The Tol Pal system integrates maintenance of the three layered cell envelope

Abstract

The rapid emergence of antibiotic-resistant superbugs poses a significant global health threat. Gram-negative bacteria are the primary culprits due to their robust, tripartite cell envelope. This review explores the emerging role of the trans-envelope Tol-Pal system in maintaining envelope integrity, by connecting envelope layers and serving as a protein interaction hub. Targeting the Tol-Pal system offers a promising approach for the development of novel envelope-disrupting antimicrobials.

Fig. 1. The OM is supported by numerous PG-bound lipoproteins ensuring its continuity and stability.

a E. coli OM is connected to PG by covalently bound lipoprotein, Lpp, and two non-covalently bound proteins lipoprotein Pal and OmpA, that both contain a OmpA-like PG binding domain,. The relative mobility of the lipoproteins, which allows them to be redistributed around the cell, is correspondingly illustrated above. b As a cell grows, divides, and responds to the changing environment, it requires constant remodelling of the OM-PG scaffold. In particular, modulations in the stabilising contacts afforded by lipoproteins. To redistribute Pal the other core proteins of Tol-Pal system form an envelope spanning complex to bind and mobilise PG-bound Pal, typically to the division site. For Lpp covalent crosslinks are removed from PG by Lpp-PG amidase.

Fig. 2. Multifarious roles of Tol-Pal complex make it an excellent antibiotic target.

Loss of Tol-Pal function results in pleiotropic phenotype resulting in lowered pathogen burden, heightened antibiotic sensitivity, and accelerated removal of bacteria by the immune system.

Fig. 3. Functionality of the Tol-Pal system depends on its recruitment to the septum.

TolA and TolQ are recruited to the divisome separately, however both require FtsN and the presence of FtsI-driven PG synthesis for transport. Whilst TolA is generally intact within cells, it has been shown that TolA domains I, II, and III can be recruited to the divisome independent of each other. TolAI requires TolQ for recruitment, TolAII trafficking is facilitated by an unknown envelope partner/s, and TolAIII requires a combination of Tol-Pal proteins including CpoB, TolB, and Pal.

Fig. 4. The Tol-Pal system interacts with a diverse array of envelope associated proteins that are involved in OM and PG synthesis.

Confirmed protein-protein interactions of the Tol-Pal system with division-related proteins are indicated by arrows. Dashed arrows represent interactions that have not been shown to be direct. Interactions involved in Tol-Pal regulation of PG synthesis are shown in blue, interactions with cytoplasmic membrane proteins are in grey, and periplasmic interactions are in purple. TolA (yellow) remains the central regulator of many interactions for both PG synthesis and division. How NlpD interacts with the system is yet to be understood.

Fig. 5. CpoB, directly links the Tol-Pal system to PG synthesis.

The Tol-Pal operon encoding seven proteins is shown with CpoB, the last protein in the sequence highlighted in purple. TolA and CpoB both modulate the activity of PBP1B, an auxiliary PG synthetase in the septum of dividing cells. Both the lipoprotein, LpoB, and TolA increase PBP1B activity, while CpoB inhibits LpoB-driven hyperactivation. Whilst TolA modulates CpoB oligomeric state, it is not known which state CpoB adopts when interacting with LpoB-PBP1B.