Understanding the biochemical arms race

In the ongoing war between mammalian cells and their bacterial invaders, an incredibly powerful mechanism has evolved to give the hosts the upper hand. Yet, with some deft biochemical trickery, Legionella is taking on evolution… and winning

Bacterial pathogens use a range of mechanisms to invade mammalian hosts and cause diseases. Many pathogenic bacteria use their secretion systems to inject dozens or hundreds of effector proteins – known as virulence factors – into host cells.

These effector proteins can play many different roles – they usually perturb a host’s signalling pathways, suppress its immune response, and help the pathogen to survive. Thus, they often play the most important role in the growth and development of bacterial pathogens.

Organisms have to fight against bacteria and other disease-causing microbes on a daily basis. To stay on top of this, cells use many ways to defend themselves. Autophagy, for example, is a process that breaks down unwanted or damaged molecules and organelles in eukaryotes. It also acts as an innate immune mechanism against infections caused by intracellular pathogens (termed xenophagy). The pathogens are sequestered, engulfed by the phagophore, the precursor to the autophagosome, and subsequently eliminated through autophagosome-lysosome fusion.

Formation of autophagosome is a complex process that requires several steps and molecules. First, cells create a growing membrane sac that collects the debris and eventually seals to form the autophagosome. One of the key proteins that expands the membrane is the protein LC3, which is located on the inner and outer side of the membrane. LC3 must be linked to phosphatidylethanolamine (PE) in order to interact with this membrane. The combined molecule LC3-PE then provides a docking station for receptor proteins that collect and deliver the debris, including bacteria, into the growing autophagosome. A specific part of these receptors called LIR, short for LC3-interacting region motif, connects the receptors to LC3.

Winning the bacteria–autophagy battle However, some virulent bacteria evolve mechanisms to bypass autophagy. One of them is Legionella pneumophila. Infection with Legionella is a common cause of community and hospital-acquired pneumonia with a death rate of up to 30%. The intracellular pathogen Legionella interferes with autophagy by delivering the effector protein RavZ to deconjugate LC3 proteins attached to PE on autophagosomal membranes. Legionella has the victory in this bacterial–autophagy battle, however, how RavZ recognizes and deconjugates LC3-PE is still not known.

To discover this mechanism, we prepared semisynthetic LC3 proteins with different C-terminal modifications using chemical ligation approaches. Using a combination of cell biological, biophysical and structure biological approaches, we have elucidated a novel mechanism of how Legionella bacteria evade host autophagy.

These findings reveal that Legionella bacteria evolved a very clever mechanism to avoid clearance by host autophagy and adapt the conditions for survival.

We find that RavZ extracts of lipid (PE) of LC3 from membrane before cleavage. RavZ specifically targets to autophagosome membranes by interaction with phosphatidylinositol 3-phosphate (PtdIns3P) via its carboxy-terminal domain and association with membranes via the hydrophobic ?3 helix. RavZ initially recognises LC3 via the LIR motif located at the amine-terminal of the protein. Then its ?3 helix associates with the membrane and facilitates extraction of the PE moiety from the membrane and docking of the lipid chains into the lipid-binding site of RavZ. This orients the C-terminal tail of LC3-PE into the active site of RavZ for cleavage.

Laying the bait Autophagy recognises bacteria through autophagy receptors that contain two crucial domains ­­– the ubiquitin-binding domain (UBD) and LC3-interacting region (LIR) motifs – which are important for cargo recognition and interaction with the LC3 proteins, respectively. The receptor binds to the ubiquitinated pathogen through its UBD and recruits it to the autophagosome membrane via the interaction of the LIR motifs with LC3 proteins. The LIR motif is composed of conserved W/Y/FxxL/I/V sequence. The binding of LIR motifs with LC3 proteins is quite conserved, with two key hydrophobic residues playing an essential role in interaction with the hydrophobic pocket of the LC3 proteins.

Three potential LIR motifs of RavZ were identified. We have demonstrated that the LIR2 motif (residue 27–32) plays an essential role in RavZ activity and the RavZ::LC3 interaction. LC3 interacts with the LIR motif of RavZ. This interaction brings LC3 and RavZ in close proximity on the membrane. The LIR::LC3 binding is usually used to recruit autophagy substrate. But now a killer for LC3-PE per se is recruited, resembling a ‘suicide’ mechanism. Therefore, RavZ LIR motif serves as bait for LC3 protein.

We have studied the structure-function relationship of LC3-PE deconjugation by RavZ using semisynthetic LC3 proteins. RavZ activity is strictly dependent on conjugated PE structures, suggesting a direct binding between RavZ and the conjugated PE. Further experiments in vitro and in vivo showed that RavZ extracts LC3-PE from the membrane before cleavage. The lipid-binding site on RavZ was also confirmed, which is structurally related to the lipid-transfer protein Sec14. However, the lipid-binding pocket is closed in the free RavZ structure, suggesting conformational changes occur upon binding to the conjugated PE.

With the knowledge of RavZ::LC3 interaction, we were able to use a peptide derived from the LIR2 sequence to compete for RavZ::LC3 binding, thereby inhibiting RavZ activity.

These findings reveal that Legionella bacteria evolved a very clever mechanism to avoid clearance by host autophagy and adapt the conditions for survival. The novel anti-autophagy mechanism of the virulent bacteria provides significant insights into host-pathogen interactions. Importantly, our study also paves a new avenue for developing drugs against infection by Legionella bacteria.