The detection of meningococcal disease through identification of antimicrobial peptides using an in silico model creation

Philosophiae Doctor - PhDNeisseria meningitidis (the meningococcus), the causative agent of meningococcal disease (MD) was identified in 1887 and despite effective antibiotics and partially effective vaccines, Neisseria meningitidis (N. meningitidis) is the leading cause worldwide of meningitis and rapidly fatal sepsis usually in otherwise healthy individuals. Over 500 000 meningococcal cases occur every year. These numbers have made bacterial meningitis a top ten infectious cause of death worldwide. MD primarily affects children under 5 years of age, although in epidemic outbreaks there is a shift in disease to older children, adolescents and adults. MD is also associated with marked morbidity including limb loss, hearing loss, cognitive dysfunction, visual impairment, educational difficulties, developmental delays, motor nerve deficits, seizure disorders and behavioural problems. Antimicrobial peptides (AMPs) are molecules that provide protection against environmental pathogens, acting against a large number of microorganisms, including bacteria, fungi, yeast and virus. AMPs production is a major component of innate immunity against infection. The chemical properties of AMPs allow them to insert into the anionic cell wall and phospholipid membranes of microorganisms or bind to the bacteria making it easily detectable for diagnostic purposes. AMPs can be exploited for the generation of novel antibiotics, as biomarkers in the diagnosis of inflammatory conditions, for the manipulation of the inflammatory process, wound healing, autoimmunity and in the combat of tumour cells. Due to the severity of meningitis, early detection and identification of the strain of N. meningitidis is vital. Rapid and accurate diagnosis is essential for optimal management of patients and a major problem for MD is its diagnostic difficulties and experts conclude that with an early intervention the patient’ prognosis will be much improved. It is becoming increasingly difficult to confirm the diagnosis of meningococcal infection by conventional methods. Although polymerase chain reaction (PCR) has the potential advantage of providing more rapid confirmation of the presence of the bacterium than culturing, it is still time consuming as well as costly. Introduction of AMPs to bind to N. meningitidis receptors could provide a less costly and time consuming solution to the current diagnostic problems. World Health Organization (WHO) meningococcal meningitis program activities encourage laboratory strengthening to ensure prompt and accurate diagnosis to rapidly confirm the presence of MD. This study aimed to identify a list of putative AMPs showing antibacterial activity to N. meningitidis to be used as ligands against receptors uniquely expressed by the bacterium and for the identified AMPs to be used in a Lateral Flow Device (LFD) for the rapid and accurate diagnosis of MD

Prediction of antimicrobial peptides using hyperparameter optimized support vector machines

Philosophiae Doctor - PhDAntimicrobial peptides (AMPs) play a key role in the innate immune response. They can be ubiquitously found in a wide range of eukaryotes including mammals, amphibians, insects, plants, and protozoa. In lower organisms, AMPs function merely as antibiotics by permeabilizing cell membranes and lysing invading microbes. Prediction of antimicrobial peptides is important because experimental methods used in characterizing AMPs are costly, time consuming and resource intensive and identification of AMPs in insects can serve as a template for the design of novel antibiotic. In order to fulfil this, firstly, data on antimicrobial peptides is extracted from UniProt, manually curated and stored into a centralized database called dragon antimicrobial peptide database (DAMPD). Secondly, based on the curated data, models to predict antimicrobial peptides are created using support vector machine with optimized hyperparameters. In particular, global optimization methods such as grid search, pattern search and derivative-free methods are utilised to optimize the SVM hyperparameters. These models are useful in characterizing unknown antimicrobial peptides. Finally, a webserver is created that will be used to predict antimicrobial peptides in haemotophagous insects such as Glossina morsitan and Anopheles gambiae.South Afric

Antimicrobial peptides from the Arctic ascidian Synoicum turgens

The rise in frequency of antibiotic resistant pathogenic bacteria makes the need for new treatment options for previously curable bacterial infections ever more important. In the process of discovering and developing antibacterial agents, one powerful approach has been borrowing wisdom from nature. Antimicrobial peptides (AMPs) are critical components of the innate immune systems found in almost all eukaryotic life forms. Their mode of action includes disruption of the bacterial membrane and to trigger supporting immune responses. Due to these properties, AMPs are considered promising lead structures that can be further developed into commercially available antibiotics to treat or prevent human diseases caused by bacteria. The work conducted in this thesis aims to discover and characterize novel antimicrobial peptides from the Arctic marine ascidian Synoicum turgens by using a marine bioprospecting approach. This includes collection, extraction and fractionation of biomass, antibacterial bioactivity testing and AMP isolation followed by chemical and biological characterization. For one isolated peptide class, truncated versions were prepared, aiming to produce shorter, linear variants with retained antimicrobial activity. In paper I, turgencin A and B and their oxidized derivatives were discovered through bioassay-guided purification. These peptides had an unusual disulfide connectivity, rarely seen in marine AMPs. Both turgencin A and B were potently active against all assayed bacterial strains. Membrane assays showed that the peptides cause bacterial membrane disruption within a few seconds. Turgencin A and B also displayed some cytotoxic activity against two human cell lines. Oxidation of the methionine present in both peptides decreased the bioactivities in all assays. Turgencin A, being the most potent AMP, was subject for sequence analysis and prediction of antimicrobial potential of different regions in paper II. Truncated and modified variants of turgencin A were synthetically produced to make smaller AMPs with the potential of being developed into antimicrobial drug leads. These 10-mer peptides, named StAMP-1–11, were made using an amino acid replacement strategy. Some of the Trp enriched peptides had similar bactericidal activity as the parent peptide turgencin A, and no cytotoxic activity against the mammalian cell lines. During turgencin isolation, a series of other smaller peptides were discovered in the same extracts presumably with antimicrobial activity. These isolated and characterized ~2 kDa, cysteine-rich peptides (CRPs) (described in paper III), were named St-CRP-1 and St-CRP-2 and contained 18-19 amino acids. The St-CRPs shared a disulfide connectivity pattern with alpha-defensins, had a neutral net charge, moderate antibacterial activity and showed no cytotoxicity. In addition, the introduction section provides background information on topics related to the thematic of the articles. This includes an introduction to bacteria, antibiotics and antibiotic resistance, AMPs, ascidians, and the marine environment

High-dimension profiling data generate a multifunctional peptide-mimic chemo-structure by connecting conserved fragments based on the neutrophil immune defense CAP37 protein as an in-silico antibacterial and woundhealing candidate agent

CAP37, a protein constitutively EXPRESSED in human neutrophils and induced in responseto infection in corneal epithelial cells, plays a significant role in host defense against infection. Initiallyidentified through its potent bactericidal activity for Gram-negative bacteria, it is now known that CAP37regulates numerous host cell functions, including corneal epithelial cell chemotaxis. Delineation of thedomains of CAP37 that define these functions and synthesize bioactive peptides for therapeutic use have alsobeen explored. Novel findings of a multifunctional domain between a 120 and 146 have also been reported.Here, in Biogenea Pharmaceuticals Ltd we for the first time generated a multifunctional peptide-mimicchemo-structure by connecting conserved fragments based on the neutrophil immune defense CAP37 proteinas an in-silico antibacterial and wound-healing canditate agent. This in silico effort was accomplished byutilizing various generated descriptors of proteins, compounds and their interactions resulting in aperformance/cost evaluation study for a GPU-based drug discovery application on volunteer computingapproaches based on Automated Structure-Activity Relationship Minings in Connecting Chemical Structureto Biological Profiles for the generation of novel Computational biomodeling of 3D drug-protein binding freeenergy evaluation

Molecular Science for Drug Development and Biomedicine

With the avalanche of biological sequences generated in the postgenomic age, molecular science is facing an unprecedented challenge, i.e., how to timely utilize the huge amount of data to benefit human beings. Stimulated by such a challenge, a rapid development has taken place in molecular science, particularly in the areas associated with drug development and biomedicine, both experimental and theoretical. The current thematic issue was launched with the focus on the topic of “Molecular Science for Drug Development and Biomedicine”, in hopes to further stimulate more useful techniques and findings from various approaches of molecular science for drug development and biomedicine

Developing an improved laboratory diagnostic test for early detection of sepsis in dogs

Dogs with sepsis – the life-threatening tissue damage resulting from an inappropriate host response to infection – often suffer up to 50% mortality, largely due to a lack of accurate clinical and laboratory tests for rapid diagnosis and guiding management. Procalcitonin (PCT) is a biomarker used for rapid and accurate diagnosis and management of sepsis in humans, thereby improving outcomes. Commercial assays for canine PCT (cPCT) are available; although prior to this project they were not validated. Different antigenicity between recombinant and native PCT was one possible explanation for poor performance of canine PCT ELISAs. Chapter One identified a need for a monoclonal antibody confirmed to detect native canine PCT. This would enable development of a reliable ELISA for procalcitonin measurement in dogs with sepsis. This project aimed to produce monoclonal antibodies (Mabs) recognizing native cPCT to develop a rapid and specific cPCT diagnostic test for sepsis. Additionally, this project aimed to better understand the epidemiology of canine sepsis. Chapter Two features an epidemiological study of sepsis in hospitalized dogs, contributing a large study to the currently limited data on clinical impact of canine sepsis. For 486 admissions of dogs, sepsis prevalence was 5.5% and incidence was 2.2%. The most common source of infection was peritonitis. Mortality was highest in the sepsis group at 35.1% (p=0.0008, χ2 test; overall mortality 15.4%), compared with dogs who had non-infectious systemic inflammatory response syndrome (SIRS) or infection. All sepsis deaths occurred within four days of diagnosis. Cost and length of hospitalisation were also highest for sepsis. Chapters Three and Four describe how native cPCT was extracted from canine thyroid glands, isolated via chromatography, identified with a commercial procalcitonin antibody (BioVendor, Asheville, NC, USA), confirmed by mass spectrometry (MS), and used to immunize mice although purity was low (≤42%). This produced two Mabs, that were assessed for PCT affinity using immunoprecipitation and MS. Unfortunately, neither monoclonal antibody demonstrated affinity to native or recombinant PCT, most likely due to insufficient purification or sameness between canine and murine PCT. However, the same screening confirmed the BioVendor polyclonal antibody does recognise native canine procalcitonin in septic dog plasma, contributing to its validation. Furthermore, native PCT was detected in septic dog plasma directly using mass spectrometry. In Chapter Five, a general discussion gives an overview of the project findings and recommends future directions. Focus should be directed towards full validation of the existing antibody in the BioVendor ELISA, calibration, and reference intervals. A monoclonal antibody should be developed to native canine PCT to enable consistent quantitation of PCT. Larger epidemiological studies can provide information on the impact of sepsis in the wider canine population

Proteomic Analysis of the Blood of \u3ci\u3eAlligator mississippiensis\u3c/i\u3e

My research focus was to investigate alligator blood using mass spectrometry-based proteomics methods to understand their innate immune systems. The first goal was to sequence peptides and proteins from the blood serum and leukocytes using tandem mass spectrometry and de novo sequencing. The second goal was to determine the function of these biological molecules and their relationship to the immune system. One- and two-dimensional gel electrophoresis was used to separate proteins from alligator leukocytes, which were enzymatically digested. The peptides were measured using reversed phase nano-high performance liquid chromatography coupled with tandem mass spectrometry (nano-HPLC-MS/MS) followed with de novo sequencing. The results, as described in Chapter 3 show that alligator leukocytes contain proteins that are similar to proteins found in other vertebrates such as mammals and reptiles that are related to immune responses. Isolation of small molecule interferences and peptides exhibiting antimicrobial activity from alligator leukocyte extracts are described in Chapters 4 and 5. Reversed-phase HPLC was used to separate the leukocyte mixture and antimicrobial activity tests were used to determine the active fractions. Interferants, EDTA and spermine were present and showed activity in early fractions. Two major peptides measured at 4.7 and 4.9 kDa in an active fraction were further separated on the basis of their charge, size and shape using ion mobility-mass spectrometry (IM-MS). Due to the limited fragmentation of the peptides using IM-MS, the peptides were isolated and fragmented using MALDI TOF/TOF MS for de novo sequencing. Lectins are a class of carbohydrate selective proteins that are part of the complement immune system. Chapter 6 presents results for a lectin isolated from alligators that have mannan and mannose binding activity. In this study, the monomeric lectin was isolated and enzymatically digested using five different proteases to create small and large peptides which were analyzed by LC-MS/MS. The peptides were determined via de novo sequencing and overlapped to generate the lectin sequence. Lectins may have varying degrees of glycosylation, therefore deglycosylation procedures suitable for mass spectrometry analysis are described in Chapter 7. Conclusions and future directions for the work in this dissertation will be summarized in Chapter 8

Conformation and dynamics of the cyclic lipopeptide viscosinamide at the water-lipid interface

Cyclic lipodepsipeptides or CLiPs from Pseudomonas are secondary metabolites that mediate a wide range of biological functions for their producers, and display antimicrobial and anticancer activities. Direct interaction of CLiPs with the cellular membranes is presumed to be essential in causing these. To understand the processes involved at the molecular level, knowledge of the conformation and dynamics of CLiPs at the water-lipid interface is required to guide the interpretation of biophysical investigations in model membrane systems. We used NMR and molecular dynamics to study the conformation, location and orientation of the Pseudomonas CLiP viscosinamide in a water/dodecylphosphocholine solution. In the process, we demonstrate the strong added value of combining uniform, isotope-enriched viscosinamide and protein NMR methods. In particular, the use of techniques to determine backbone dihedral angles and detect and identify long-lived hydrogen bonds, establishes that the solution conformation previously determined in acetonitrile is maintained in water/dodecylphosphocholine solution. Paramagnetic relaxation enhancements pinpoint viscosinamide near the water-lipid interface, with its orientation dictated by the amphipathic distribution of hydrophobic and hydrophilic residues. Finally, the experimental observations are supported by molecular dynamics simulations. Thus a firm structural basis is now available for interpreting biophysical and bioactivity data relating to this class of compounds