Biology subjects

Potempa, J.

Publications and source records attributed to Potempa, J..

16 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

Chryseobacterium indologenes mediates resistance to osimertinib by activating the IGF1R pathway in EGFR-mutant lung adenocarcinoma

IntroductionLung cancers harboring sensitizing epidermal growth factor receptor (EGFR) mutations typically exhibit an initial response to osimertinib; however, the development of resistance is inevitable, and there are currently no approved targeted therapies available once resistance emerges. Accumulating evidence indicates that intra-tumoral bacteria can influence tumor biology and contribute to therapy resistance in several cancer types, including pancreatic and colorectal cancer. Despite these findings, whether intra-tumoral bacteria play a role in modulating response and resistance to osimertinib in EGFR-mutant lung adenocarcinoma remains largely unexplored. This study aims to investigate the contribution of intra-tumoral bacteria to osimertinib resistance, thereby providing new insights into resistance mechanisms and identifying potential therapeutic strategies. MethodsBacteria previously identified in lung cancer tissue samples were cultured in liquid growth medium, and their preconditioned medium (PCM) was collected. PC9 cells were treated with PCM in the presence or absence of osimertinib to screen for bacteria capable of mediating resistance. Cell viability was assessed using Cell Counting Kit-8 assays. To investigate potential mechanisms, Western blotting and receptor tyrosine kinase (RTK) phosphorylation arrays were performed. Control groups included cells treated with osimertinib alone, PCM alone, or vehicle. Statistical analyses were conducted using Students t-test as appropriate, with p < 0.05 considered statistically significant. Data represent mean {+/-} SD from at least three independent experiments. ResultsThe addition of PCM from Chryseobacterium indologenes restored cell viability in EGFR-mutant lung adenocarcinoma cells treated with osimertinib. PCM exposure markedly increased insulin-like growth factor-1 receptor (IGF1R) phosphorylation levels. PCM did not enhance cell viability when IGF1R was silenced or inhibited with linsitinib, demonstrating the pathways essential role. Proteinase K treatment abolished the ability of PCM to protect cells from osimertinib, and removal of proteins potentially interacting with IGF1R further diminished its efficacy. Finally, PCM also conferred resistance to osimertinib in patient-derived EGFR-mutant lung adenocarcinoma cell lines. ConclusionOur study demonstrates that the intra-tumoral bacteria C. indologenes may significantly influence sensitivity to osimertinib or impart resistance in EGFR-mutant lung adenocarcinoma by activating the IGF1R signaling pathway.

cancer biology↗

bioRxiv2025

Effect of a bacterial Glutaminyl Cyclase inhibitor on multi-species-biofilms

Modifying bacterial virulence could be an interesting alternative to antibiotics. The study aimed to examine the effects of an inhibitor targeting bacterial glutaminyl cyclase (which is selectively present in Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), and Prevotella intermedia (Pi)) on various multispecies biofilms. Two multi-species biofilms--one containing four species (including Tf) and another with 12 species (including Tf, Pg, and Pi)--were cultured in the presence of 31.25-500 {micro}M of a [4,5-c]pyridine-based inhibitor. After 24 hours, bacterial counts, biofilm mass, metabolic activity, and, when Pg was included, Arg-gingipain activity were measured. Additionally, the biofilms were exposed to monocytic cells; here, the release of interleukin (IL)-1{beta} and IL-10 was analyzed. The data were analyzed using a one-way analysis of variance (ANOVA) with a post-hoc comparison performed using the Bonferroni correction. In all biofilms, total bacterial counts and those of Pg and Tf remained unaffected by the inhibitor. In the 12-species biofilm, both mass" and total metabolic activity decreased at high inhibitor concentrations (500 {micro}M to 75.2{+/-}6.5% and 87.2{+/-}5.8%, respectively; each p<0.001). The arginine-specific amidolytic activities of Rgp declined dose-dependently, down to 60.4{+/-}10.2% (p<0.001) at 500 {micro}M of the inhibitor. Consequently, Pg colonies lost pigmentation as inhibitor concentrations increased. The inhibitor also reduced IL-1{beta} release from monocytic cells stimulated by the 12-species biofilm. The studied [4,5-c]pyridine-based inhibitor is able to modify virulence of a multispecies biofilm. It might have the potential to be a promising approach in periodontal prevention and therapy.

microbiology↗

bioRxiv2025

Innocent until proven guilty: Tannerella forsythia may attenuate the virulence of Porphyromonas gingivalis

Periodontitis is caused by a dysbiotic microbiome beneath the gum line, primarily driven by the major pathobionts Porphyromonas gingivalis (Pg) and Tannerella forsythia (Tf). Their virulence depends on the excessive, uncontrolled activity of secreted proteases that sustain chronic inflammation, leading to the destruction of tissues supporting the tooth. Paradoxically, Tf also encodes multiple protease inhibitors, including miropin, a serpin with a broad range of targets. Here, we demonstrate that both native and recombinant miropin effectively inhibit lysine-specific gingipain (Kgp) and thiol protease (Tpr), impairing the growth of Pg in peptide-limited media and reducing its virulence in vivo. A rationally designed variant, RVK-miropin, also inhibited both lysine-specific and arginine-specific gingipains, fully suppressing Pg proliferation and virulence in a mouse infection model. Miropin is abundant on the surface of Tf and forms covalent inhibitory complexes with Pg proteases. In an oral gavage model of periodontitis, coinfection with wild-type Tf (but not a miropin-deficient mutant) and Pg significantly reduced alveolar bone loss caused by Pg alone. Miropin thus counteracts Pg virulence factors and host inflammatory responses, revealing an unexpected protective role for Tf. This challenges the traditional view of Tf as a primary periodontal pathogen, suggesting a context-dependent role as a microbial modulator within the dysbiotic biofilm. Beyond periodontal disease, the unique ability of miropin to inhibit structurally diverse proteases makes it a promising candidate for the development of new therapies that restore proteolytic balance in the periodontium.

microbiology↗

bioRxiv2025

Infection-Specific Reprogramming of Microglia Reveals Distinct Virulence Pathways Linking Periodontal Pathogens to Alzheimer's Disease

Microglial dysregulation is increasingly recognized as a driver of Alzheimers disease (AD), yet how pathogen-specific cues sculpt microglial diversity remains unclear. Here we integrate high-dimensional single-cell cytometry in vitro with spatial proteomics in vivo to dissect the impact of two major periodontal pathogens on microglia. Using a 36-marker CyTOF panel, we exposed SIM-A9 microglia to wild-type Porphyromonas gingivalis (Pg) or Tannerella forsythia (Tf) and to gingipain-deficient or S-layer-deficient mutants, resolving 38 clusters. Virulence-factor "switches" redirected cells from homeostatic states toward i) oxidative, antigen-presenting programmes driven by Pg gingipains and ii) an immunosuppressive, exhausted-like state driven by the Tf S-layer. Complementary 37-marker imaging mass cytometry of 5xFAD x hTau knock-in mice chronically infected with Pg identified 21 microglial subclusters. The cortex--but not hippocampus--lost two Arg1/IL-10 immunoregulatory subsets (>2-fold decrease) while NADPH-oxidase-high microglia accumulated around amyloid-{beta} and tau aggregates. These data demonstrate pathogen-specific reprogramming of microglia across model systems and brain regions, linking virulence-factor activity to AD-relevant neuroinflammation. By pinpointing gingipains and the bacterial S-layer as molecular "switches," our study highlights tractable therapeutic targets for limiting infection-driven microglial dysfunction in Alzheimers disease.

neuroscience↗

bioRxiv2025

Virion stripping: A new role for bacterial proteases

We demonstrate that proteases produced by the oro-pharyngeal bacterial colonizer Porphyromonas gingivalis (Pg) reduce viral burden and modulate host interferon responses during respiratory syncytial virus (RSV) infection. Several oral bacteria, including Pg, have been shown to translocate to the upper airways through sub-clinical micro-aspiration. Our findings reveal that Pg, upon translocating to this new niche, significantly attenuated lung damage by reducing viral loads during respiratory viral infections in the lungs of wild-type mice. This protective effect was attributed to the activity of gingipains, cysteine endopeptidases produced by Pg, which cleaved envelope glycoproteins on RSV as well as on related murine-specific Sendai virus (SeV), thereby impairing their infectious capacity. Notably, the reduction in viral loads was independent of interferon lambda (IFN-{lambda}) signaling, which is actively suppressed by Pg in airway epithelial cells. However, the complete absence of IFN-{lambda} signaling resulted in a stronger inflammatory response despite a low viral load. Thus, we show a previously undescribed role for the oro-respiratory bacterial colonizer Pg in creating bottlenecks to viral infection by the activity of its proteases. SIGNIFICANCE STATEMENTReciprocal interactions between microbial colonizers and host epithelial cells are critical for providing initial defense against viral infections. However, our understanding of this phenomenon has been limited to microbiota-derived ligands that activate host pattern recognition receptors (PRRs), inducing basal interferon expression and downstream antiviral genes. Here, we present a novel mechanism that relies on microbial proteases to directly reduce viral load. Specifically, we discovered that the infectious capacity of the Respiratory Syncytial Virus (RSV) was significantly inhibited upon contact with the proteases (gingipains) produced by the oropharyngeal colonizer Porphyromonas gingivalis. Gingipains caused proteolytic degradation of the RSV envelope and attachment proteins, rendering them inactive. This preemptive reduction in viral infectious capacity consequently diminished the severity of respiratory viral infections in an IFN-independent manner.

microbiology↗

bioRxiv2025

Tyrosine phosphorylation coupling of one carbon metabolism and virulence in an endogenous pathogen

Endogenous pathogens can constrain virulence to ensure survival in the host. Pathogenic state can be controlled by metabolic responses to the prevailing microenvironment; however, the coupling and effector mechanisms are not well understood. Flux through the One Carbon Metabolism (OCM) pathway can modulate virulence of the oral pathobiont Porphyromonas gingivalis, and here we show that this is controlled by tyrosine phosphorylation-dependent differential partitioning of gingipain proteases. The OCM essential precursor pABA inhibits the low molecular weight tyrosine phosphatase Ltp1, and consequently relieves inhibition of its cognate kinase, Ptk1. We found that in the absence of pABA, reduced Ptk1 kinase activity blocks extracellular release of gingipains. Surface retention of gingipains confers resistance to neutrophil mobilization and killing, and virulence in animal models of disease is elevated. Reciprocally, Ptk1 and gingipains are required for maximal flux through OCM, and Ptk1 can phosphorylate the OCM pathway enzymes GlyA and GcvT. Further, ALP, an alkaline phosphatase involved in synthesis of DHPPP, which combines with pABA to make DHP, is phosphorylated and activated by Ptk1. We propose, therefore, that although the primary function of Ptk1 is to maintain OCM balance, it mechanistically couples metabolism with tunable pathogenic potential through directing the location of proteolytic virulence factors.

microbiology↗

bioRxiv2024

Periodontopathogens interfere with the human renin-angiotensin system through a surface-attached protease

The renin-angiotensin system (RAS) executes its functions through biologically active peptides, angiotensins (Ang). Angiotensinogen-derived precursor, Ang I is cleaved by angiotensin-converting enzyme (ACE) into proinflammatory Ang II, which increases blood pressure. In the alternate pathway performed by neprilysin and ACE II, Ang 1-7 is produced from Ang I with activities opposite to Ang II. Here, we show that Porphyromonas gingivalis (Pg) and Tannerella forsythia (Tf), endogenous oral pathogens, direct RAS into generation of Ang 1-7 through endopeptidases O, PgPepO and TfPepO, respectively. PepOs are thermophilic metalloproteases inhibited by cation chelators, but not by specific ACE and neprilysin inhibitors. PgPepO and TfPepO prefer large hydrophobic amino acids at the carbonyl side of scissile peptide bonds (P1 position), and TfPepO, contrary to all known homologous proteases, hydrolyzes substrates away from both terminuses. Solved crystal structures show that exceptionally wide entrance to the catalytic cleft explains unique properties of TfPepO. Furthermore, the different nature of subsites S1 and S2 in the substrate binding site explains refractory of PepOs to inhibitors of human homologous proteases. Multiple immunoassays clearly show that PepOs are attached to the bacteria cell surface and are released in outer membrane vesicles. Moreover, PepO is responsible for Ang I hydrolysis by Pg and Tf. Finally, PepO deletion reduced only the virulence of Tf in the Galleria mellonella model. Thus, our data show that Pg and Tf interfere with RAS through a PepO protease.

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↗

bioRxiv2024

Inhibition of SARS-CoV-2 infection by Porphyromonas gingivalis and the oral microbiome

The COVID-19 pandemic persists despite the availability of vaccines, and it is therefore crucial to develop new therapeutic and preventive approaches. In this study, we investigated the potential role of the oral microbiome in SARS-CoV-2 infection. Using an in vitro SARS-CoV-2 pseudovirus infection assay, we found a potent inhibitory effect exerted by Porphyromonas gingivalis on SARS-CoV-2 infection mediated by known P. gingivalis compounds such as phosphoglycerol dihydroceramide (PGDHC) and gingipains as well as by unknown bacterial factors. We found that the gingipain-mediated inhibition of infection is likely due to cytotoxicity, while PGDHC inhibited virus infection by an unknown mechanism. Unidentified factors present in P. gingivalis supernatant inhibited SARS-CoV-2 likely via the fusion step of the virus life cycle. We addressed the role of other oral bacteria and found certain periodontal pathogens capable of inhibiting SARS-CoV-2 pseudovirus infection by inducing cytotoxicity on target cells. In the human oral cavity, we observed the modulatory activity of oral microbial communities varied among individuals in that some saliva-based cultures were capable of inhibiting while others were enhancing infection. These findings contribute to our understanding of the complex relationship between the oral microbiome and viral infections, offering potential avenues for innovative therapeutic strategies in combating COVID-19.

microbiology↗

bioRxiv2021

Acriflavine, a clinically aproved drug, inhibits SARS-CoV-2 and other betacoronaviruses

SO_SCPLOWUMMARYC_SCPLOWThe COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identified acriflavine as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

microbiology↗

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↗

bioRxiv2020

Kallikrein-related peptidase 14 activates zymogens of membrane type matrix metalloproteinases (MT-MMPs) - a CleavEx library-based analysis

Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix (ECM), thus modulating the pericellular environment in physiology and excessively in pathologies like cancer. However, the interconnection between these groups of proteases remains elusive. To test this hypothesis, we have developed a peptide library-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences specifically. Initial assessment of the library identified a total of ten MMP activation domain sequences which were validated by Edman degradation. The CleavEx analysis revealed that membrane-type (MT) MMPs are likely targeted by KLK14 for activation. Correspondingly, commercially available proMT-MMPs, namely proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT MMPs were yielded and confirmed by Edman degradation. In addition, the productivity of proMMP activation was analyzed by gelatin zymography, which indicated the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts stably overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation within the ECM. As both, KLKs and MT-MMPs are implicated in cancer, the activation described herein may constitute an important factor in tumor progression and metastasis.

biochemistry↗

bioRxiv2020

Kallikrein 13: a new player in coronaviral infections.

Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but in vitro model for virus replication is lacking. Interaction between the coronaviral spike (S) protein and its receptor is the major determinant of virus tissue and host specificity, but virus entry is a complex process requiring a concerted action of multiple cellular elements. Here, we show that KLK13 is required for the infection of the human respiratory epithelium and is sufficient to mediate the entry of HCoV-HKU1 to non-permissive RD cells. We also demonstrated HCoV-HKU1 S protein cleavage by KLK13 in the S1/S2 region, proving that KLK13 is the priming enzyme for this virus. Summarizing, we show for the first time that protease distribution and specificity predetermines the tissue and cell specificity of the virus and may also regulate interspecies transmission. It is also of importance that presented data may be relevant for the emerging coronaviruses, including SARS-CoV-2 and may help to understand the differences in their zoonotic potential.

microbiology↗

bioRxiv2020

Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells

Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Intracellularity is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death by intracellular S. aureus after translocation into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. Our study suggests that staphopain A is utilized by S. aureus to mediate escape from the host cell and thus contributes to tissue destruction and dissemination of infection. Author SummaryStaphylococcus aureus is a well-known antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium asymptomatically colonizes the upper respiratory tract and skin of about one third of the human population and takes advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus is not regarded as a professional intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.

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↗