Cracking Antimicrobial Resistance

Antimicrobial resistance is one of the biggest threats to global health, requiring immediate and concerted action across multiple disciplines. In our work and we seek to break antimicrobial resistance with interdisciplinary approaches from systems and synthetic biology. In a first, top-down approach we aim at deriving a systems-level understanding of antimicrobial resistance networks in Gram-positive bacteria. To this end, we combine experiments and mathematical modelling to develop systems-level descriptions of key metabolic processes in the cell and study the interplay between antibiotic perturbations and the compensatory regulation of resistance mechanisms. Our key long-term goals are the identification of novel targets for therapeutic intervention and the prediction of synergistic drug-drug interactions. Complementing these top-down efforts of understanding natural resistance networks, in a second, bottom-up approach we exploit this knowledge to rationally engineer new ways of interfering with these resistance networks, thereby disarming resistant bacteria and thus re-sensitising them to antibiotics that have turned ineffective.

Understanding cell wall antibiotic action

Cell wall antibiotics are crucial for combatting the emerging wave of resistant bacteria. Yet, our understanding of antibiotic action is limited, as many strains devoid of all resistance determinants display far higher antibiotic tolerance in vivo than suggested by the antibiotic-target binding affinity in vitro. To resolve this conflict, we developed a comprehensive theory for the bacterial cell wall biosynthetic pathway and study its perturbation by antibiotics. It turned out that the closed-loop architecture of the lipid II cycle of wall biosynthesis features a highly asymmetric distribution of pathway intermediates, showing that antibiotic tolerance scales inversely with the abundance of the targeted pathway intermediate. We formalize this principle of minimal target exposure as an intrinsic resistance mechanism and predict how cooperative drug-target interactions can mitigate resistance. The theory accurately predicts the in vivo efficacy of various cell wall antibiotics in different Gram-positive bacteria and contributes to a systems-level understanding of antibiotic action.

The theory also helps us to understand how different active resistance mechanisms are orchestrated within the intricate cell envelope stress response network of bacteria. For instance, when challenging Bacillus subtilis with the cell wall antibiotic bacitracin, we find a high level of cross-regulation between the major resistance modules. To rationalize these data, an expansion of our theory for the lipid II cycle captures the complex interplay between these modules and the cell wall biosynthetic cycle and accurately predicts the minimal inhibitory bacitracin concentration in mutant cell lines deficient in different resistance modules. This highlights how bacterial resistance emerges from an interlaced network of redundant homeostasis and stress response modules.

Based on these insights our group seeks to break resistance by developing novel strategies to interfere with the resistance gene expression network, using tools from systems and synthetic biology available in our lab. The current focus is to target cell wall antibiotic resistance in Gram-positive bacteria, including the model organism B. subtilis and the human pathogen Enterococcus faecalis.

The lipid II cycle of Gram-positive bacteria is a prime target for antibiotics.

Selected publications

Piepenbreier H., Diehl A., and Fritz G. (2019), Minimal exposure of lipid II cycle intermediates triggers cell wall antibiotic resistance. Nature Communications, 10, 2733

Piepenbreier H., Sim A., Kobras C.M., Radeck J., Mascher T., Gebhard S. and Fritz G. (2020) From modules to networks: A systems-level analysis of the bacitracin stress response in Bacillus subtilis. mSystems, 5, e00687-19

Radeck J., Fritz G., Mascher T. (2017). The cell envelope stress response of Bacillus subtilis: From static signaling devices to dynamic regulatory network. Current Genetics 63, 79-90

Radeck J., Orchard P.S., Kirchner M., Höfler C., Gebhard S., Mascher T., and Fritz G. (2016). Anatomy of the bacitracin resistance network in Bacillus subtilis. Molecular Microbiology 100, 607–620

Fritz G., Dintner S., Treichel N., Radeck J., Gerland U., Mascher T., Gebhard S. (2015). A new way of sensing: Need-based activation of antibiotic resistance by a flux-sensing mechanism. mBio 6, e00975-15

Engineering tools for Synthetic Biology

Synthetic biology applies rational engineering principles, including standardisation, modularisation and modelling, to equip living organisms with exciting functionalities. In our work, we develop genetic and computational tools to facilitate the adoption of these engineering principles in biology. This includes the construction of modular cloning toolboxes featuring hundreds of characterised DNA parts, enabling standardised work with Escherichia coli, Bacillus subtilis and Vibrio natriegens. Within this framework, we explore the potential of alternative σ factors as orthogonal transcriptional regulators, and how they can convey robust, context-independent control over gene expression. This work is guided through the development of bioinformatics tools, allowing us to analyze σ factor phylogenies and predict their target promoters, putative regulators and σ factor/anti-σ factor interactions.

ECF σ factors play an important role in natural stress responses (top) and serve as ideal building blocks for the construction of synthetic gene expression programs to introduce temporal control in biosynthetic pathway activation (bottom).

Building orthogonal gene expression programs

Despite rational engineering ambitions underlying synthetic biology, our limited quantitative understanding of complex biological processes in living cells can hamper the successful forward design of synthetic circuits. In fact, orthogonality, while at the heart of any classical engineering effort, remains a major challenge in synthetic biology, due to the high interconnectivity of all parts within biological cells. In our work, we seek to overcome these limitations by harnessing extracytoplasmic σ factors (ECFs) as orthogonal regulators for synthetic circuit design. These alternative σ factors are subunits of the RNA polymerase and are found in almost all bacterial species. They regulate diverse processes and often respond to perturbations of extracytoplasmic functions – hence their name. Today, our bioinformatic work has revealed more than 150 phylogenetically distinct ECF groups, which often recognize group-specific target promoter motifs, making them ideal building blocks for developing multiple, orthogonal switches that can be simultaneously used in a bacterial cell. We use those switches to build higher-level circuits, for example, those that can serve as autonomous "timer circuits" to control biosynthetic pathway activation with defined time delays. Such temporal control on gene expression can be advantageous, e.g. if the final product of the pathway is toxic to the heterologous host cell.

Selected publications

Pinto D.*, Vecchione S.*, Wu H., Mauri M., Mascher T., and Fritz G. (2018), Engineering orthogonal synthetic timer circuits based on extracytoplasmic function σ factors. Nucleic Acids Research 46, 7450–7464

Casas-Pastor D., Müller R.R., Becker A., Buttner M., Gross C., Mascher T., Goesmann A., and Fritz G. (2021), Expansion and re-classification of the extracytoplasmic function (ECF) σ factor family. Nucleic Acids Research 49, 986-1005

Iyer S.C., Casas-Pastor D., Kraus D., Mann P., Schirner K., Glatter T., Fritz G., Ringgaard S. (2020), Transcriptional regulation by σ-factor phosphorylation in bacteria. Nature Microbiology 5, 395–406

Casas-Pastor D., Diehl A., Fritz G. (2020) Coevolutionary Analysis Reveals a Conserved Dual Binding Interface between Extracytoplasmic Function σ Factors and Class I Anti-σ Factors. mSystems, 5, e00310-20

Wu H., Liu Q., Casas-Pastor D., Dürr F., Mascher T. & Fritz G. (2019), The role of C-terminal extensions in controlling ECF σ factor activity in the widely conserved groups ECF41 and ECF42. Molecular Microbiology 112, 498-514

Building Applications for Biotechnology

Under construction...

Research Infrastructure

Located in the iconic Bayliss Building on UWA Crawley campus, our lab provides and shares access to excellent in-house research infrastructure:

  • High-throughput liquid handling robotics facility including

    • Beckman & Coulter Echo 650 acoustic nanoliter dispenser

    • Integra Assist Plus microliter pipetting platform

    • Multiple microplate readers

  • Biomolecular interactions facility

  • Protein production and structure facility

  • Mass spectrometry facility

  • High-performance computer cluster

  • etc.