Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-negative Bacterial Membranes.

The double-membrane cell envelope of Gram-negative bacteria is a sophisticated barrier that facilitates the uptake of nutrients and protects the organism from toxic compounds. An antibiotic molecule must find its way through the negatively charged lipopolysaccharide layer on the outer surface, pass through either a porin or the hydrophobic layer of the outer membrane, then traverse the hydrophilic peptidoglycan layer only to find another hydrophobic lipid bilayer before it finally enters the cytoplasm, where it typically finds its target. This complex uptake pathway with very different physico-chemical properties is one reason that Gram-negative are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target-based screening programs have failed to deliver novel antibiotics for these organisms. Due to the lack of general methods available for quantifying the flux of drugs into the cell, little is known about permeation rates, transport pathways and accumulation at the target sites for particular molecules. Here we summarize the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate

Predicting bacterial accumulation of anti-infectives by measuring permeability across surrogates of the Gram-negative bacterial cell envelope

The complex Gram-negative bacterial cell envelope is an important factor of intrinsic and acquired antibiotic resistance and explains the limited treatment options for infections caused by such pathogens. To support the discovery of highly permeating and thus potentially more active compounds, in vitro models based on permeable well plate inserts have been developed, advanced, and characterized. Advancing an approach by F. Graef[1], which mimics the total envelope structure of Gram-negative bacteria, similarities to the actual cell envelope structure have been revealed by CLSM and x-ray microtomography. Commercially available antibiotics have been tested with nalidixic acid permeating fastest. A second model was obtained by exploring if polysaccharide gels allow to distinguish high from low accumulating antibiotics. With 20% (w/v) starch gel performing best, the preparation was automated, structure-permeation relationships investigated and validated by machine learning. A third model is based on extracellular vesicles of Escherichia coli. These vesicles and the model derived thereof have been characterized by electron microscopy, while the performance of the model was investigated by comparing in vitro data to in bacterio accumulation and to permeability data from liposome-based models. Lacking porins, the total envelope model was limited to predict porin-independent permeation. This was, however, better achieved by using the starch-based and vesicle-based models.Die Zellmembran gramnegativer Bakterien ein wichtiger Faktor für intrinsische und erworbene Antibiotikaresistenzen. Um die Entdeckung von gut permeierenden und folglich potenziell wirksameren Antibiotika zu fördern, wurden in vitro-Modelle basierend auf Wellplatten mit durchlässiger Membran (weiter-)entwickelt und charakterisiert. Bei der Weiterentwicklung eines Ansatzes von Gräf et al.[1], der die Gesamtstruktur der gramnegativen Zellmembran nachbildet, wurde die Ähnlichkeit zum tatsächlichen gramnegativen Membranaufbau durch CLSM und Röntgenmikrotomographie festgestellt. Kommerziell erhältliche Antibiotika wurden getestet, wobei Nalidixinsäure am schnellsten permeierte. Ein zweites Modell wurde erhalten, als explorativ untersucht wurde, ob Polysaccharidgele im Stande sind, gute von schlechtakkumulierenden Antibiotika zu unterscheiden. Mit 20%igem (m/v) Stärkegel, welches am besten abschnitt, wurde die Modellherstellung automatisiert, Struktur-Wirkungsbeziehungen untersucht und diese durch maschinelles Lernen validiert. Ein drittes Modell basiert auf extrazellulären Vesikeln von Escherichia coli. Die Vesikel und das Modell wurden durch Elektronenmikroskopie charakterisiert, während die Leistung durch Vergleich der erhaltenen Permeabilitätsdaten Daten mit bakteriellen Akkumulationsdaten und Permebilitätsdaten von liposomenbasierten Modellen überprüft wurde. Durch die fehlenden Porine im ersten Ansatz war dieser lediglich auf die Vorhersage von porinunabhängiger Permeation beschränkt. Die Vorhersage von porinabhängiger Permeation wurde besser durch die stärkebasierten und vesikelbasierten Modelle erzielt

Expanding the toolbox for the study of antimicrobial peptides

There is an urgent lack of new antibiotics in the face of an ever-expanding antimicrobial resistance crisis. The fact that fewer new classes of antibiotics are being developed, and resistance soon follows newly available antibiotics, only serves to underline the urgency of the matter. There is a clear need of a paradigm shift with regards to antibiotics, and one such hope is antimicrobial peptides (AMPs). AMPs are an integral part of the innate immune systems of most organisms within the domains of life; since their discovery they have become of significant interest as a new type of antimicrobial agent, due in part to the low capacity of bacteria to develop resistance mechanisms towards them. Despite their potential, and lengthy study so far, establishing the specifics of the mechanism of action of many AMPs remains difficult– particularly of those that target the bacterial cell membrane. This lack of understanding limits the ability to rationally design new AMPs with a view to developing new antimicrobial agents. The aim of this work was to help identify new potential hit compounds through NMR structure elucidation, and to develop new methods that would give greater insight into the activity of membrane active AMPs. This in turn could help enable the rational design of new AMPs. WIND-PVPA, a method to quantify permeabilities of water and ions as a means to evaluate the disruptive capabilities of AMPs, was developed. This was demonstrated on a number of AMPs, and it was shown that WIND-PVPA can identify AMPs that have strong, selective, membrane disruptive activities such as the AMP WRWRWR, as well as more modestly disruptive AMPs such as KP-76. The WIND-PVPA was further used with a non-AMP membrane active natural product – lulworthinone – that was characterised over the course of the project. The findings of the study helped classify lulworthinone as a non-disruptive membrane active agent. In addition, microscale thermophoresis (MST) was shown to be a viable method by which the binding and partition coefficients of Trp-rich AMPs can be determined, and it was shown that the derived lipid-bindings of the AMPs correlates well with their bactericidal activity. Both WIND-PVPA and MST have expanded the toolbox available to the study of AMP-lipid interactions and can be used synergistically to give greater insight into the possible mechanism by which AMPs act, by helping to identify interesting cases, such as non-disruptive AMPs with potent activities. In summary, the methods developed have great potential that can be further refined into robust methods that can greatly assist in the deconvolution of AMP activity and can open up possibilities of the rational design of membrane active AMPs as a new generation of antimicrobial agents.

Regulatory Mechanisms of Bacterial Stress Responses

Bacterial growth and survival critically hinges on the ability to rapidly adapt to ever-changing environmental conditions. Elaborated stress response systems allow bacteria to sensitively detect and adequately respond to fluctuations in environmental conditions, such as pH, temperature, osmolarity, or the concentrations of nutrients and harmful substances. Often, bacterial stress responses towards a specific stressor involve multiple interconnected mechanisms - controlled by a sophisticated network involving signal-transduction cascades, metabolic pathways and gene expression regulation. In this thesis, bacterial stress responses towards two different environmental stressors are analysed; mainly focussing on the regulatory mechanisms that give rise to the overall cellular response. The first part of this thesis addresses the heme stress response in Corynebacterium glutamucim. Heme is an essential cofactor and alternative iron source for almost all bacterial species but can cause severe toxicity when present in elevated concentrations. Consequently, heme homeostasis needs to be tightly controlled. Therefore, one important challenge is to understand how bacteria regulate heme stress responses to both benefit from heme while simultaneously eliminating the associated toxicity. It is shown that C. glutamicum induces a heme detoxification mechanism (mediated via the heme exporter HrtBA) and a heme utilization mechanism (mediated via the heme ogygenase HmuO) in a temporal hierarchy, with prioritisation of detoxification over utilization. A combined approach of experimental reporter profiling and computational modelling reveals how differential biochemical properties of the two two-component systems that sense heme in C. glutamicum - ChrSA and HrrSA - and an additional regulator (the global iron-regulator DtxR) control this hierarchical expression of the two stress response modules. This analysis sheds light on the multi-layered heme stress response that contributes to overall heme homeostasis in C. glutamicum and adds on to the understanding of bacterial strategies to deal with the Janus-faced nature of heme. The second part of this thesis focusses on bacterial response strategies towards cell wall antibiotics, which play a key role in bacterial antibiotic resistance. To combat resistance evolution, it is important to understand how cell wall antibiotics affect bacterial cell wall biosynthesis and how bacteria orchestrate stress response mechanisms to protect themselves from cell wall damage. The first question is addressed through a comprehensive mathematical model describing the bacterial cell wall synthetic pathway - the lipid II cycle - and its systems-level behaviour under antibiotic treatment. It is found that the lipid II cycle features a highly asymmetric distribution of pathway intermediates and that the efficacy of antibiotics in vivo scales directly with the abundance of targeted pathway intermediates: The lower the relative abundance of a lipid II intermediate within the lipid II cycle, the lower the in vivo efficacy of an antibiotic targeting this intermediate. This leads to the formulation of a novel principle of ‘minimal target exposure’ as an intrinsic bacterial resistance mechanism and it is demonstrated that cooperativity in drug-target binding can mitigate the associated resistance. The development of new drugs to counteract antibiotic resistance clearly benefit from these insights. The second question is then addresses by an experimental-based expansion of the model, which allows the analysis of the interplay between multiple stress response mechanisms that protect against a single antibiotic - focussing here on the well-studied response of Bacillus subtilis towards the cell wall antibiotic bacitracin. This study reveals that the properties of the lipid II cycle itself control the interaction between the primary bacitracin stress response determinant BceAB mediating bacitracin detoxification, and the secondary determinant BcrC, which contributes to cell wall homeostasis under bacitracin treatment. By elucidating regulatory mechanisms of the multi-layered response towards bacitracin, this analysis contributes to an advanced understanding of bacterial antibiotic resistance

The role of the DSB system in antimicrobial resistance

Extensive use of antibiotics in medicine and agriculture has led to increasing emergence of antimicrobial resistance in bacterial populations. Dwindling resources in the discovery of novel active compound leads and the increasing demands for safety and efficacy of new drugs mean that we are now faced with treatment failures due to multi-drug resistant pathogens. In the quest for new targets that will enable us to counter antibiotic resistance, it is often ignored that many resistance mechanisms precede the clinical use of antibiotics. Instead, the ability to adapt, survive and bypass the toxicity of many chemical compounds is wired within the bacterial genome. Continuous inter-strain and inter-species competition have given microorganisms tools to thrive under conditions of chemical warfare. Recognising this is important when characterising mechanisms underpinning bacterial antimicrobial resistance, as it can lead to novel strategies that can help us bypass it. The work described here explores the connection between the disulfide bond formation system, a key oxidative protein folding pathway in the cell envelope of Gram-negative bacteria, and two widespread antimicrobial resistance mechanisms, b-lactamase catalysed hydrolysis of b-lactam antibiotics and efflux-mediated drug expulsion. It is demonstrated that oxidative-protein-folding-mediated proteostasis is crucial for both resistance mechanisms, and its inhibition can sensitise multidrug-resistant pathogens to existing antibiotics. Preliminary results from an experimental evolution approach, set the scene for future exploration of the importance of disulfide linkages for the capacity of b-lactamase enzymes to evolve under selective pressure. Together, these findings aim to address the mechanistic basis of a new avenue for antibiotic adjuvant therapy, whereby targeting a non-essential process would allow us to potentiate existing antibiotics towards previously resistant bacterial strains. With novel essential targets against bacteria being scarce, adjuvant approaches like this one could prolong the use and efficacy of existing drugs against some of the most resistant Gram-negative pathogens.Open Acces