The Role of Antimicrobial Peptides as Antimicrobial and Antibiofilm Agents in Tackling the Silent Pandemic of Antimicrobial Resistance

Just over a million people died globally in 2019 due to antibiotic resistance caused by ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The World Health Organization (WHO) also lists antibiotic-resistant Campylobacter and Helicobacter as bacteria that pose the greatest threat to human health. As it is becoming increasingly difficult to discover new antibiotics, new alternatives are needed to solve the crisis of antimicrobial resistance (AMR). Bacteria commonly found in complex communities enclosed within self-produced matrices called biofilms are difficult to eradicate and develop increased stress and antimicrobial tolerance. This review summarises the role of antimicrobial peptides (AMPs) in combating the silent pandemic of AMR and their application in clinical medicine, focusing on both the advantages and disadvantages of AMPs as antibiofilm agents. It is known that many AMPs display broad-spectrum antimicrobial activities, but in a variety of organisms AMPs are not stable (short half-life) or have some toxic side effects. Hence, it is also important to develop new AMP analogues for their potential use as drug candidates. The use of one health approach along with developing novel therapies using phages and breakthroughs in novel antimicrobial peptide synthesis can help us in tackling the problem of AMR

Discovery and characterization of novel RNA repair systems

RNA is one of the major macromolecules essential for all known forms of life. RNA in the cell is subject to many types of damage as DNA, and the ubiquitous RNA degradation by surveillance machinery is an important way to respond RNA damage. However, RNA repair is an alternative way for cells to deal with RNA damage, which may play an important role in maintenance of cellular RNAs and even for cell survival. RNA repair is the mechanism that rectifies the purposeful RNA damage during RNA processing or cellular stress. To counter the unexpected RNA breakage, RNA repair systems have evolved in some organisms to restore the normal structure and function of RNA. The first RNA repair system was found in T4phage, in which two proteins T4Pnkp and T4Rnl carried out RNA healing and sealing, respectively. The bacterial Pnkp/Hen1 complex has also been shown to repair ribotoxin-cleaved RNAs in vitro, but it was distinguished from T4 systems by performing 3’-terminal 2’-O-methylation during RNA repair, which prevents the repaired RNA from repeated cleavage at the same site. Bacterial Pnkp and Hen1 appear pair-wise in the same operon in approximately 5% of known bacterial species. Although the bacterial Pnkp has been shown to possess kinase, phosphatase for RNA healing and all the signature motifs of a RNA ligase, it alone is not able to carry out RNA repair. In our study, we crystallized an active RNA ligase consisting of the C-terminal half of Pnkp (Pnkp-C) and the N-terminal half of Hen1 (Hen1-N) from Clostridium thermocellum, and provided the molecular basis for the ligase activation of bacterial Pnkp by Hen1. We also carried out a detailed functional study to pinpoint the activation step during RNA ligation. Guided by the sequence and structure, we created a series of point mutants for this new ligase and carried out biochemical assays. These studies provide additional insight into the mechanism of RNA ligation by Pnkp/Hen1. Based on a comprehensive Blast search, a novel RNA repair system composed of three proteins (Pnkp1, Rnl and Hen1) has been found in 10 bacterial species. This new system possesses the features from both T4Pnkp/Rnl and bacterial Pnkp/Hen1 systems. Efficient in vitro RNA repair only occurred in the presence of all three proteins. We showed that these three proteins formed a heterohexamer in vitro that contains two copies of each active site (kinase, phosphatase, methyltransferase and ligase). We crystallized and solved the strucuture of the heterohexamer to gauge four different enzymatic activities. Based on the structural and biochemical studies, we propose a novel mechanism of processive RNA repair with efficient 2’-O-methylation. The study of bacterial Pnkp1/Rnl/Hen1 complex may shed light on the bacterial Pnkp/Hen1 system due to their similarities. Both structural and biochemical assays indicated bacterial RNA repair systems have a broad range of RNA substrates, but we still have little knowledge of their biological functions in vivo. We therefore introduced genes encoding toxins and RNA repair proteins into bacteria through tightly controlled plasmids, and tested the inhibition and recovery of cellular growth upon the induction of gene expression. This method helped to identify potential toxin genes predicted by sequence alignment, and also facilitated the in vivo study by mimicking the environment the bacteria might be exposed to. If the RNA repair system provides surviving advantage for bacteria to defend themselves under stress, targeting this system may have the potential to limit the growth of pathogenic bacteria possessing the RNA repair systems. In addition to bacterial RNA repair, I am also interested in important NTase fold protein, which constitutes a large and highly diverse superfamily of proteins. Almost all known members of NTase transfer NMP to a hydroxyl group of substrates including proteins, nucleic acids and small molecules. A newly identified subgroup of this superfamily, Mab21, is shown to be essential for development or immune response. Particularly, Mab21D1 (cGAS) is important for interferon response activation by sensing dsDNA in cytosol and generating a second messenger molecule cGAMP. I crystallized and solved the structure of a close member Mab21D2, which is highly conserved in vertebrates but with unknown function. Through structural comparison to other NTase fold members as well as preliminary biochemical assays, we propose that it might work differently than cGAS

Structural basis of antibacterial peptide transport across membranes

Microcins are gene encoded antibacterial peptides secreted by enterobacteria in the gastrointestinal tract and play an important role in the control of bacterial populations. They present an attractive prospect in our effort to minimize the problem of bacterial drug resistance. Microcin J25 (MccJ25) is a 2 kDa plasmid encoded, ribosomally synthesized antimicrobial peptide comprised of 21 amino acid residues. MccJ25 undergoes post-translational modification and has a unique lasso structure. McjD, an ABC exporter, confers immunity to the producing strains by exporting the mature MccJ25 out of the cell. Studies have been designed to look into the transport mechanism of this peptide, which uses the siderophore receptor FhuA and ABC transporter McjD. MccJ25 uses the Trojan horse strategy by hijacking the iron import machineries as a mode of transport into the cell and acts as a transcription inhibitor by binding to RNA polymerase. Iron is an important nutrient for bacteria cell survival. To date, there is limited structural evidence on the import and extrusion mechanism of this antimicrobial peptide in Gram-negative bacteria. We have obtained a high-resolution structure of MccJ25 with its outer membrane receptor FhuA at 2.3 Å. FhuA is monomeric 22-strand antiparallel ß-barrel protein with the N-terminal domain folded inside to form a plug domain. MccJ25 binds to FhuA with hydrogen bonds and hydrophobic interactions with the extracellular loops of FhuA and its plug domain. We have also identified key residues that might play a role in MccJ25 translocation. Overall the structure provides information on how MccJ25 hijacks the iron uptake pathway to get into bacteria. Ligand binding studies and biochemical analysis demonstrate the functionality of McjD and its interaction with its natural ligand, MccJ25. The high substrate specificity and known cavity make McjD an excellent model for interaction studies.Open Acces

Delivery and activity of toxic effector domains from contact-dependent growth inhibition systems in Escherichia coli

Bacteria are ubiquitous in nature and have evolved a variety of communication and competition systems to survive in dense, complex environments. Contact-dependent growth inhibition (CDI) is a microbial competition system that is widespread throughout Gram-negative bacteria. CDI is mediated by the CdiB/CdiA two-partner secretion system, which displays the large CdiA exoprotein on the surface of CDI+ inhibitor cells. The C-terminal domain of CdiA (CdiA-CT) is toxic and inhibits cell growth after delivery into target bacteria. CDI+ cells are protected from auto-inhibition by expression of a cognate immunity protein (CdiI), which binds to the CdiA-CT and inactivates toxicity. CdiA-CT/CdiI pairs are highly divergent across species, indicating that CDI systems are capable of deploying a variety of toxins.This thesis explores several aspects of CDI, including delivery of CdiA-CTs into target cells, the toxic activities that lead to growth inhibition by CdiA-CT domains, and target cell stress responses that may influence CDI populations in natural environments. In Chapter I, we provide a general introduction to both diffusible and contact-dependent bacterial competition systems. This provides an overview of our current knowledge of CDI systems and also highlights key features of other secretion systems that contribute to population dynamics within bacterial communities. We then present a genetic study characterizing pathways of CdiA-CT translocation into target cells in Chapter II. In Chapter III, we focus on one protein, YciB, which is required for translocation of CdiA-CTo11EC869, a DNase toxin from E. coli, and examine the role of YciB in E. coli physiology outside of CDI. Chapter IV explores genetic responses that occur inside target cells after delivery of CdiA-CTo11EC869; in Chapter V, we present a study characterizing CdiA-CT/CdiI modules related to CdiA-CT/CdiIo11EC869. In Chapter VI, we discuss unpublished work examining the role of the translation factor EF-Tu as a co-factor required for activity by numerous CdiA-CT toxins. Chapter VII describes a collaborative project that utilized principle components of CDI systems as synthetic biology tools. Finally, we discuss research questions of significant interest in the field of CDI in Chapter VIII

Repression of btuB gene transcription in Escherichia coli by the GadX protein

<p>Abstract</p> <p>Background</p> <p>BtuB (B twelve uptake) is an outer membrane protein of <it>Escherichia coli</it>, it serves as a receptor for cobalamines uptake or bactericidal toxin entry. A decrease in the production of the BtuB protein would cause <it>E. coli </it>to become resistant to colicins. The production of BtuB has been shown to be regulated at the post-transcriptional level. The secondary structure switch of 5' untranslated region of <it>butB </it>and the intracellular concentration of adenosylcobalamin (Ado-Cbl) would affect the translation efficiency and RNA stability of <it>btuB</it>. The transcriptional regulation of <it>btuB </it>expression is still unclear.</p> <p>Results</p> <p>To determine whether the <it>btuB </it>gene is also transcriptionally controlled by trans-acting factors, a genomic library was screened for clones that enable <it>E. coli </it>to grow in the presence of colicin E7, and a plasmid carrying <it>gadX </it>and <it>gadY </it>genes was isolated. The <it>lacZ </it>reporter gene assay revealed that these two genes decreased the <it>btuB </it>promoter activity by approximately 50%, and the production of the BtuB protein was reduced by approximately 90% in the presence of a plasmid carrying both <it>gadX </it>and <it>gadY </it>genes in <it>E. coli </it>as determined by Western blotting. Results of electrophoretic mobility assay and DNase I footprinting indicated that the GadX protein binds to the 5' untranslated region of the <it>btuB </it>gene. Since <it>gadX </it>and <it>gadY </it>genes are more highly expressed under acidic conditions, the transcriptional level of <it>btuB </it>in cells cultured in pH 7.4 or pH 5.5 medium was examined by quantitative real-time PCR to investigate the effect of GadX. The results showed the transcription of <it>gadX </it>with 1.4-fold increase but the level of <it>btuB </it>was reduced to 57%.</p> <p>Conclusions</p> <p>Through biological and biochemical analysis, we have demonstrated the GadX can directly interact with <it>btuB </it>promoter and affect the expression of <it>btuB</it>. In conclusion, this study provides the first evidence that the expression of <it>btuB </it>gene is transcriptionally repressed by the acid responsive genes <it>gadX </it>and <it>gadY</it>.</p

Structural studies of toxins and toxin-like proteins

Toxins are an ancient mechanism of interaction between cohabiting organisms: basal concentrations serve as an informal cue, enough as a warning signal; too much and the dialog is over. As such, the evolutionary race to arms led to the development of a vast trove of molecular unique biochemical mechanisms, from small molecules to protein toxins. The study of these mechanisms is not only essential for the treatment of toxin-related pathologies, but also as the potential source for novel therapeutic drugs. In this thesis, a series of studies of different toxins and toxin-like proteins are compiled. To further understand the biological function and relevance of each toxin, their detailed study and characterization were pursued. Here are described the advances made using a combination of different complementary biophysical and structural methods, chosen in each case to specifically target each molecule characteristics. In the first chapter, the general biological theme of this thesis is introduced: toxins, particularly protein toxins, their description, and classification, as well as the role of structural biology in the study of proteins in general. To set the theoretical background of the following chapters, are also described the general principles of two of the most prominent methods for the study of proteins in structural biology: nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction. In the second chapter, the interaction between human FKBP12 chaperone protein and two similar bacterial small molecule toxins is detailed: rapamycin initially used as an anti-fungal before the discovery of its potent immunosuppressive properties as a mTOR inhibitor; and mycolactone, a bacterial toxin responsible for the disease Buruli ulcers in humans. In the third chapter, the cell-free protein expression system is introduced as a technique best suited for the expression of cytotoxic proteins and otherwise difficult targets, as explored further in the following chapters. In the fourth chapter, advancements towards the structural and conformational characterization of the membrane-inserted state of two similar pore-forming toxins are detailed: the bacterial Colicin Ia protein; and the human Bax protein, an apoptosis effector; using X-ray crystallography, solution NMR and solid-state NMR. Finally, in the fifth chapter, two FIC-domain bacterial toxins are investigated: the bacterial VbhTA toxin-antitoxin protein complex, and the structural determination with its cognate target, DNA GyraseB enzyme; and the auto-activation of the bacterial NmFIC protein; in both cases using a combination of X-ray crystallography and NMR spectroscopy, as well as other biophysical techniques

Structural studies of toxins and toxin-like proteins

Toxins are an ancient mechanism of interaction between cohabiting organisms: basal concentrations serve as an informal cue, enough as a warning signal; too much and the dialog is over. As such, the evolutionary race to arms led to the development of a vast trove of molecular unique biochemical mechanisms, from small molecules to protein toxins. The study of these mechanisms is not only essential for the treatment of toxin-related pathologies, but also as the potential source for novel therapeutic drugs. In this thesis, a series of studies of different toxins and toxin-like proteins are compiled. To further understand the biological function and relevance of each toxin, their detailed study and characterization were pursued. Here are described the advances made using a combination of different complementary biophysical and structural methods, chosen in each case to specifically target each molecule characteristics. In the first chapter, the general biological theme of this thesis is introduced: toxins, particularly protein toxins, their description, and classification, as well as the role of structural biology in the study of proteins in general. To set the theoretical background of the following chapters, are also described the general principles of two of the most prominent methods for the study of proteins in structural biology: nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction. In the second chapter, the interaction between human FKBP12 chaperone protein and two similar bacterial small molecule toxins is detailed: rapamycin initially used as an anti-fungal before the discovery of its potent immunosuppressive properties as a mTOR inhibitor; and mycolactone, a bacterial toxin responsible for the disease Buruli ulcers in humans. In the third chapter, the cell-free protein expression system is introduced as a technique best suited for the expression of cytotoxic proteins and otherwise difficult targets, as explored further in the following chapters. In the fourth chapter, advancements towards the structural and conformational characterization of the membrane-inserted state of two similar pore-forming toxins are detailed: the bacterial Colicin Ia protein; and the human Bax protein, an apoptosis effector; using X-ray crystallography, solution NMR and solid-state NMR. Finally, in the fifth chapter, two FIC-domain bacterial toxins are investigated: the bacterial VbhTA toxin-antitoxin protein complex, and the structural determination with its cognate target, DNA GyraseB enzyme; and the auto-activation of the bacterial NmFIC protein; in both cases using a combination of X-ray crystallography and NMR spectroscopy, as well as other biophysical techniques

Spaceflight Alters Bacterial Gene Expression and Virulence and Reveals Role for Global Regulator Hfq

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the spaceflight environment has never been accomplished due to significant technological and logistical hurdles. Moreover, the effects of spaceflight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared to identical ground control cultures. Global microarray and proteomic analyses revealed 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground based microgravity culture model. Spaceflight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during spaceflight missions and provide novel therapeutic options on Earth