Interdisciplinary Initiatives Program Round 12 - 2024
Project Investigators:
Tino Pleiner, Molecular & Cellular Physiology
Alex Gao, Biochemistry
Abstract:
The development of antibiotics stands out as one of the most extraordinary achievements of modern science that helped save millions of lives from once deadly infectious diseases. The excessive overuse of antibiotics, however, has given rise to many antibiotic-resistant bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA), a human pathogen that poses a significant public health threat. MRSA infections alone result in over 100,000 global deaths annually and contribute to escalating healthcare costs. There is a pressing need to identify new targets for a next generation of more potent and selective antibiotics.
In this proposal, we describe how bacterial anti-phage defense proteins, present in both gram-positive and gram-negative bacterial membranes and cytosol, can be repurposed as novel antibiotic targets. Many defense proteins share a common activation mechanism: recognition of target bacteriophage proteins triggers oligomerization (e.g. tetramerization) activating a downstream catalytic domain that induces cell death and stops the phage infection from spreading to the rest of the population. The discovery of this altruistic mechanism represents a timely opportunity to leverage anti-phage defense proteins as druggable kill-switches.
To activate these kill-switches, we plan to develop genetically encoded single-domain antibodies, or nanobodies, from alpacas against specific MRSA anti-phage defense proteins. By fusing nanobodies to engineered oligomerization domains, we can arrange MRSA defense proteins into oligomeric assemblies, mimicking phage-induced activation and causing cell death. To identify the most potent nanobodies, we will screen oligomeric nanobody libraries using high-throughput cell death assays in Escherichia coli that use deep sequencing as a read-out. Successful nanobodies will undergo in vitro characterization, including functional assays and structural analysis by cryo-electron microscopy, before testing them in Staphylococcus aureus.
Since nanobodies can be cheaply produced in gram-scale as purified proteins or delivered into cells in DNA or mRNA form, they should allow for cost-efficient mass production in reproducible quality. We envision that selective nanobodies against extracellular and intracellular anti-phage defense proteins of human pathogens like MRSA can be developed into a new class of antibiotics. Beyond their use as antibiotics, we also foresee that bacterial species or even strain-selective cell-death inducing nanobodies can be used to modulate the composition of complex bacterial communities like the gut microbiome for applications in both basic research and as therapeutics.
