# High-Throughput Analysis of the Flagella FliK-Dependent Surfaceome and Secretome in Bacillus thuringiensis

## TL;DR

This study explores how a protein called FliK in Bacillus thuringiensis affects the bacteria's ability to resist immune defenses and secrete harmful proteins.

## Contribution

The study identifies FliK's role in regulating the secretome and surfaceome of B. thuringiensis, revealing new candidate proteins for combating antibiotic resistance.

## Key findings

- FliK deficiency significantly alters the secreted protein profile of B. thuringiensis.
- The absence of FliK impacts resistance to antimicrobial peptides and virulence.
- The study identifies candidate proteins for further investigation into bacterial resistance mechanisms.

## Abstract

Bacteria employ diverse virulence strategies to invade host tissues and damage them while evading immune defenses. Growing evidence indicates that flagella structures, primarily involved in motility, also contribute to different aspects of virulence beyond movement/motility. In particular, the secretion apparatus of the flagellum, which governs its assembly, also plays a crucial role in the secretion of virulence factors. Using Bacillus thuringiensis (B. thuringiensis), a bacterium known for its use as a biocontrol agent, we have recently identified FliK, a key component of the flagella export apparatus, as essential for resistance to antimicrobial peptides, which act at the forefront of highly conserved immune defenses. To better understand the role of FliK, we conducted a large-scale comparative analysis of the protein composition secreted by a fliK-deficient strain and its reference counterpart. Our findings reveal that the absence of FliK and of a functional flagellar apparatus significantly alters the secreted protein profile of B. thuringiensis. Most importantly, our study identifies promising candidate proteins for further investigation, potentially unveiling new strategies to combat antibiotic resistance.

Bacterial pathogens employ multiple strategies to invade and damage host tissues while evading immune defenses. Recent studies highlight flagella as crucial contributors to bacterial virulence, not only by facilitating motility, but also by regulating the secretion of virulence factors. However, the role of the flagella-dependent secretome remains largely unexplored. We have recently shown that FliK, a key regulator that defines substrate specificity in the flagellar export apparatus, is essential for the resistance of Bacillus thuringiensis (B. thuringiensis) against antimicrobial peptides (AMPs) and its virulence in a Drosophila infection model. In this study, we used liquid chromatography–tandem mass spectrometry to conduct a large-scale comparative analysis of the proteins secreted in culture supernatant or associated with the cell wall of the ΔfliK mutant and its reference strain. Our results reveal significant differences in the secretome and surfaceome of the ΔfliK mutant compared to the reference strain. These findings emphasize the role of FliK in regulating the production and secretion of several proteins, underscoring the importance of flagella in controlling various biological processes. This work provides valuable insights into the functional characterization of potential candidate proteins involved in B. thuringiensis virulence and AMP resistance mechanisms. Overall, these results open new perspectives for understanding the molecular processes that govern bacterial resistance to AMPs.