Engineering a genome‐reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo . We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro , ex vivo , and in vivo . To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections. Synopsis A non‐pathogenic strain of Mycoplasma pneumoniae is engineered to express biofilm dispersing agents as well as bactericidal peptides against Staphylococcus aureus . The engineered strain efficiently dissolves S. aureus biofilms in vitro and in vivo . Mycoplasma pneumoniae offers unique features that might be of interest for a bacterial‐based therapeutic vector. Here, an attenuated version of this bacterium is generated after studying the role of different pathogenic factors. Using strong synthetic promoters and native secretion signals, a genetic platform is designed coding for antibiofilm and bactericidal enzymes. In vitro , ex vivo and in vivo studies confirmed the ability of the engineered M. pneumoniae strain to efficiently dissolve S. aureus biofilms. Graphical Abstract A non‐pathogenic strain of Mycoplasma pneumoniae is engineered to express biofilm dispersing agents as well as bactericidal peptides against Staphylococcus aureus . The engineered strain efficiently dissolves S. aureus biofilms in vitro and in vivo .