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Protein secretion systems are vital for prokaryotic life, as they enable bacteria to acquire nutrients, communicate with other species, defend against biological and chemical agents, and facilitate disease through the delivery of virulence factors. In this review, we will focus on the recently discovered type IX secretion system (T9SS), a complex translocon found only in some species of the phylum. T9SS plays two roles, depending on the lifestyle of the bacteria. It provides either a means of movement (called gliding motility) for peace-loving environmental bacteria or a weapon for pathogens. The best-studied members of these two groups are , a commensal microorganism often found in water and soil, and , a human oral pathogen that is a major causative agent of periodontitis. In and some other periodontopathogens, T9SS translocates proteins, especially virulence factors, across the outer membrane (OM). Proteins destined for secretion bear a conserved C-terminal domain (CTD) that directs the cargo to the OM translocon. At least 18 proteins are involved in this still enigmatic process, with some engaged in the post-translational modification of T9SS cargo proteins. Upon translocation across the OM, the CTD is removed by a protease with sortase-like activity and an anionic LPS is attached to the newly formed C-terminus. As a result, a cargo protein could be secreted into the extracellular milieu or covalently attached to the bacterial surface. T9SS is regulated by a two-component system; however, the precise environmental signal that triggers it has not been identified. Exploring unknown systems contributing to bacterial virulence is exciting, as it may eventually lead to new therapeutic strategies. During the past decade, the major components of T9SS were identified, as well as hints suggesting the possible mechanism of action. In addition, the list of characterized cargo proteins is constantly growing. The actual structure of the translocon, situated in the OM of bacteria, remains the least explored area; however, new technical approaches and increasing scientific attention have resulted in a growing body of data. Therefore, we present a compact up-to-date review of this topic.

Fucoidanases are endo-fucoidanases (also known as endo-fucanases) that catalyze hydrolysis of α-glycosidic linkages in fucoidans, a family of sulfated fucose-rich polysaccharides primarily found in the cell walls of brown seaweeds. Fucoidanases are promising tools for producing bioactive fucoidan oligosaccharides for a range of biomedical applications. High sulfation degree has been linked to high bioactivity of fucoidans. In this study, a novel fucoidanase, Fhf2, was identified in the genome of the aerobic, Gram-negative marine bacterium Formosa haliotis . Fhf2 was found to share sequence similarity to known endo-α(1,4)-fucoidanases (EC 3.2.1.212) from glycoside hydrolase family 107. A C-terminal deletion mutant Fhf2∆484, devoid of 484 amino acids at the C-terminus, with a molecular weight of approximately 46 kDa, was constructed and found to be more stable than the full-length Fhf2 protein. Fhf2∆484 showed endo-fucoidanase activity on fucoidans from different seaweed species including Fucus evanescens , Fucus vesiculosus , Sargassum mcclurei , and Sargassum polycystum . The highest activity was observed on fucoidan from F. evanescens . The Fhf2∆484 enzyme was active at 20–45°C and at pH 6–9 and had optimal activity at 37°C and pH 8. Additionally, Fhf2∆484 was found to be calcium-dependent. NMR analysis showed that Fhf2∆484 catalyzed hydrolysis of α(1,4) linkages between L-fucosyl moieties sulfated on C2 (similar to Fhf1 from Formosa haliotis ), but Fhf2∆484 in addition released oligosaccharides containing a substantial amount of 2,4-disulfated fucose residues. The data thus suggest that the Fhf2∆484 enzyme could be a valuable candidate for producing highly sulfated oligosaccharides applicable for fucoidan bioactivity investigations.

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins.

The type 9 secretion system (T9SS) is fundamental to bacterial gliding motility, pathogenesis, and surface colonization. Our findings reveal that the C-terminal region of the conveyor belt-associated protein GldJ functions as a molecular switch which is capable of reversing the rotational direction of T9SS. Through the coordinated actions of the T9SS stator units (akin to a driving motor), the GldK ring (the gear that converts rotational energy into linear movement), and GldJ, this machinery forms a smart conveyor belt system reminiscent of flexible or cognitive mechanical conveyors. Such advanced conveyors can alter their direction to adapt to shifting demands. Here, we show that the bacterial T9SS similarly adjusts its rotational bias based on feedback from the conveyor belt-associated protein GldJ. This dual-role feedback mechanism underscores an evolved, controllable biological snowmobile, offering new avenues for studying how bacteria fine-tune motility in dynamic environments.

Riemerella anatipestifer infection is a critical disease that is a major threat to the poultry industry worldwide. The adhesion and invasion of host cells are key steps in the primary stages of bacterial infection. However, the outer membrane proteins that mediate these events in R. anatipestifer are poorly characterized. In this study, the PorV and PosF proteins, as well as the previously described OMP71 protein, were identified as important mediators of the adhesion and invasion of duck embryo fibroblast (DEF) cells by R. anatipestifer . Affinity chromatography-based surface proteomics was used to screen for adhesion proteins. The surface proteins on DEF cells were labelled with biotin-avidin to enrich for outer membrane proteins of R. anatipestifer, which generated 11 candidate proteins that were tested further. Protein adhesion and blocking assays and polyclonal antiserum inhibition analysis revealed that the PorV, PosF, and OMP71 proteins are adhesion factors. Knockout of porV or posF reduced the adhesion and invasion of R. anatipestifer in DEF cells. Moreover, the pathogenicity of the mutant strains was significantly attenuated, which supports the hypothesis that PorV and PosF are important virulence factors required for the pathogenicity of R. anatipestifer . The PorV protein is a key component of the type IX secretory system and is responsible for transporting effector substrates to the extracellular environment, whereas PosF belongs to the porin superfamily of barrel-shaped transmembrane proteins. This is the first description that PorV is an adhesin involved in host‒microbial interactions, which represents a breakthrough in pathogenicity studies of R. anatipestifer and other members of Flavobacteriaceae .

Bacteria 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. Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis , the CTD of 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 β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro . Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of 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 β-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface. IMPORTANCE Bacteria 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 higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer- infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

Flavobacterium columnare causes columnaris disease in freshwater fish in both natural and aquaculture settings. This disease is often lethal, especially when fish population density is high, and control options such as vaccines are limited. The type IX secretion system (T9SS) is required for F. columnare virulence, but secreted virulence factors have not been fully identified. Many T9SS-secreted proteins are predicted peptidases, and peptidases are common virulence factors of other pathogens. T9SS-deficient mutants, such as Δ gldN and Δ porV , exhibit strong defects in secreted proteolytic activity. The F. columnare genome has many peptidase-encoding genes that may be involved in nutrient acquisition and/or virulence. Mutants lacking individual peptidase-encoding genes, or lacking up to ten peptidase-encoding genes, were constructed and examined for extracellular proteolytic activity, for growth defects, and for virulence in zebrafish and rainbow trout. Most of the mutants retained virulence, but a mutant lacking 10 peptidases, and a mutant lacking the single peptidase TspA exhibited decreased virulence in rainbow trout fry, suggesting that peptidases contribute to F. columnare virulence.

Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 β-sandwich (β1–β7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus (‘motif C-t.’) and the loop connecting strands β3 and β4 (‘motif Lβ3β4’) as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS , which shares the same general topology as in Porphyromonas CTDs. However, motif Lβ3β4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.

Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, can rapidly degrade crystalline cellulose through direct cell-to-substrate contact. Most of its cellulases are secreted by the Type IX secretion system (T9SS) and anchored to the cell surface. Our previous study proved that the C-terminal domain (CTD) of the T9SS substrate cellulase Cel9A is glycosylated in C. hutchinsonii. However, its glycosylation mechanism has remained elusive. In this study, we found that chu_3394, which encodes UDP-glucose 6-dehydrogenase (Ugd), was important for the glycosylation of large amounts of periplasmic and outer membrane proteins in C. hutchinsonii. The contents of mannose, glucose, galactose, and xylose were detected to be reduced in the glycoproteins of the ∆ugd mutant compared to that of wild-type. They might be essential monosaccharides that contribute to the structure and function of glycans attached to proteins in C. hutchinsonii. The depletion of mannose, glucose, galactose, and xylose indicates a decrease in glycosylation modifications in the ∆ugd mutant strain. Then, we found that the deletion of ugd resulted in weakened glycosylation modification of the recombinant green fluorescent protein-tagged CTD of Cel9A. Additionally, the outer-membrane localization of Cel9A was affected in the mutant. Besides this, Ugd was also important for the synthesis of O-antigen of lipopolysaccharide (LPS). Thus, Ugd was involved in the synthesis of glycans in both glycoproteins and LPS in C. hutchinsonii. Moreover, the deletion of ugd affected the cellulose degradation, cell motility, and stress resistance of C. hutchinsonii.