Zinc Metalloprotease ProA from Legionella pneumophila Inhibits the Pro-Inflammatory Host Response by Degradation of Bacterial Flagellin

Abstract

The environmental bacterium Legionella pneumophila is an intracellular pathogen of various protozoan hosts and able to cause Legionnaires’ disease, a severe pneumonia in humans. By encoding a wide selection of virulence factors, the infectious agent possesses several strategies to manipulate its host cells and evade immune detection. In the present study, we demonstrate that the L. pneumophila zinc metalloprotease ProA functions as a modulator of flagellin-mediated TLR5 stimulation and subsequent activation of the pro-inflammatory NF-κB pathway. We found ProA to be capable of directly degrading immunogenic FlaA monomers but not the polymeric form of bacterial flagella. These results indicate a role of the protease in antagonizing immune stimulation, which was further substantiated in HEK-BlueTM hTLR5 Detection assays. Addition of purified proteins, bacterial suspensions of L. pneumophila mutant strains as well as supernatants of human lung tissue explant infection to this reporter cell line demonstrated that ProA specifically decreases the TLR5 response via FlaA degradation. Conclusively, the zinc metalloprotease ProA serves as a powerful regulator of exogenous flagellin and presumably creates an important advantage for L. pneumophila proliferation in mammalian hosts by promoting immune evasion.

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

“…Purified and active ProA demonstrably abolished the receptor activation caused by monomeric flagellin. Moreover, continuative experiments displayed diminished recognition of a flaA deletion mutant but increased values with a proA deletion strain compared to the L. pneumophila wild type using bacterial suspensions as well as HLTE infection supernatants (Scheithauer et al, 2022). Conclusively, ProA leads to a significant reduction of flagellin-dependent TLR5 stimulation in a physiological human background.…”

“…Forty years after discovery, the role of ProA was specified recently in human lung tissue explants (HLTEs) using purified protease as well as proA mutant strains. This detailed elucidation evidenced protease-dependent tissue destruction with swelling and disintegration of alveolar septa as well as increased bacterial proliferation, transmigration, and immune evasion of ProA-expressing Legionella strains in the physiological human background (Figure 1) (Scheithauer et al, 2021;Scheithauer et al, 2022).…”

“…Nevertheless, ProA can also regulate excess of certain proteins by simple degradation. For example, it reduces free immunogenic flagellin monomers in the extracellular space, which are otherwise recognized by the host immune system (Mascarenhas and Zamboni, 2017;Scheithauer et al, 2022).…”

“…The finding that the flagella subunit FlaA from L. pneumophila is a physiological substrate of the zinc metalloprotease ProA might be of high relevance during the infection of human lung tissue. Interestingly, only monomeric flagellin was shown to be digested, while polymerized FlaA assembling the filament remains stable (Scheithauer et al, 2022). This is important to preserve its crucial functions for the intracellular life cycle.…”

Concept about the Virulence Factor of Legionella

“…An effector protein called ProA is a zinc metalloprotease that is translocated by the type II secretion system and has been demonstrated to suppress the immune response by modifying PPR ligands during the transmissive phase. It can prevent flagellin-TLR5-mediated NF-κB pathway activation by degrading immunogenic FlaA monomers [ 172 , 216 ]. Besides that, several effectors translocated by the T4SS secretion system were proven to participate in host immune signaling inhibition.…”