Are teichoic acids responsible for formation of the Gram-positive bacterial pseudo-periplasm?

Gram-positive (monoderm) bacteria, including clinically important pathogens such as Streptococci, are characterized by a single cell membrane surrounded by a thick cell wall. This proposal will address a crucial puzzle: how do these bacteria, which maintain a massive internal pressure of 30 atmospheres, avoid having their cell membrane compressed tightly against the cell wall — a scenario that would immobilise all transmembrane proteins and thus prove fatal to the cell? To address this, the existence of a pseudo-periplasmic space between the membrane and cell wall has been proposed, in which proteins and other large molecules can move freely. This is supported by electron microscopy observations of a low density “inner-wall zone” region of the Gram-positive cell envelope in multiple species. Recently, it was hypothesised that teichoic acids, highly expressed poly-electrolyte polymers which are essential in almost all Gram-positive bacteria, may form the pseudo-periplasm by acting as gel-like spacer layer. Teichoic acids have multiple known functions in the Gram-positive cell envelope, but why they are essential remains a mystery. Demonstrating that they are responsible for formation of the pseudo-periplasm would be a major advance in our understanding of these polymers. This would ultimately support efforts to design teichoic acid-targeting antibiotics or vaccines. Aims and objectives: We will test the central hypothesis that teichoic acids are responsible for the formation of the Gram-positive pseudo-periplasm and determine the core biophysical properties of this enigmatic spacer layer. First, we will execute two parallel work packages focussed on the genetically tractable Bacillus subtilis, where we uniquely benefit from established simultaneous knockouts/depletions of all teichoic acid synthases: WP1: Computationally determine the physical and biochemical feasibility of the teichoic acids acting as a pseudo-periplasm spacer layer. Key methodologies: Coarse-grained computational modelling (Molecular Dynamics using Martini 3, and Brownian Dynamics using LAMMPS). WP2: Experimentally determine the physical properties of the pseudo-periplasm and the role of teichoic acids in pseudo-periplasm formation. Key methodologies: single molecule tracking, quantitative light microscopy, cryo-electron tomography, molecular microbiology. Second, we will apply these methodologies to investigate maintenance of the pseudo-periplasm in the clinically important pathogen Streptococcus pneumoniae, whose teichoic acids lack any overall charge, unlike most other Gram-positive bacteria: WP3: Determine how uncharged teichoic acids in S. pneumoniae change the biophysical properties of the periplasmic space. Outcomes: This fundamental bioscience study will elucidate the properties of the Gram-positive pseudo-periplasm and thus substantially improve our understanding of the cell envelope of most Gram-positive bacteria. This fits BBSRC priorities of understanding the rules of life, particularly antimicrobial resistance and engineering biology, and developing a highly-skilled bioscience workforce. Tangible benefits and potential impacts: Developing a comprehensive understanding of the pseudo-periplasm and teichoic acid function will be broadly useful for bacterial cell envelope biology topics such as osmoregulation and cell wall synthesis. As teichoic acids are essential and represent promising targets for antimicrobials and vaccines, uncovering their fundamental role in Gram-positive bacteria will pave the way for innovative approaches in the rational design of next-generation therapeutics. Understanding of the molecular basis of the pseudo-periplasm – particularly in the industrial workhorse Bacillus subtilis - may offer new biotechnology opportunities for strain optimisation in industrial fermentation and protein secretion. The new coarse-grained computational modelling framework will be adaptable to other biopolymers containing sugar molecules, such as glycans, expanding beyond the extensively studied DNA and protein polymers.