The global spread of multi- and pan-resistant bacteria has triggered research to identify novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages. In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally, plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed the production of satisfactory peptide quantities. When these sequences were integrated into the T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select phage genome, expression was below detection limits and an effect on the growth of potentially phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we were able to show that apidaecins can be integrated into the T7Select phage genome to induce their expression in host cells, but further research is required to optimize the engineered T7Select phages for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures.

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Reflection absorption infrared spectroscopy was used as the primary analysis technique to study the interfacial chemistry of surfaces relevant to chemical and biological warfare agent defense. Many strategies utilized by the military to detect and decompose chemical and biological warfare agents involve their interaction with surfaces. However, much of the chemistry that occurs at the interface between the agents and surfaces of interest remains unknown. The surface chemistry plays an important role in efficacy of both detection and decontamination technology, and by obtaining a deeper understanding of that chemistry, researchers might be able to develop more sensitive detection devices and more effective decontamination strategies. Our efforts have focused on three different areas of surface chemistry relevant to chemical and biological warfare agent defense: 1) The development of a surface synthesis strategy to create and control the structure of antibacterial self-assembled monolayers (SAMs). Our work demonstrated a successful strategy for creating SAMs that contain long-chain quaternary ammonium groups, which were synthesized and subsequently characterized using RAIRS and X-ray photoelectron spectroscopy (XPS). 2) The determination of the surface conformation, orientation, and relative surface density of immobilized antimicrobial peptides. Our results revealed that the peptides consisted of tilted (50-60°), α-helices on the surface, regardless of solution conditions. 3) The design and construction of a new ultrahigh vacuum surface science instrument that allows for the study of gas-surface reactions with up to three gases simultaneously. 4) The study of the adsorption of chemical warfare agent simulants to silica nanoparticulate films. Our work demonstrated that the adsorbate structure was dependent on the number of hydrogen-bonding groups, and the adsorption consists of a pressure-dependent two part mechanism. The results presented here will help increase the understanding of the surface chemistry of three interfaces relevant to chemical and biological defense. Future researchers may apply the new information to develop more effective detection and decontamination strategies for chemical and biological warfare agents. / Ph. D.

The interaction of the protein Rev with Rev Response Element (RRE) RNA is critical to the HIV-1 life cycle as this complex is required for the export of singly-spliced and unspliced mRNAs from the nucleus to the cytoplasm. Disruption of this interaction is considered to be a powerful strategy towards the development of HIV-1 therapeutics. Therefore, we have developed several branched peptide libraries containing unnatural amino acids to target the high-affinity binding site of RRE RNA (RRE IIB), with the idea that branching in peptides can provide multivalent contacts with folded RNA structures and boost binding affinity and selectivity for the target. Unnatural amino acids were incorporated into the library design to encourage non-canonical interactions with the RNA and to improve proteolytic stability. The on-bead high-throughput screening of our first branched peptide library (46,656 sequences) against HIV-1 RRE RNA generated hit peptides with binding affinities in the low micromolar range. We demonstrated that branching in the peptide is required for efficient binding and selectivity towards the RNA, and that the peptides bind a large surface area of RRE IIB. Introduction of boronic acids into branched peptides boosted selectivity of the peptides for RRE IIB, and proved to be a novel and tunable mode of binding towards RNA. Additionally, we revealed that these branched peptide boronic acids (BPBAs) were cell permeable and non-toxic. One BPBA (BPBA3) bound RRE IIB selectively and was able to inhibit HIV-1 replication in vitro, revealing enzymatic cleavage of the RNA upon binding. A second generation BPBA library that introduced acridinyl lysine as an intercalator (4,096 sequences) was screened against RRE IIB. Several hit compounds bound in the low nanomolar regime, and a significant number of compounds inhibited HIV-1 replication in vitro. These BPBAs were also found to severely inhibit the microbial growth of bacteria and fungus, with MICs as low as 1 µg/mL against Staphylococcus aureus, Candida albicans, and Escherichia coli. These compounds were also found to significantly inhibit biofilm formation and growth, and were non-hemolytic. High-throughput screening of a third generation BPBA library containing all unnatural amino acids (46,656 sequences) revealed several hits that bound RRE IIB RNA in the nanomolar range. Sequence motifs present in the hit peptides suggested that the location and composition of amino acids within the branched peptide structure were important for recognizing the RNA target. In particular, lead compounds 2C5 and 4B3 demonstrated selectivity towards RRE, and footprinting experiments combined with SHAPE experiments revealed different interactions of the peptides with the RNA Toxicity assays revealed no impact on cell viability for the majority of hit sequences tested up to 100 µM, and several compounds also demonstrated inhibition of HIV-1 replication. / Ph. D.

No / This review focuses on treatment targets for the most common inflammatory skin diseases, eczema and psoriasis with an emphasis on cytokines expressed in the uppermost layer of the skin which is easily accessible for diagnostic and therapeutic approaches. Recently, a significant body of research has highlighted the influence of the skin barrier and the patients’ microbiome on skin inflammatory responses and we will comment on their impact on mediator regulation. Itch is a prominent dermatology symptom which is influenced by cytokines and can via itch–scratch cycle impact on the skin barrier and mediator expression associated with damage. Taking the contribution of pruritus and superficial skin damage into account, we address cytokines as targets for stratified treatment approaches in subgroups of eczema and psoriasis.

The rapid progression of Klebsiella pneumoniae towards antibiotic resistance is a significant concern, primarily due to its protective extracellular polysaccharide (EPS) capsule that shields the bacteria from host immunity. Our previous research demonstrated that antimicrobial peptides could disrupt the EPS capsule of K. pneumoniae. Further analysis identified Bac7 (1-35), a proline-rich antimicrobial peptide (PrAMP), as having the greatest ability to aggregate with the K. pneumoniae EPS capsule, exhibiting potent antimicrobial activity. However, the relationship between key features facilitating EPS and membrane interactions, as well as antimicrobial efficacy, remains poorly understood. Here, we used natural PrAMPs from diverse organisms to investigate their interactions with the cell envelope of K. pneumoniae. Apidaecin Cd3+, Tur1A, and PR-39 peptides demonstrated activity against all tested strains, with a minimum inhibitory concentration ≤ 1 µg/mL. These peptides shared a proline content exceeding 36% and a charge greater than +5. Active PrAMPs induced membrane depolarization in K. pneumoniae, with the extent of depolarization directly correlating with peptide charge, suggesting membrane depolarization as a potential mechanism for PrAMP entry into the cell. Checkerboard assays of active PrAMPs with PepC, an inactive peptide, suggested the membrane actions of PrAMPs have potential to rescue a therapeutic unable to access the bacterial membrane. Consistent with our findings with bac7(1-35) truncated analogs, both active and inactive PrAMPs aggregated with K. pneumoniae EPS, suggesting that the antimicrobial activities and polysaccharide aggregation potential of this class of peptides can be studied independently. Furthermore, the treatment of biofilms with active peptides revealed unique structure-based biofilm changes, with Tur1A causing more structural collapse than PR-39. Our findings highlight a potential membrane mediated peptide uptake into the cell which is dependent on the charge of the peptide. Differential biofilm interactions between similar peptides and EPS aggregation of inactive peptides warrant these attributes of PrAMPs to be further studied independently.