Linke, L. | Search bioRxiv

bioRxiv2025

Bottlenecks and Resistance Evolution against an Antimicrobial 1 Peptide

Bottlenecks - a severe reduction in population size--are ubiquitous for bacteria in natural systems. For example, during the complete metamorphosis of insects, the gut microbiota is bottlenecked due to a massive secretion of immune effectors such as antimicrobial peptides (AMPs) into the gut. However, the effect of natural bottlenecks on resistance evolution of bacteria to AMPs is currently unknown. Here we measured the bottlenecking of the gut microbiota of G. mellonella, the greater wax moth. Based on these population estimates, we tested in vitro the influence of population size on the adaptation of E. coli against an AMP. We used wild-type E. coli and mutator strains with a 100-fold increase in mutation rates to partly disentangle population size from mutant supply. We found that large E. coli mutator strain populations evolved higher resistance than small populations. Population size, however, did not affect adaptation in wild-type strains. They were not able to evolve resistance under our experimental conditions. This shows that natural bottlenecks can benefit insect hosts by combating the evolution of AMP resistance to their resident gut microbiota, but this should also be applicable to infection in other organisms that use AMPs as an immune defense.

evolutionary biology↗

bioRxiv2021

An optimized live bacterial delivery platform for the production and delivery of therapeutic nucleic acids and proteins

There is an unmet need for delivery platforms that realize the full potential of next-generation therapeutic and vaccine technologies, especially those that require intracellular delivery of nucleic acids. The in vivo usefulness of the current state-of-the-art delivery systems is limited by numerous intrinsic weaknesses, including lack of targeting specificity, inefficient entry and endosomal escape into target cells, undesirable immune activation, off-target effects, a small therapeutic window, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we present our characterization of a delivery platform based on the use of engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli strain SVC1) for intracellular cargo delivery. The SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to escape the endosome upon intracellularization, and to have minimal immunogenicity. Here we report findings on key features of this system. First, we demonstrated that bacterial delivery of a short hairpin RNA (shRNA) can target and silence a gene in an in vitro mammalian respiratory cell model. Next, we used an in vivo mouse model to demonstrate that SVC1 bacteria are invasive to epithelial cells of various tissues and organs (eye, nose, mouth, stomach, vagina, skeletal muscle, and lungs) via local administration. We also showed that repeat dosing of SVC1 bacteria to the lungs is minimally immunogenic and that it does not have adverse effects on tissue homeostasis. Finally, to validate the potential of SVC1 bacteria in therapeutic applications, we demonstrated that bacterial delivery of influenza-targeting shRNAs to the respiratory tissues can mitigate viral replication in a mouse model of influenza infection. Our ongoing work is focused on further refining this platform for efficient delivery of nucleic acids, gene editing machinery, and therapeutic proteins, and we expect that this platform technology will enable a wide range of advanced therapeutic approaches.

microbiology↗