Spatial and temporal dynamics of pneumococcal cell wall under host immune response revealed by super-resolution microscopy

Introduction: The cell wall of Streptococcus pneumoniae is a complex, multi-layered structure that surrounds the bacterial cell, providing rigidity, and protection. It also plays a crucial role in host-pathogen interactions by anchoring key proteins involved in immune evasion. The cell wall is composed of a thick peptidoglycan (PG) layer attached to teichoic acids, and lipoteichoic acids. The PG itself is formed of long chains of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) units, which are cross-linked by short peptide chains.

Objectives: Understanding the organization and synthesis of the PG layer and its interactions with cell wall anchored proteins is essential for comprehending how the organism is protected against external cues.

Methods: To explore the relationship between cell wall protein anchoring and the rate of cell wall synthesis, we examined the spatial and temporal distribution of the surface-exposed pneumococcal protein PspA on the reference strain D39 (WT). The subcellular localization was accessed by super-resolution structured illumination microscopy (SIM). The super-resolution 3D image stacks were processed using HuygensPro and Imaris, whereas the 2D images were analysed using custom-developed workflows written in the graphical image processing language JIPipe. We then compared the PG layer synthesis rates between the WT and a PspA mutant strain (ΔpspA). To spatially distinguish between new and old PG and thusly address the temporal resolution, we sequentially labelled new PG with three various fluorescent D-amino acid (FDAA) probes. Throughout the experiments, we grew bacteria in a regular media and under an immunological challenging condition (i.e., presence of serum).

Results: PspA showed a homogenous distribution along the cell surface with the exception of the septal area. Interestingly, when bacteria were exposed to human serum, the PspA relocated to a predominantly mid-cell, septal region. We observed that PG was asymmetrically synthesized. Marked differences occurred in the labelling pattern of ΔpspA when growing in the serum-containing media. There was minimal overlap between old and new PG in the WT strain but this was different in the ΔpspA strain.

Conclusions: We observed for the first time a dynamic spatial positioning of pneumococcal proteins during an infection scenario, thus opening new ways to reassess how pathogens exploit their protein machineries to evade the immune system.