# Direct and indirect pathways linking the Lon protease to motility behaviors in the pathogen Pseudomonas aeruginosa

## TL;DR

This study identifies new proteins degraded by the Lon protease in Pseudomonas aeruginosa, linking Lon to motility and pathogenicity.

## Contribution

The study identifies multiple new Lon substrates in P. aeruginosa, including key motility and cell division regulators.

## Key findings

- Lon degrades several motility-related proteins like FliG, FliS, and FlgE in P. aeruginosa.
- Lon-dependent degradation of the cell division inhibitor SulA is essential for motility and growth.
- Lon regulates flagellar and type IV pilus-mediated motility through targeted protein degradation.

## Abstract

The ATP-dependent cytoplasmic protease Lon has critical functions in protein quality control and cellular regulation in organisms across the three domains of life. In the opportunistic pathogen Pseudomonas aeruginosa, lon loss-of-function mutants exhibit multiple phenotypic defects in motility, virulence, antibiotic tolerance and biofilm formation. However, only a couple of native substrate proteins of Lon are described in P. aeruginosa until now and most of the phenotypes associated with Lon remain unexplained. Here, we searched for novel Lon substrates in P. aeruginosa by analyzing proteome-wide changes in protein levels and stabilities following lon overexpression. Our search yielded a large number of putative Lon substrates with diverse cellular functions, including metabolic enzymes, stress proteins and a significant fraction of motility-related proteins. In vitro degradation assays confirmed the metabolic protein SpeH, the heat shock protein IbpA as well as seven proteins involved in flagella- and type IV pilus-mediated motility as novel substrates of Lon. The new motility-associated substrates include both key regulators of motility (FliA, RpoN, AmrZ) as well as structural flagellar components (FliG, FliS and FlgE). Further, by isolating suppressor mutations bypassing the motility defect of lon- cells, we reveal that Lon-dependent degradation of the specific substrate SulA, a cell division inhibitor, is crucial for ensuring proper cell division and motility under optimal conditions. In sum, our work highlights Lon’s regulatory role in degrading functional proteins involved in critical cellular processes and contributes to a better molecular understanding of the pathways underlying Pseudomonas pathogenicity.

Proteases specifically degrade other proteins inside cells and constitute promising drug targets due to their central roles in cell physiology. In the human pathogen Pseudomonas aeruginosa, the protease Lon has been implicated in the regulation of various processes that are linked to the ability of P. aeruginosa to tolerate antibiotic exposure and to establish infections. Here, we have determined the group of proteins that are directly degraded by Lon in P. aeruginosa. Among these proteins, we found several proteins known to be involved in swimming, swarming and twitching motility, which are critical behaviors of P. aeruginosa during host colonization. Further, we found that by degrading a cell division inhibitor protein, Lon ensures proper cell division, growth and motility under optimal growth conditions. Our work sheds light onto the molecular mechanisms linking the Lon protease to bacterial pathogenicity, which is critical for exploring the potential of Lon and other cellular proteases as antibiotic drug target.