Biology subjects

Madej, M.

Publications and source records attributed to Madej, M..

5 recordsLinked to original sources

bioRxiv2026

A moonlighting serpin of Tannerella forsythia compromises the bactericidal activity of neutrophils

Serine protease inhibitors (serpins) are extremely rare in prokaryotes, and their role in immune evasion remains unclear. We identified a bifunctional TLR2-binding serpin (miropin) produced by Tannerella forsythia that broadly suppresses neutrophil antimicrobial capacity. Miropin binding to TLR2 primed neutrophils, enhancing phagocytic capacity yet, counterintuitively, promoting bacterial persistence. Mechanistically, like other serpins, miropin acted as an irreversible suicide inhibitor of neutrophil serine proteases (NSPs), blocking their activity upon granule mobilization and NETosis. Unexpectedly, miropin, independent of its protease-inhibitory activity, bound and inhibited myeloperoxidase (MPO), thereby blocking the oxidative burst. This simultaneous suppression of oxidative and non-oxidative bactericidal mechanisms promoted bacterial survival in vivo without significantly altering the tissue inflammatory response. Thus, we identified novel moonlighting roles (TLR2 signaling and MPO inhibition) for an unorthodox prokaryotic serpin and provided context for how T. forsythia and other oral pathogens survive within the subgingival niche, an environment highly enriched in activated neutrophils.

immunology↗

bioRxiv2025

Discovery of a distinct BAM complex in the Bacteroidetes

The BAM ({beta}-barrel assembly machinery) complex is an evolutionarily conserved, multiprotein machine that catalyses the folding and membrane insertion of newly synthesised {beta}-barrel outer membrane (OM) proteins in Gram-negative bacteria. Based on Proteobacteria, bacterial BAM is also structurally conserved, with an essential BamAD core and up to three auxiliary periplasmic lipoproteins of poorly defined function. Here we show, using structural biology, quantitative proteomics and functional assays, that the BAM complex is radically different within the Bacteroidetes, a large and important phylum widely distributed within the environment and animal microbiomes. Cryogenic electron microscopy (cryo-EM) structures of BAM complexes from the human gut symbiont Bacteroides thetaiotaomicron and the human oral pathogen Porphyromonas gingivalis show similar, seven-component complexes of [~]325 kDa in size with most of the mass in the extracellular space. In addition to canonical BamA and BamD, the complexes contain an integral OM protein named BamF that is essential and intimately associated with BamA, as well as four surface-exposed lipoproteins (SLPs) named BamG-J. Together, BamF-J form a large, extracellular dome that likely serves as an assembly cage for the {beta}-barrel-SLP complexes that are a hallmark of the Bacteroidetes. Our data suggest that BAM functionality in Bacteroidetes is substantially expanded from that in Proteobacteria and underscores the importance of studying other phyla for a more complete understanding of fundamental biological processes.

microbiology↗

bioRxiv2024

Unveiling the Molecular Mechanisms of the Type-IX Secretion System's Response Regulator: Structural and Functional Insights

The Type-IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo. First, our structural studies revealed PorX harbours a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorXs effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorXs dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the non-pathogenic Flavobacterium johnsoniae, but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.

biochemistry↗

bioRxiv2020

The essential Porphyromonas gingivalis type IX secretion system component PorZ delivers anionic-lipopolysaccharide to the PorU sortase for transpeptidase processing of cargos

Cargo proteins of the type IX secretion system (T9SS) in human pathogens from phylum Bacteroidetes invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of selected cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ, and found that it consists of two {beta}-propeller moieties followed by a CTD. In this study, we performed structure-based modelling suggesting that PorZ is a carbohydrate-binding protein. We found that recombinant PorZ specifically binds A-LPS. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of the A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the attachment complex composed of PorU, PorZ, and the integral OM {beta}-barrel proteins PorV and PorQ, which is involved in post-translational modification and retention of T9SS cargos on the bacterial surface. Author summaryBacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis. Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS- bound PorZ interacts with sortase with significantly greater affinity, facilitating modification of cargo proteins by the cell-surface attachment complex of the T9SS.

microbiology↗

bioRxiv2019

Dynamic oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis

Porphyromonas gingivalis, an asaccharolytic Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases, such as rheumatoid arthritis, cardiovascular disease and Alzheimers disease. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how those peptides enter the cell is not clear. Here we identify RagAB as the outer membrane importer for peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2 complex with the RagB substrate binding surface-anchored lipoprotein forming a closed lid on the TonB-dependent transporter RagA. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a \"pedal bin\" nutrient uptake mechanism. Together with mutagenesis, peptide binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic OM oligopeptide acquisition machine with considerable substrate selectivity that is essential for the efficient utilisation of proteinaceous nutrients by P. gingivalis.

microbiology↗