation and masking strategy, we found the degree of bacterial immobilization was dependent on HD6 concentration. Finally, using a single amino acid variant of HD6, HD6F2A, which retains the ability to bind flagellin but cannot self-assemble, we showed that the bacterial immobilization was dependent on HD6's ability to self-assemble. Together, these results suggest a specialized role of HD6 in targeting and inhibiting bacterial motility. To interrogate the effect of flagellar arrangement and post-translational glycosylation HD6-mediated motility inhibition, future studies can utilize other motile bacteria, such as Pseudomonas aeruginosa. Additionally, outlined within are different mouse models that aim to explore role of HD6 in modulating the commensal microbiota and the adaptive immune system. In summary, this work expands our understanding of the effects of HD6 on flagellated bacteria, and more broadly provides evidence supporting a specific role of the host immune system in targeting bacterial motility.

Rising to dangerously high levels all over the world, antibiotic resistance is now a critical global problem. To inhibit or kill bacteria, most antibiotics target the growth process of bacterial cells. Therefore, most antibiotics are ineffective for slowly growing bacteria with lower cellular influx. Current therapeutic approaches most commonly utilize broad-spectrum antibiotics and/or drug cocktails to target a wide range of bacteria. Unfortunately, broad-spectrum antibiotics can target both beneficial and pathogenic bacteria. This nonspecificity imparts significant pressure on bacterial species to evolve into resistant strains. In addition, beneficial bacteria do not always fully recover, increasing susceptibility to infections and diseases. It is not practical to develop antibiotics for all species of interest. To mitigate these issues, we must engineer new antibiotics in a drastically different way to prevent running out of effective therapies. Bacteria are capable of producing defensive molecules. These secreted multi-functional molecules inhibit other closely related strains competing for the same environmental niches. We were inspired by the defensive behavior of bacteria to engineer novel antibiotics featuring tunable functionalityThis tunability allows for an adjustable bandwidth of antimicrobial activity against multiple species within a specific set of environmental conditions. Moreover, we produced antibiotics able to inhibit pathogenic bacteria without harming host cells or beneficial commensal bacteria. We were interested in antimicrobial peptides (AMPs) as antibiotics, since they target generic features common to the outer membrane of many pathogenic species. These antibiotics allow us to regulate complex microbial communities by inhibiting or killing detrimental bacteria without harming host-associated microbial communities, that have beneficial impacts on human health. Additionally, these new antibiotics help beneficial bacteria win the competition over environmental niches. Therefore, the development of resistance is significantly inhibited compared to that of conventional antibiotics. To engineer new antimicrobials, we leveraged recently developed sequence design rules, using both positive charge and hydrophobicity as necessary conditions for antimicrobial activity. These hybrid antimicrobial molecules can be used for treating resistant infections and regulating species distribution in microbial communities. We engineered tunable antimicrobial peptides with a ‘threshold’ activity profile using pH-switchable charge. To tune the antimicrobial activity of α−helical AMPs, we modified functional groups on the side chain of specific basic amino acids (lysine and arginine). By incorporating electron withdrawing and/or hydrophobic compounds on the side chains of arginine and lysine residues, the pKa at which these residues become positively charged can be lowered. Therefore, by incorporating different masking groups in different locations, we adjust the activity of the antimicrobial peptides by turning them on at specific and tunable pH values.

Abstract: The race towards uncovering and developing novel antibiotics requires a multipronged, interdisciplinary approach. Highlighted in this thesis are biochemical and structural studies on peptides, small naturally occurring molecules that have innate bioactivities, such as antimicrobial characteristics. These include: faerocin MK, a potent anti-listerial molecule; teixobactin, a peptide with the ability to inhibit Gram-positive bacteria; tridecaptin A1, an inhibitor of Gram-negative bacteria; and microcin J25, a lasso peptide with activity against Gram-negative foodborne pathogens. Faerocin MK was uncovered after the genome sequencing of a newly discovered probiotic bacterium, Enterococcus faecium M3K31. The sequence encoding for faerocin MK contained two components: a structural gene and an immunity protein. Heterologous expression studies led to the production, isolation, and characterization of this new peptide. These studies showed that this peptide is active against Gram-positive organisms, with potent activity against Listeria spp., a genus of foodborne pathogens responsible for listeriosis. Teixobactin is a peptide isolated in 2015 and has gained much attention due to its potent activity against Gram-positive bacteria and unique structural features. With the use of analogues, the binding of teixobactin and its bacterial target, lipid II, a peptidoglycan precursor present within bacterial membranes, was investigated. Furthermore, the activity of teixobactin was expanded to reach Gram-negative organisms through synergistic assays with membrane-disrupting peptides. Tridecaptin A1 is part of a class of peptides which are potent against Gram-negative bacteria. They are known to bind to lipid II, the same peptidoglycan precursor targeted by teixobactin and a number of other antimicrobial peptides. In order to better understand the tridecaptin A1 mechanism of action, in particular its interaction with lipid II at the bacterial membrane surface, nuclear magnetic resonance experiments were employed. The structures of tridecaptin A1 and lipid II were studied in a new series of membrane-mimicking micelles. These micelle-forming dodecylphosphocholine compounds were synthesized, with heteroatom replacements at strategic positions, and structural data was compared amongst analogues. Microcin J25 is a lasso peptide from Escherichia coli active against Gram-negative, foodborne pathogens such as Salmonella species and E. coli. The unique structure of this class of peptides, wherein the C-terminal is threaded through an N-terminal macrocycle, confers on them desirable attributes such as thermal stability and protease resistance, along with potent innate activities against bacteria and viruses. The lasso shape has long been regarded as a valuable scaffold for pharmaceutical development, yet these peptides have thus far been accessed through biological means as a facile synthetic approach has not been developed. A method to synthesize this interlocked, stable structure was initiated, providing a potential facile means of accessing microcin J25 and other lasso peptides in the future.

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell-directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell-directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.

Salmonella and Campylobacter are leading bacterial agents both nationally and globally, making them high public health concerns. Both are significant important zoonotic pathogens commonly found in livestock. Poultry and poultry products are the most common sources of human infections. Infected poultry show little to no clinical signs, and risk the chance of entering the food system, potentially contaminating consumers. Direct links between the high load of bacteria in the chicken intestinal tract and the high contamination of poultry carcasses support the need for pre-harvest control. Previously, antibiotics were used to control bacterial infections and growth; but increasing occurrence of antibiotic resistant bacteria has caused laws and practices to shift. Targeting the control of foodborne pathogens in the pre-harvest stage can improve animal welfare and public health. Alternative treatment methods are needed to combat Salmonella and Campylobacter in production animals, improve antibiotic stewardship, and subsequently strengthen the economy.We discovered the antimicrobial efficacy of Lactobacillus acidophilus (LA), Lactobacillus rhamnosus GG (LGG), and Bifidobacterium animalis subsp. lactis (Bb12) in vitro. More importantly, we showed LGG significantly reduced Salmonella in the chicken cecum by 1.9 logs (PSalmonella serovars in an agar well diffusion assay. Characterization of antibacterial activity of LGG revealed that although organic acids are present, the most crucial part of inhibition are the heat and protease stable peptides that were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).After which, we tested the efficacy of Lactobacillus rhamnosus GG (LGG) derived small peptides (P1-NPSRQERR, P2- PDENK, and P4-MLNERVK) against Salmonella Typhimurium (ST) in chickens and examined their antibacterial effects against Salmonella Enteritidis (SE) and other Salmonella serovars in vitro. The antagonistic effects of peptides were heat and protease resistant and antibacterial activity is likely due to their effect on Salmonella membranes. Although, P1, P2, and P4 inhibited ST, SE, and other Salmonella serovars in vitro at 18mM, 15mM, and 18mM respectively, they did not affect the growth of Gram-positive commensal bacteria. P1and P2 at 50mg/kg successfully reduced the colonization of ST by 2.2 logs and 1.8 logs, respectively (P< 0.05), in chickens 7 days post infection. P1, P2, or P4 had no adverse effects on chicken cecum microbial communities, but birds not treated and challenged with Salmonella showed differences in microbial richness and evenness compared to the unchallenged birds. Confocal and transmission electron microscopy revealed that peptides attacked the membrane of Salmonella causing its flaccidity, sloughing, or disruption.Lastly, we analyzed the ability of Salmonella Typhimurium derived recombinant attenuated Salmonella vaccines (RASV) to reduce the colonization of C. jejuni in chickens. One-day-old Salmonellaand Campylobacter free chickens were orally immunized with RASV constructs (2x108 CFU/ bird), and a booster was given 10 days later. Birds were challenged with a cocktail of 5 C. jejuni strains on day 18. Colonization load was determined by enumerating C. jejuni in the cecum of the birds. Four trials were conducted analyzing the RASVs effect on Campylobacter load in the cecum. Our data suggested that vaccine efficacy was dependent on amount of challenge dose and antigens present in vaccine. RASV90+201 and RASV131 showed consistent reduction across trials 2,3, and 4. The best reduction in Campylobacter load occurred in trial 3 with 103 Campylobacter challenge dose. At 17 days post infection, pG8R-86 (cjaA), pG8R-90+201 (FlaA), and pG8R-131 (FlaA) low challenge dose groups yielded significant reductions in Campylobacter load (approximately 3.7, 2.4, and 1.7 logs, respectively, compared to vector control). Additionally, in multiple trials RASV-90 showed a significant induction of IgY antibody titers compared to the untreated and unchallenged vector control group. Overall, these studies revealed that antibiotic independent novel therapeutics and vaccines are promising alternatives to control Salmonella and Campylobacter in chickens.

Avian pathogenic E. coli (APEC), an extra-intestinal pathogenic E. coli (ExPEC), is one of the most common bacterial pathogens affecting poultry, including broilers, layers, breeders, turkeys and many other avian species. It causes high morbidity and mortality (up to 20%), decrease in production and increase in condemnation of carcasses (up to 43%) during slaughter, thus resulting in substantial economic losses to the poultry industry worldwide. Recent reports have suggested APEC as a source of human extra-intestinal infections, including urinary tract infections and sometimes meningitis. Further, APEC is also considered as a source of antibiotic resistance genes (ARGs) to human pathogens. Therefore, APEC is a pathogen of significant importance to both animal and human health. Currently, antibiotics are commonly used to control APEC infections; however, the increasing emergence of resistance to antibiotics and FDA (Food and Drug Administration) restrictions on using antibiotics in food-producing animals necessitate the development of new and effective antibacterials that can circumvent the resistance problem. Antibacterials targeting the outer membrane (OM) of bacteria can evade the problem of resistance in Gram-negative bacteria such as APEC. To this end, we discovered and evaluated small molecule (SM) growth inhibitors (GIs) and antimicrobial peptides (AMPs) affecting OM of APEC. We uncovered their antibacterial targets in the OM of APEC and assessed their impact on gut microbiome. We further demonstrated the potential of GIs as adjuvants to current antibiotics, including one of the last-resort antibiotics, colistin. A total of 11 GIs (GI1 – GI11) with bactericidal activity against APEC were identified through high throughput screening of pre-selected enriched small molecule library. Eight GIs that were effective and showed low toxicity in vitro in cultured epithelial and macrophage cells, red blood cells, and in vivo in wax moth (Galleria mellonella) larva model were selected for evaluation in chickens. GIs were evaluated by administering orally (1 mg/kg body wt.) in chickens followed by subcutaneous APEC challenge. Three GIs (GI-7>GI-10>GI-6) reduced the mortality (up to 71.42%), lesions severity (up to 62%), and APEC load (up to 2.6 logs) in chickens. Further, administration of GI-7, a most effective GI, in drinking water at optimal dose (60 mg/L), for seven days, also reduced the mortality (42%), APEC load (2.0 logs) and lesions severity (29.5%), which is comparable to currently used antibiotic sulfadimethoxine. The abundance of Lactobacillus was increased in gut of GI-7 treated chickens. The expression of OM lipopolysaccharide (LPS) transporter genes (lptD/E) were downregulated with low levels of LptE and formation of non-functional LptD intermediate in APEC treated with GI-7. in silico docking studies revealed that GI-7 interacts with Tyr244, Lys234 and Glu733 residues of LptD which are essential for LPS transport, thus likely interfering in LPS transport to OM. This was further confirmed by lower levels of LPS in the OM of GI-7 treated APEC. We investigated the potential of identified GIs (GI1 – GI11) to act as adjuvants to current antibiotics used in treating APEC infection in poultry (colistin, tetracycline and ciprofloxacin). Checkerboard assay followed by in vivo evaluation in wax moth larva model was performed. The combination of GIs (GI-2/SM-2 and GI-3/SM-3) synergistically reduced the minimum bactericidal concentration of colistin by at least 10-fold. In larvae, the GIs combination increased the efficacy of colistin by two-fold with enhanced (>50%) survival and reduced (>4 logs) APEC load. The impact of GIs combination on colistin resistance evolution was investigated by analyzing whole genome sequences of APEC isolates passaged with colistin alone or in combination with GIs. The combined administration of GIs decreased the frequency (5/6 to 1/6) of colistin resistance evolution. Previously unknown mutations in pmrB (L14Q, T92P) and pmrA (A80V), which are predicted deleterious, were identified in the colistin-resistant APEC isolates when passaged with colistin alone but not in combination with GIs. Further, the expression of pmrCAB and pmrH genes was downregulated in APEC isolates passaged with colistin in combination with GIs.In a separate study, we tested the anti-APEC activity of multiple commensal and probiotic bacteria in an agar gel diffusion assay and identified Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) producing strong zone of inhibition against APEC. In co-culture assay, LGG and Bb12 completely killed the APEC by 24 h. Further investigation revealed that antibacterial products secreted/released in culture supernatants were responsible for anti-APEC activity. The analysis of culture supernatants using LC-MS/MS identified multiple novel bioactive peptides (VQAAQAGDTKPIEV, AFDNTDTSLDSTFKSA, VTDTSGKAGTTKISNV, and AESSDTNLVNAKAA) in addition to lactic acid. The oral administration (108 CFU/chicken) of LGG, but not Bb12, significantly (PP1>P3) at 50 mg/kg and 100 mg/kg doses reduced the cecum colonization (0.5 to 1.3 logs) and translocation of APEC to internal organs of chickens. Cecal microbiota analysis revealed that two peptides (P1 and P2) decreased Enterobacteriaceae (Escherichia-Shigella) abundance. Confocal fluorescence and transmission electron microscopy revealed peptides affecting the APEC membrane integrity either by causing membrane shedding, rupturing or flaccidity. Further, gene expression analysis revealed that peptide treatment downregulated the expression of ompC (>13.0 folds), ompF (>11.3 folds) and mlaA (>4.9 folds) genes responsible for maintenance of OM lipid asymmetry in APEC. In immunoblot analysis performed using anti-OmpC and anti-MlaA polyclonal antibodies, peptides treatment decreased the levels of OmpC and MlaA proteins, suggesting OmpC/F-MlaA complex as the likely target of these peptides.Overall, these studies identified novel antibacterial agents that can be developed as anti-APEC therapeutics for poultry in the future. Further, small molecule adjuvants identified from this study can be developed as anti-evolution drugs that can slow down colistin resistance development and possibly other antibiotics. Moreover, validation of these druggable targets can help in future drug development against pathogens related to APEC such as human ExPECs and other Gram-negative bacteria.

The number of newly approved antimicrobial compounds has been steadily decreasing over the past 50 years emphasizing the need for novel antimicrobial substances. Besides, current antimicrobial therapies become increasingly ineffective due to the rapid spread of drug-resistant pathogens, and even the most potent small-molecule antibiotics may eventually fail to cure infections with highly resistant bacteria. Peptides are a promising class of potential drug substances, as they are still underexplored, offer high structural complexity, and at least partly cover the chemical space left open between biologics and small molecule drugs. Ribosomally produced antimicrobial peptides are of particular interest, as they already act as effective antimicrobials in the defense against invading pathogens of most organisms. Consequently, they are currently being investigated for potential use to fight infectious diseases in humans. The fact that antimicrobial peptides are produced using transcription and translation makes them particularly attractive as they can be produced recombinantly and their modification can be simply achieved by straightforward manipulation of the synthesis template at the DNA level. In this thesis, we developed a high-throughput method for the discovery of antimicrobial peptides which is based on the principle of self-screening: Different peptides are expressed in a recombinant Escherichia coli strain, and their effect on growth rate is recorded. In our case, this is done by next-generation sequencing of expression plasmids in the bacterial culture, enabling the recording of massive numbers of growth curves for single strains in a single flask. We termed this technology massively parallelized growth assays (Mex). We applied this method to discover novel candidates for antimicrobial peptides by screening a library of ~12’000 naturally occurring peptides with a length between 5 and 42 amino acids and diverse properties. Analysis of thousands of growth curves allowed us to identify more than 1,000 previously unknown antimicrobials. Additionally, by incorporating the kinetics of growth inhibition, we were able to obtain a first indication of the mode of action, with important implications for the ultimate usefulness of the peptide in question. We chemically synthesized the most promising peptides of the screen and determined their activity when applied externally. Notably, the results indicated that 10 out of 15 investigated peptides efficiently eradicated bacteria at a minimal inhibitory concentration in the upper nM / lower µM range. We think that this work represents a step-change in the high-throughput discovery of functionally diverse antimicrobial peptides. Next, we applied a simplified version of Mex to optimize a single antimicrobial peptide in high-throughput. As a model peptide, we optimized the 23 amino acid version of the well-researched and highly active antimicrobial peptide Bac71-23 using deep mutational scanning, consisting of a first random and then a semi-rational approach. The random library of ~600,000 different Bac71-23 variants allowed us to derive a fitness landscape of the peptide and to identify residues that are essential for growth inhibition and residues with potential for activity optimization. A smaller semi-rational library of ~160,000 Bac71 23 variants enabled us to extract the most beneficial amino acid combinations, thereby generating an antimicrobial peptide that, if synthesized chemically, is non-toxic and superior to Bac71-23 against a large panel of bacterial pathogens. We thus created a new-to-nature peptide lead with a great potential to be further developed in pre-clinical stages. To our best estimation, these novel methods exceed competing approaches in terms of throughput, hit-rate and sensitivity, while also offering an opportunity for the direct functional characterization of large libraries. These methods will accelerate the discovery and optimization of antimicrobials drastically, and may thus provide a path forward to master one of today’s most urgent challenges, the antimicrobial resistance crisis.

Cell-penetrating peptides containing arginine as positively charged residues and tryptophan or diphenylalanine as hydrophobic residues were synthesized. The synthesis was accomplished through the Fmoc solid-phase peptide synthesis in the presence of HBTU and DIPEA. The side-chain protected linear peptides were cleaved from the resin and cyclized in the presence of DIC and HOAt in the solution phase overnight. MALDI-TOF mass spectrometry was used to characterize the peptides. The cytotoxicity of the synthesized peptides was determined in CCRF-CEM (human, lymphoblast peripheral blood), and HEK-293 (human, embryonic epithelial kidney healthy) cells using the MTS assay. A concentration of 10 µM was found to have minimal cytotoxic effects. CCRF-CEM and SK-OV-3 (human, epithelial ovary adenocarcinoma) cells were employed to measure the cellular uptake of fluorescence-labeled phosphopeptide (F´-GpYEEI), stavudine, lamivudine, emtricitabine, and siRNA in the presence of peptides with flow cytometry. The cellular uptake studies showed that [DipR]5 significantly improved the uptake of F´-GpYEEI, by approximately 110-folds after 3 h incubation. Confocal microscopy demonstrated the improved delivery of F´-GpYEEI (6-folds), which was mostly localized in the cytosol, in the presence of [DipR]5 for MDA-MB-231 cells. To determine the mechanism of the cellular uptake, the physical mixture of F´-GpYEEI and [DipR]5 was used to measure the fluorescence in the treated CCRF-CEM cells and in a concentration- and time-dependent manner. A study was conducted in the presence of endocytosis inhibitors such as nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin suggesting that the uptake for [DipR]5 may not be endocytosis-dependent. Circular dichroism, dynamic light scattering, and transmission electron microscopy were utilized to determine the secondary structure, particle sizes, and morphology of peptides, respectively. The data indicates that [DipR]5 and Fluorescence-labeled [DipR]5 can act as a molecular transporter of several compounds including the cell-impermeable phosphopeptide and siRNA specifically. The antibacterial activities of peptides were evaluated against pathogens. The minimum inhibitory concentrations (MIC) of peptides were determined by micro-broth dilution protocol against Methicillin-Resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae, and Bacillus subtilis. [DipR]5 and((DipR)2(WR)3) showed promising MIC values of 0.39-6.25 µM against Gram-positive bacteria strains and 3.13-25 µM against Gram-negative bacteria strains.

Non-traditional antimicrobials have been an area of great interest due to the increasing prevalence of antimicrobial resistance (AMR) in bacteria. Antimicrobial peptides (AMPs) & ionic liquids (ILs) are two examples that have been investigated as a potential solution. Most AMPs are naturally derived & exhibit high selectivity against bacterial targets over host cells. The venom-derived peptide, ponericin L1 from Neoponera goeldii, was used to investigate the role of cationic residues & net charge on peptide activity. Using both in vitro & microbiological methods, L1 peptide & derivatives exhibited an alpha-helical conformation with enhanced binding to lipid vesicles containing anionic lipids & low hemolytic activity. Net charge & identity of the cationic groups on the peptide were shown to play a significant role in antimicrobial activity. ILs have also been investigated in combination with antimicrobial compounds to fight AMR. Imidazolium chloride-based ILs with differing alkyl tails & commercially available antibiotics were used to examine the potential for synergistic effects on multiple bacteria. Analysis of the IL data indicates that length of the alkyl chain plays a crucial role in antimicrobial activity & cytotoxicity. A synergistic effect was exhibited while showing low cytotoxicity when tested in a mammalian cell culture model.

Antimicrobial peptides (AMPs) are innate host defense peptides that protect against pathogenic microbes by neutralizing toxins or via a direct killing mechanism. AMPs are classified based on their physical properties such as charge, structure, and binding motifs. Here we investigated the antimicrobial and immune-modulating effects of the Regenerating Islet-Derived Protein 3 (REG3) family and LL-37 REG3 peptides are C-type lectins and have been demonstrated to have antimicrobial activity against Gram-positive bacteria by binding to sugars on the peptidoglycan membrane of these bacteria. A similar strategy is also employed by the lectin Surfactant Protein-D which has been shown to bind and neutralize Influenza A Virus (IAV). REG3 peptides were shown to be expressed in the lungs of mice infected with IAV. We observed reduction of IAV infected cells when IAV was pre-incubated with an Escherichia coliexpressed recombinant version of human REG3A peptide. This peptide also modified interaction of IAV with primary human neutrophils. However, these effects were lost when using a mammalian cell expressed recombinant REG3A. A second member of the REG3 family, REG3G, showed minimal inhibition of IAV infection. While the mechanism remains unclear, LL-37 has demonstrated killing activity against a spectrum of microbes including IAV. Previous work from our group identified the core domain of LL-37 responsible for IAV neutralization. In addition, our group showed that LL-37 modulates interaction of IAV with neutrophils. Here we tested three modified versions of LL-37 that retain the overall size and charge of LL-37, but with modifications in the core domain reducing hydrophobicity. We observed that these mutants retain IAV killing activity across multiple strains. In addition, these mutants retain the modulation of IAV induced neutrophil responses. We also found that the compounds sodium butyrate and Entinostat, which can upregulate endogenous expression of LL-37, have variable effects in IAV infection. We believe these findings will aid in the development of LL-37 derivatives to expand the repertoire of antimicrobials.

Approximately 80% of microbial infections including device and non-device-associated infection are biofilms mediated. Pseudomonas aeruginosa and Staphylococcus aureus frequently colonise medical devices and cause biofilm related infections. Antimicrobial peptides (AMPs), owing to the minimal possibility of development of bacterial resistance and broad-spectrum activity at low concentrations, can be used to prevent such infections. The present study investigates how two AMPs melimine and Mel4 interact with P. aeruginosa and S. aureus and prevent biofilm formation. AMPs were immobilized on glass using azido benzoic acid linker. Subsequent experiments examining membrane interactions and its impact on the leakage of cellular contents were performed and compared with free peptides. The mechanism of action of both free and immobilized peptides against P. aeruginosa was predominantly membrane disruption via depolarization and permeabilization. Disruption of cell membranes was associated with release of ATP and DNA/RNA. Like P. aeruginosa, depolarization of the cytoplasmic membrane of S. aureus occurred more slowly when the AMPs were bound to glass. Loss of membrane depolarization resulted in slower release of ATP. Similar to P. aeruginosa, free or immobilised melimine permeabilized the cytoplasmic membrane of S. aureus and released nucleic acid. Mel4 when either free or bound transiently permeabilized the cytoplasmic membrane of S. aureus, this effect was not associated with release of nucleic acids. The mechanism of action of free and immobilized Mel4 was involved in release of autolysins along with minimum effect on cell membrane of S. aureus. Both AMPs either in solution or surface immobilized followed similar sequence of events of membranes interaction against P. aeruginosa and S. aureus. However, membranes disruption kinetics was slower for immobilized peptides at their minimum inhibitory concentration but was faster at equivalent concentrations. Neither P. aeruginosa nor S. aureus could develop resistance to melimine and Mel4. Both AMPs inhibited biofilm formation and disrupted pre-formed biofilms of ciprofloxacin resistance and sensitive isolates of P. aeruginosa and S. aureus and showed enhanced antibiofilm effect in combination with ciprofloxacin. This study has provided a significant increase in the understanding of the mechanism of action of AMPs bound to surfaces and against biofilms of these two medically important bacteria.

Human host defense against mycobacteria depends on a functioning immune system. While it is currently established that Th1 cells are major players in host defense against mycobacteria, other cell types such as Th17 cells also correlate strongly with the protective forms of disease. However, the role of Th17 cells in the context of intracellular infection are incompletely understood. Recent work has shown that Th17 cells can secrete an antimicrobial protein IL-26, which can directly lyse extracellular bacteria. Given the established role of antimicrobial peptides against mycobacterial infection, we decided to further investigate IL-26 and determine whether it is antimicrobial against mycobacteria. We find that IL-26 can be secreted by PBMCs and purified T cells in response to IL-1β in the absence of T cell receptor activation. This process is also more rapid than TCR stimulation. Among helper T cells, we show that IL-1RI expression was necessary for this response and identified IL-1RI+ Th17 cells as a cell type that can secrete IL-26 in response to IL-1β. Furthermore, we establish that IL-26 secreted in response to IL-1β is functionally antimicrobial.We also examined whether IL-26 is antimicrobial against intracellular bacteria. We find that IL-26 is differentially expressed between clinical forms of leprosy, with higher expression in tuberculoid leprosy, the resistant form of the disease, as compared to lepromatous leprosy, the progressive form of the disease. Incubation of IL-26 with M. leprae and attenuated M. tuberculosis strain H37RA led to dose dependent killing, both with bacteria in culture and, importantly, while the bacteria resided within infected macrophages. Additionally, we find that IL-26 treatment of infected macrophages stimulates the autophagy response and enhances phagolysosome fusion in a STING dependent manner. Altogether, this work uncovers a novel mechanism by which Th17 cells contribute to defense against mycobacterial infection.

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics, which have a serious resistance crisis. We studied the mechanisms of three different types of AMPs using computational and experimental methods. First, computer simulations are performed to study the AMP microcin J25 (MJ25), a 21-mer peptide with an unusual lasso structure and high activity against Gram-negative bacteria. MJ25 has intracellular targets. The initial step for MJ25 acquisition in bacterial cells is binding to the outer membrane receptor FhuA. Molecular dynamics simulation is implemented to study the binding mechanism of MJ25 to FhuA and to search for important binding residues. The absolute binding free energy calculated from combined free energy perturbation (FEP) and thermodynamic integration (TI) methods agrees well with experimental data. In addition, computational mutation analysis reveals that the His5 is the key residue responsible for MJ25 and FhuA association. We find that the number of hydrogen bonds is essential for MJ25 binding to FhuA. This atomistic, quantitative insight sheds light on the mechanism of action of MJ25, and may pave a path for designing active MJ25 analogues. Second, protegrin-1 (PG-1) is a cationic arginine-rich AMP. It is widely accepted that PG-1 induces membrane disruption by forming pores that lead to cell death. However, the insertion mechanism for these highly cationic peptides into the hydrophobic membrane environment is still poorly understood at the molecular scale. It has previously been determined that the association of arginine guanidinium and lipid phosphate groups results in strong bidentate bonds that stabilize peptide-lipid complexes. It has also been suggested that arginine residues are able to drag phosphate groups as they insert inside the membrane to form a toroidal pore. However, whether bidentate bonds play a significant role in inducing a pore formation remains unclear. To investigate the role of bidentate complexes in PG-1 translocation, we conducted molecular dynamics simulations. Two computational electroporation methods were implemented to examine the translocation process. We found PG-1 could insert into the membrane provided the external electric potential is large enough to first induce a water column or pore within the lipid bilayer membrane. We also found that the highly charged PG-1 is capable in itself of inducing molecular electroporation. Substitution of arginines with charge-equivalent lysines showed a markedly reduced tendency for insertion. This indicates the guanidinium group likely facilitates PG-1 translocation. Potential of mean force calculations suggest that peptide insertion inside the hydrophobic environment of the membrane core is not favored. We found that formation of a water column or pore might be a prerequisite for PG-1 translocation. We also found PG-1 can stabilize the pore after insertion. We suggest that PG-1 could be a pore inducer and stabilizer. This work sheds some light on PG-1 translocation mechanisms at the molecular level. Methods presented in this study may be extended to other arginine-rich antimicrobial and cell-penetrating peptides. Third, oncocin is a proline-rich antimicrobial peptide that inhibits protein synthesis by binding to the bacterial ribosome. In this work, the antimicrobial activity of oncocin was improved by systematic peptide mutagenesis and activity evaluation. We found that a pair of cationic substitutions (P4K and L7K/R) improves the activity by 2-4 fold (p