Protein sociology of ProA, Mip and other secreted virulence factors at the Legionella pneumophila surface

Abstract

The pathogenicity of L. pneumophila, the causative agent of Legionnaires' disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted "protein sociology" of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

Keywords: Legionella pneumophila; computational biology; interactomics; macrophage infectivity potentiator; secreted effectors; surface-associated proteins; zinc metalloprotease ProA.

Figure 1

ProA mediates virulence regulation, host tissue degradation and immune evasion. The virulence factor ProA can interfere with a wide variety of infection-associated processes via the diverse range of substrates. During Legionnaires’ disease, it significantly mediates tissue degradation due to structural host targets such as collagen IV of the basal lamina and the cell adhesion protein vitronectin. ProA also cleaves α₁-antitrypsin, important for controlling of host proteases. In addition, ProA exhibits hemolytic properties and contributes significantly to immune evasion. The zinc metalloprotease not only cleaves humoral serum components and chemokines (IL-2, IL-6, TNF-α), but also influences the recruitment and proliferation of various immune cells via these signaling molecules of specific surface receptors. However, ProA also attacks cellular structures directly, for example, by degrading the CD4 receptor. Aside from host targets, the protease also affects other bacterial proteins that play an important role during the intracellular life cycle of L. pneumophila. Via cleavage of exogenous flagellin, the TLR5-mediated immune response and thus the cytokine expression are reduced. Through direct processing, ProA can additionally activate virulence factors such as PlaA/PlaC and LapA, which contribute to lipid degradation at the LCV and amino acid synthesis (created with BioRender.com).

Figure 2

Preproenzyme composition, structure and catalytic center of L. pneumophila M4 zinc metalloprotease ProA. (A) Schematic overview of the ProA preproenzyme containing a signal- and a propeptide (60 kDa, 543 amino acids). Secretion and autoprocessing at residue Glu 208 results in a 38 kDa mature peptidase unit (teal and green). Important residues for cofactor coordination (red) and proteolytic activity (pink) are highlighted within the zinc-binding motifs. Especially the HExxH motif is highly conserved among the M4 family of TLPs. (B) Two domain structure of ProA (PDB code: 6YA1) with N-terminal β-sheets (teal) and C-terminal α-helices (green). A close-up of the active center displays the zinc cofactor as well as amino acid residues for coordination and catalysis (created with PyMOL 2.0 and BioRender.com).

Figure 3

AlphaFold v2.2 multimer modeling of the interaction between L. pneumophila ProA and FlaA. (A) Complex of the protease ProA in magenta and monomeric FlaA in marine blue. The binding interface with the most contacts is localized within the D₀ polymerization domain of flagellin. (B) Modeling of ProA (magenta) and generic polymerized FlaA (marine blue). The flagellar filament was generated by aligning the predicted L. pneumophila FlaA structure on the known Cryo-EM structure of the P. aeruginosa filament (PDB ID: 5WK6) (Karagöz et al., 2022). ProA was directly integrated into a deranged flagellar structure, due to steric clashes in the FlaA polymerization region. These clashes were quantified using the repulsive Lennard-Jones (LJ) Energy score from the Rosetta scoring modeling suite according to Alford et al., 2017 and corroborated that this is not a possible protein-protein interaction (Alford et al., 2017). (C) Typically assembled filament without access for ProA (ProA placed randomly in vicinity). The conserved cleavage sites of FlaA are hidden within the polymerized flagellar filament.

Figure 4

Mip mediates virulence regulation, host tissue degradation and immune evasion. The virulence factor Mip interferes with a wide variety of infection-associated processes via different interaction partners. During Legionnaires’ disease, it is important that bacteria migrate through different tissue barriers. This is facilitated by binding of Mip to collagen IV, which is subsequently degraded by a serine protease. Mip binds to SspB, Lpc2061 and FlaA, and promotes flagellation by its PPIase domain that can be inhibited by FK506 and rapamycin. Binding of FK506 and rapamycin to PPIases modulates immunometabolism and interferes with bacterial immune evasion. Mip regulates stress response and infectivity in bacteria together with other PPIases and promotes Phospholipase C (PLC) activity (created with BioRender.com).

Figure 5

Docking of FK506 to Mip and competitive inhibition of Lpc2061 binding. (A) Docking of FK506 (grey) to the Mip homodimer (green and cyan) results in binding to the dimerization and the two PPIase domains. (B) Lpc2061 (magenta) binding to Mip is inhibited by FK506, since the inhibitor blocks this triangle. (C) Overlapping binding regions of Lpc2061 and FK506 after rotation by 180°. Molecular docking was performed using DiffDock (Corso et al., 2022).

Figure 6

Surface plot visualization of the tripartite complex of Mip, FlaA and Lpc2061. Modeling was perfomed by docking of FlaA (marine blue) to the best pose of Lpc2061 (magenta) and the Mip homodimer (green and cyan). While Lpc2061 binds to the Mip dimerization region, monomeric FlaA interacts with the C-terminus of the PPIase domain. This tripartite complex was found to be more stable than the binary Mip-Lpc2061 interaction (Karagöz et al., 2022).