Justyna Kozlowska | Kings College London (original) (raw)

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Papers by Justyna Kozlowska

Systems biology studies are becoming increasingly important as the need to study organisms in a h... more Systems biology studies are becoming increasingly important as the need to study organisms in a holistic manner, instead of looking at processes in isolation, is being recognised. This is especially true for the study of host-pathogen interactions where the responses from bacteria are complex and overlap extensively. This thesis explores the application of H NMR metabolomics to the study of bacteriahost interactions and seeks to identify its strengths and weaknesses with a view to integrating this technique into a combined approach that can provide an unprecedented, sophisticated understanding of host-pathogen interactions that we believe is intractable by other methodologies. The BBSRC CASE studentship that supported this work was awarded in conjunction with Procarta Biosystems Ltd who have produced a new generation of antibiotics with a novel mechanism of action. The final objective for the studentship therefore, was to develop a validated systems approach capable of defining the ...

Scientific Reports, 2016

The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteri... more The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteria is a key determinant of their abilities to exert diverse bactericidal effects. Here we present a molecular level understanding of the initial target membrane interaction for two cationic α-helical AMPs that share structural similarities but have a ten-fold difference in antibacterial potency towards Gram-negative bacteria. The binding and insertion from solution of pleurocidin or magainin 2 to membranes representing the inner membrane of Gram-negative bacteria, comprising a mixture of 128 anionic and 384 zwitterionic lipids, is monitored over 100 ns in all atom molecular dynamics simulations. The effects of the membrane interaction on both the peptide and lipid constituents are considered and compared with new and published experimental data obtained in the steady state. While both magainin 2 and pleurocidin are capable of disrupting bacterial membranes, the greater potency of pleuroci...

Journal of Biological Chemistry, 2012

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010

The interactions of cationic amphipathic antimicrobial peptides (AMPs) with anionic biological me... more The interactions of cationic amphipathic antimicrobial peptides (AMPs) with anionic biological membranes have been the focus of much research aimed at improving the activity of such compounds in the search for therapeutic leads. However, many of these peptides are thought to have other polyanions, such as DNA or RNA, as their ultimate target. Here a combination of fluorescence and circular dichroism (CD) spectroscopies has been used to assess the structural properties of amidated versions of buforin II, pleurocidin and magainin 2 that support their varying abilities to translocate through bacterial membranes and bind to double stranded DNA. Unlike magainin 2 amide, a prototypical membrane disruptive AMP, buforin II amide adopts a poorly helical structure in membranes closely mimicking the composition of Gram negative bacteria, such as Escherichia coli, and binds to a short duplex DNA sequence with high affinity, ultimately forming peptide-DNA condensates. The binding affinities of the peptides to duplex DNA are shown to be related to the structural changes that they induce. Furthermore, CD also reveals the conformation of the bound peptide buforin II amide. In contrast with a synthetic peptide, designed to adopt a perfect amphipathic α-helix, buforin II amide adopts an extended or polyproline II conformation when bound to DNA. These results show that an α-helix structure is not required for the DNA binding and condensation activity of buforin II amide.

Scientific Reports, 2018

A correction to this article has been published and is linked from the HTML and PDF versions of t... more A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Metabolomics, 2013

Chronic polymicrobial lung infections in adult cystic fibrosis patients are typically dominated b... more Chronic polymicrobial lung infections in adult cystic fibrosis patients are typically dominated by high levels of Pseudomonas aeruginosa. Determining the impact of P. aeruginosa growth on airway secretion composition is fundamental to understanding both the behaviour of this pathogen in vivo, and its relationship with other potential colonising species. We hypothesised that the marked differences in the phenotypes of clinical isolates would be reflected in the metabolite composition of spent culture media. 1 H NMR spectroscopy was used to characterise the impact of P. aeruginosa growth on a synthetic medium as part of an in vitro CF lower airways model system. Comparisons of 15 CF clinical isolates were made and four distinct metabolomic clusters identified. Highly significant relationships between P. aeruginosa isolate cluster membership and both patient lung function (FEV 1) and spent culture pH were identified. This link between clinical isolate growth behaviour and FEV 1 indicates characterisation of P. aeruginosa growth may find application in predicting patient lung function while the significant divergence in metabolite production and consumption observed between CF clinical isolates suggests dominant isolate characteristics have the potential to play both a selective role in microbiota composition and influence pseudomonal behaviour in vivo.

PLoS Pathogens, 2014

Obtaining an in-depth understanding of the arms races between peptides comprising the innate immu... more Obtaining an in-depth understanding of the arms races between peptides comprising the innate immune response and bacterial pathogens is of fundamental interest and will inform the development of new antibacterial therapeutics. We investigated whether a whole organism view of antimicrobial peptide (AMP) challenge on Escherichia coli would provide a suitably sophisticated bacterial perspective on AMP mechanism of action. Selecting structurally and physically related AMPs but with expected differences in bactericidal strategy, we monitored changes in bacterial metabolomes, morphological features and gene expression following AMP challenge at sub-lethal concentrations. For each technique, the vast majority of changes were specific to each AMP, with such a plastic response indicating E. coli is highly capable of discriminating between specific antibiotic challenges. Analysis of the ontological profiles generated from the transcriptomic analyses suggests this approach can accurately predict the antibacterial mode of action, providing a fresh, novel perspective for previous functional and biophysical studies.

Scientific Reports, 2014

Despite the fundamental contribution of the gut microbiota to host physiology, the extent of its ... more Despite the fundamental contribution of the gut microbiota to host physiology, the extent of its variation in genetically-identical animals used in research is not known. We report significant divergence in both the composition and metabolism of gut microbiota in genetically-identical adult C57BL/6 mice housed in separate controlled units within a single commercial production facility. The reported divergence in gut microbiota has the potential to confound experimental studies using mammalian models. R esearchers using animal models are becoming increasingly aware of possible influences of the gut microbiota on physiology. Murine models have been used to demonstrate relationships between the gut microbiota and obesity 1 , metabolic disease 2 , cardiovascular health 3 , nervous system development 4 , diabetes 5 , and immune function 6 , hepatic function 7 , inflammatory bowel conditions 8 , and carcinogenesis 9 , highlighting the potential impact that differences in the microbiome of mice from different animal facilities could have on research. However, most researchers assume that genetically-identical mice derived from a single supplier will have an equivalent microbiome. To test this assumption we studied the faecal microbiome and metabolome of genetically-identical C57BL/6 mice housed in four separate controlled units within a single facility of a commercial supplier of animals for research. Faecal samples were collected at eight weeks of age from twenty mice, with five mice sampled in each of four barrier rooms. These mice were separated by no more than ten generations. Methods Murine faecal samples. Faeces were collected from eight week old C57BL/6 at the Charles River commercial facility (Margate, UK) under commercial licence, with all mice kept in accordance with protocols approved by The Animal Health and Welfare Board for England. Samples were collected from 20 mice, housed in four separate barrier rooms within the facility, fed the same chow (a VRF1 diet, SDS). The five mice sampled in each room were housed in separate cages. The five mice from each of the four rooms were taken from separate cages i.e. no two mice came from the same cage. Mice in this study were handled by individuals wearing gloves for cage cleaning purposes on a weekly basis. Mice were not housed exclusively with litter mates, with 27 individuals housed per room. Samples consisted of individual faecal pellets taken from individual mice. After collection, pellets were placed into separate collection tubes and frozen prior to analysis. Microbiota. Nucleic acid extractions were carried out using a combination of physical disruption and phenol/chloroform extraction methods, described previously 10. 16S rRNA gene universal Bacterial primers 27F-519R (27F 59-AGRGTTTGATCMTGGCTCAG, 519R 59-GTNTTACNGCGGCKGCTG) were used in a single-step 30 cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) performed under the following conditions: 94oC for 5 minutes, followed by 28 cycles of: 94oC for 30 seconds, 53oC for 40 seconds, and 72oC for 1 minute. Amplification was followed by a final elongation step at 72oC for 5 minutes. Following PCR, all amplicon products from different samples were mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA). Samples were sequenced utilizing Roche 454 FLX titanium instruments and reagents following manufacturer's guidelines. A total of 165,934 16S rRNA gene sequences were obtained from the 20 faecal sample extracts. Following curation, an average of 4,356 sequences was obtained for each of the samples. For analysis of alpha and beta diversity, samples were normalised to 2,179 sequences per sample. Sequence data analysis was carried out. Here, the Q25 sequence data derived from the sequencing process was processed using standard analysis pipeline processes (MR DNA, Shallowater, USA). Sequences were depleted of barcodes and primers then short sequences ,200 bp removed, as were sequences with ambiguous base calls removed, and sequences with homopolymer runs exceeding 6 bp, sequences were denoised and chimeras removed 11-17. Operational taxonomic units were defined after removal of singleton sequences, clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using BLASTn against a curated databased derived from GreenGenes, NCBI and RDP databases 18. Normalized and de-noised files were then rarefied and run through QIIME 19 to generate alpha and beta diversity data. Additional statistical analyses were performed with NCSS2007 (NCSS, UT) and XLstat 2012 (Addinsoft, NY).