Bio-oligomers as antibacterial agents and strategies for bacterial detection

In this thesis I examined the potential of Bio-Oligomers such as peptoids, peptides and aptamers, as therapeutic and diagnostic entities. Therapeutic Bio-Oligomers; A series of peptoid analogs have been designed and synthesised using solid phase synthesis. These peptoids have been subjected to biological evaluation to determine structure-activity relationships that define their antimicrobial activity. In total 13 peptoids were synthesised. Out of 13 different peptoids, only one peptoid called Tosyl-Octyl-Peptoid (TOP) demonstrated significant broad-spectrum bactericidal activity. TOP kills bacteria under non-dividing and dividing conditions. The Minimum Inhibitory Concentrations (MIC) values of TOP for S. epidermidis, E. coli and Klebsiella were 20 μM, whereas Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) were 40 μM. The highest MIC values were observed for Pseudomonas aeruginosa (PAO1) at 80 μM. The selectivity ratio (SR) or Therapeutic index (TI) was calculated, by dividing the 10% haemolysis activity (5 mM) by the median of the MIC (50 μM) yielding a TI for TOP as 100. This TI is well above previously reported peptidomimetics TI of around 20. TOP demonstrates selective bacterial killing in co-culture systems and intracellular bacterial killing activity. Diagnostic Bio-Oligomers; In the second part of my thesis, I investigated aptamer and peptide-based molecular probes to detect MRSA. As well as screening aptamers and peptide probes against whole MRSA, I over-expressed and purified PBP2A protein. This purified protein was used as a target for aptamer and peptide probes to detect MRSA. Two different aptamer libraries were initially screened for utility. In-vitro conditions for SELEX were optimised. Biopanning with a phage derived peptides was also performed. Target sequences for both methods were identified and chemically synthesised. Evaluation of fluorescently labelled sequences with flow cytometry and confocal imaging showed no specificity for MRSA detection with either method. The Bio-Oligomers and the in-vitro selection methodology require further refinement to improve diagnostic utility

Supramolecular structure in the membrane of Staphylococcus aureus

The fundamental processes of life are organized and based on common basic principles. Molecular organizers, often interacting with the membrane, capitalize on cellular polarity to precisely orientate essential processes. The study of organisms lacking apparent polarity or known cellular organizers (e.g., the bacterium Staphylococcus aureus) may enable the elucidation of the primal organizational drive in biology. How does a cell choose from infinite locations in its membrane? We have discovered a structure in the S. aureus membrane that organizes processes indispensable for life and can arise spontaneously from the geometric constraints of protein complexes on membranes. Building on this finding, the most basic cellular positioning system to optimize biological processes, known molecular coordinators could introduce further levels of complexity. All life demands the temporal and spatial control of essential biological functions. In bacteria, the recent discovery of coordinating elements provides a framework to begin to explain cell growth and division. Here we present the discovery of a supramolecular structure in the membrane of the coccal bacterium Staphylococcus aureus, which leads to the formation of a large-scale pattern across the entire cell body; this has been unveiled by studying the distribution of essential proteins involved in lipid metabolism (PlsY and CdsA). The organization is found to require MreD, which determines morphology in rod-shaped cells. The distribution of protein complexes can be explained as a spontaneous pattern formation arising from the competition between the energy cost of bending that they impose on the membrane, their entropy of mixing, and the geometric constraints in the system. Our results provide evidence for the existence of a self-organized and nonpercolating molecular scaffold involving MreD as an organizer for optimal cell function and growth based on the intrinsic self-assembling properties of biological molecules

MicroRNA-142 Critically Regulates Group 2 Innate Lymphoid Cell Homeostasis and Function

Innate lymphoid cells are central to the regulation of immunity at mucosal barrier sites, with group 2 innate lymphoid cells (ILC2s) being particularly important in type 2 immunity. In this study, we demonstrate that microRNA(miR)-142 plays a critical, cell-intrinsic role in the homeostasis and function of ILC2s. Mice deficient for miR-142 expression demonstrate an ILC2 progenitor_biased development in the bone marrow, and along with peripheral ILC2s at mucosal sites, these cells display a greatly altered phenotype based on surface marker expression. ILC2 proliferative and effector functions are severely dysfunctional following Nippostrongylus brasiliensis infection, revealing a critical role for miR-142 isoforms in ILC2-mediated immune responses. Mechanistically, Socs1 and Gfi1 expression are regulated by miR-142 isoforms in ILC2s, impacting ILC2 phenotypes as well as the proliferative and effector capacity of these cells. The identification of these novel pathways opens potential new avenues to modulate ILC2-dependent immune functions

MicroRNA-142 Critically Regulates Group 2 Innate Lymphoid Cell Homeostasis and Function.

From PubMed via Jisc Publications RouterHistory: received 2020-06-01, accepted 2021-03-19Publication status: ppublishFunder: Wellcome Trust; Grant(s): 204394Funder: British Heart Foundation; Grant(s): PG/12/36/29444Funder: Medical Research Council; Grant(s): MR/M003493/1, MR/R024812/1Innate lymphoid cells are central to the regulation of immunity at mucosal barrier sites, with group 2 innate lymphoid cells (ILC2s) being particularly important in type 2 immunity. In this study, we demonstrate that microRNA(miR)-142 plays a critical, cell-intrinsic role in the homeostasis and function of ILC2s. Mice deficient for miR-142 expression demonstrate an ILC2 progenitor-biased development in the bone marrow, and along with peripheral ILC2s at mucosal sites, these cells display a greatly altered phenotype based on surface marker expression. ILC2 proliferative and effector functions are severely dysfunctional following infection, revealing a critical role for miR-142 isoforms in ILC2-mediated immune responses. Mechanistically, and expression are regulated by miR-142 isoforms in ILC2s, impacting ILC2 phenotypes as well as the proliferative and effector capacity of these cells. The identification of these novel pathways opens potential new avenues to modulate ILC2-dependent immune functions. [Abstract copyright: Copyright © 2021 The Authors.

Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation

Defective silencing of retrotransposable elements has been linked to inflammageing, cancer and autoimmune diseases. However, the underlying mechanisms are only partially understood. Here we implicate the histone H3.3 chaperone Daxx, a retrotransposable element repressor inactivated in myeloid leukaemia and other neoplasms, in protection from inflammatory disease. Loss of Daxx alters the chromatin landscape, H3.3 distribution and histone marks of haematopoietic progenitors, leading to engagement of a Pu.1-dependent transcriptional programme for myelopoiesis at the expense of B-cell differentiation. This causes neutrophilia and inflammation, predisposing mice to develop an autoinflammatory skin disease. While these molecular and phenotypic perturbations are in part reverted in animals lacking both Pu.1 and Daxx, haematopoietic progenitors in these mice show unique chromatin and transcriptome alterations, suggesting an interaction between these two pathways. Overall, our findings implicate retrotransposable element silencing in haematopoiesis and suggest a cross-talk between the H3.3 loading machinery and the pioneer transcription factor Pu.1