Biology subjects

Lauber, F.

Publications and source records attributed to Lauber, F..

5 recordsLinked to original sources

bioRxiv2025

Identification of receptor-binding domains of Bacteroidales antibacterial pore-forming toxins

Bacteroidales are abundant Gram-negative bacteria present in the gut microbiota of most animals, including humans, where they carry out vital functions for host health. To thrive in this competitive environment, Bacteroidales use sophisticated weapons to outmatch competitors. Among these, BSAPs (Bacteroidales Secreted Antimicrobial Proteins) represent a novel class of bactericidal pore-forming toxins that are highly specific to their receptor, typically targeting only a single membrane protein or lipopolysaccharide. The molecular determinants conferring this high selectivity remain unknown. In this study, we therefore investigated the model protein BSAP-1 and determined which of its domains is involved in providing receptor specificity. We clearly demonstrate that receptor recognition is entirely driven by the C-terminal domain (CTD) of BSAP-1 using a combination of in vivo competition assays and in vitro protein binding studies. Specifically, we show that deletion of the CTD abrogates BSAP-1 bactericidal activity by preventing receptor binding, while grafting the CTD to unrelated carrier proteins enables CTD-driven interaction with the BSAP-1 receptor. Building upon this discovery, we show that BSAPs can be categorized according to the structure of their CTD and that BSAPs within the same cluster are likely to target the same type of receptor. Additionally, we show that the CTD of BSAP-1 can be repurposed to generate probes for fluorescent labelling of membrane proteins in live cells. In summary, our research demonstrates that BSAP receptor recognition is driven by their CTD and that these can be engineered to develop novel tools for the investigation of Bacteroidales biology.

microbiology↗

bioRxiv2024

TamL is a key player of the outer membrane homeostasis in Bacteroidetes.

In Proteobacteria, the outer membrane protein TamA and the inner membrane-anchored protein TamB form the Translocation and Assembly Module (TAM) complex, which facilitates the transport of autotransporters, virulence factors, and likely lipids across the two membranes. In Bacteroidetes TamA is replaced by TamL, a TamA-like lipoprotein with a lipid modification at its N-terminus that likely anchors it to the outer membrane. This structural difference suggests that TamL may have a distinct function compared to TamA. However, the role of TAM in bacterial phyla other than Proteobacteria remains unexplored. Our study aimed to elucidate the functional importance of TamL in Flavobacterium johnsoniae, an environmental Bacteroidetes. Unlike its homologues in Proteobacteria, we found that TamL and TamB are essential in F. johnsoniae. Through genetic, phenotypic, proteomic, and lipidomic analyses, we discovered that TamL depletion severely compromises outer membrane integrity, as evidenced by reduced cell viability, altered cell shape, increased susceptibility to membrane-disrupting agents, and elevated levels of outer membrane lipoproteins. Notably, we did not observe any impact on outer membrane lipid composition. Via pull-down protein assays, we confirmed that TamL interacts with TamB in F. johnsoniae, likely forming the TAM complex. Furthermore, our in silico analysis revealed that the presence of TamL and TamB monocistronic genes is a shared genetic feature among Bacteroidetes members, including the human pathogen Capnocytophaga canimorsus where we also confirmed the essentiality of the TamL and TamB homologs. To our knowledge, this study is the first to provide functional insights into a TAM subunit beyond Proteobacteria. SignificanceIn Proteobacteria, the outer membrane (OM) protein TamA forms with the inner membrane (IM)-anchored protein TamB the Translocation and Assembly Module Complex (TAM). which contributes to efficient biogenesis of the OM. In Bacteroidetes TamA is replaced by TamL, a TamA-like lipoprotein of unknown role. In this work, we studied TamL in the Bacteroidetes Flavobacterium johnsoniae. We found that TamL and TamB are essential for cell viability, and that TamL depletion disrupts outer membrane stability, increases outer membrane vesicle size, and lead to higher sensitivity to OM stressors. These findings highlight TamL critical role in maintaining OM structure in Bacteroidetes. To our surprise, we also identified multiple TamL, TamB and TamA homologs in Bacteroidetes. Altogether, our findings extend the current knowledge on TAM and provide novel insights into a field of research barely investigated outside Proteobacteria.

microbiology↗

bioRxiv2024

LolA and LolB are conserved in Bacteroidetes and are crucial for gliding motility and Type IX secretion.

In Gram-negative bacteria, lipoproteins are major components of the outer membrane (OM) where they play a variety of roles, from the involvement in membrane biogenesis to virulence. Bacteroidetes, a widespread phylum of Gram-negative bacteria, including free-living organisms, commensals and pathogens, encode an exceptionally high number of outer membrane lipoproteins. These proteins are crucial in this phylum mainly because they are key components of SUS-like nutrient acquisition systems as well as of the Type 9 secretion (T9SS) and gliding motility machineries. The transport of lipoproteins to the OM has mainly been studied in E. coli and relies on the Lol system, composed of the inner membrane extraction machinery LolCDE, the periplasmic carrier LolA and the OM lipoprotein LolB. While most Lol proteins are essential and conserved across Gram-negative bacteria, to date, no LolB homologs have been identified outside of {gamma}- and {beta}-proteobacteria. How lipoproteins reach and are inserted in the OM of Bacteroidetes is not known. Here we identified LolB homologs in Bacteroidetes and disclosed the co-existence of several LolA and LolB in several species. We provide evidence that one LolA (LolA1) and one LolB (LolB1) of F. johnsoniae are devoted to targeting gliding and T9SS lipoproteins to the OM. A proteomic analysis of the OM composition of the lolA1 and lolB1 mutants supports this evidence. Furthermore, we show that, while LolB1 and LolA1 have conserved functions in Bacteroidetes, they are functionally different from their E. coli counterparts. We also show that surface lipoprotein transport is LolA and LolB independent. Finally, the finding that, in the absence of LolA and LolB homologs, lipoproteins still localize to the OM, suggests the presence in Bacteroidetes of yet unidentified LolAB-alternative lipoprotein transport pathways. In conclusion, Bacteroidetes have evolved different and more complex lipoprotein transport pathways than other Gram-negative bacteria and further research is required to uncover their complexity. SignificanceIn Gram-negative bacteria, lipoproteins are key components of the outer membrane (OM), essential for functions like membrane biogenesis and virulence. Bacteroidetes, a widespread phylum, encode a high number of OM lipoproteins crucial for nutrient acquisition, Type IX secretion, and gliding motility. While lipoprotein transport in E. coli depends on the Lol system, LolB homologs were previously unidentified outside {gamma}- and {beta}-proteobacteria. Here we identify LolB homologs in Bacteroidetes, revealing the co-existence of multiple LolA and LolB proteins in various species. In F. johnsoniae, LolA1 and LolB1 specifically target gliding and Type 9 secretion system lipoproteins to the OM. Despite this, lipoproteins still localize to the OM without LolA and LolB, suggesting alternative transport pathways. These findings indicate that Bacteroidetes have evolved more complex lipoprotein transport mechanisms than other Gram-negative bacteria, requiring further research to fully understand them.

microbiology↗

bioRxiv2024

Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

The discovery of Toll-like receptors (TLRs) represented a significant breakthrough that paved the way for the study of hostpathogen interactions in innate immunity. However, there are still major gaps in understanding TLR function, especially the early dynamics of downstream TLR pathways remains less clear. Here, we present a label-free optical biosensor-based assay as a powerful method for detecting TLR activation in a native and label-free environment and defining the dynamics of TLR pathway activation. This technology is sufficiently sensitive to detect TLR signaling and readily discriminates between different TLR signaling pathways. We define pharmacological modulators of cell surface and endosomal TLRs and downstream signaling molecules and uncover previously unrecognized TLR signaling signatures, including biased receptor signaling. These findings highlight that optical biosensor assays complement traditional assays that use a single endpoint and have the potential to facilitate the future design of selective drugs targeting TLRs and their downstream effector cascades.

pharmacology and toxicology↗

bioRxiv2020

Structure of a proton-powered molecular motor that drives protein transport and gliding motility

Ion-driven motors are rare in biology. The archetypes of the three classes identified to date are ATP synthase, the bacterial flagellar motor, and a proton-driven motor that powers gliding motility and protein secretion in Bacteroidetes bacteria. Whilst the molecular mechanism of ATP synthase is now well understood, structural information is lacking for the other two classes of motor. Here we present the structure of the Bacteroidetes gliding motility motor determined by cryo-electron microscopy. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM proteins are positioned within a ring of five GldL proteins. Combining mutagenesis and single-molecule tracking, we identify protonatable amino acid residues within the transmembrane domain of the complex that are important for motor function. Our data imply a mechanism in which proton flow leads the periplasm-spanning GldM dimer to rotate with respect to the intra-membrane GldL ring to drive processes at the bacterial outer membrane. This work provides a molecular basis for understanding how the gliding motility motor is able to transduce the energy of the inner membrane protonmotive force across the bacterial cell envelope.

biochemistry↗