Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood-brain barrier damage in ducks during Riemerella anatipestifer infection.

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.

Proteomic characterization of Tenacibaculum dicentrarchi under iron limitation reveals an upregulation of proteins related to iron oxidation and reduction metabolism, iron uptake systems and gliding motility.

A strategy for vaccine design involves identifying proteins that could be involved in pathogen-host interactions. The aim of this proteomic study was to determine how iron limitation affects the protein expression of Tenacibaculum dicentrarchi, with a primary focus on virulence factors and proteins associated with iron uptake. The proteomic analysis was carried out using two strains of T. dicentrarchi grown under normal (control) and iron-limited conditions, mimicking the host environment. Our findings revealed differences in the proteins expressed by the type strain CECT 7612T and the Chilean strain TdCh05 of T. dicentrarchi. Nonetheless, both share a common response to iron deprivation, with an increased expression of proteins associated with iron oxidation and reduction metabolism (e.g., SufA, YpmQ, SufD), siderophore transport (e.g., ExbD, TonB-dependent receptor, HbpA), heme compound biosynthesis, and iron transporters under iron limitation. Proteins involved in gliding motility, such as GldL and SprE, were also upregulated in both strains. A negative differential regulation of metabolic proteins, particularly those associated with amino acid biosynthesis, was observed under iron limitation, reflecting the impact of iron availability on bacterial metabolism. Additionally, the TdCh05 strain exhibited unique proteins associated with gliding motility machinery and phage infection control compared to the type strain. These groups of proteins have been identified as virulence factors within the Flavobacteriaceae family, including the genus Tenacibaculum. These results build upon our previous report on iron acquisition mechanisms and could lay the groundwork for future studies aimed at elucidating the role of some of the described proteins in the infectious process of tenacibaculosis, as well as in the development of potential vaccines.

Genome-Wide Analysis and Characterization of the Riemerella anatipestifer Putative T9SS Secretory Proteins with a Conserved C-Terminal Domain.

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Recent studies have shown that the R. anatipestifer type IX secretion system (T9SS) acts as a crucial virulence factor. We previously identified two T9SS component proteins, GldK and GldM, and one T9SS effector metallophosphoesterase, which play important roles in bacterial virulence. In this study, 19 T9SS-secreted proteins that contained a conserved T9SS C-terminal domain (CTD) were predicted in R. anatipestifer strain Yb2 by searching for CTD-encoding sequences in the whole genome. The proteins were confirmed with a liquid chromatography-tandem mass spectrometry analysis of the bacterial culture supernatant. Nine of them were reported in our previous study. We generated recombinant proteins and mouse antisera for the 19 predicted proteins to confirm their expression in the bacterial culture supernatant and in bacterial cells. Western blotting indicated that the levels of 14 proteins were significantly reduced in the T9SS mutant Yb2ΔgldM culture medium but were increased in the bacterial cells. RT-qPCR indicated that the expression of these genes did not differ between the wild-type strain Yb2 and the T9SS mutant Yb2ΔgldM. Nineteen mutant strains were successfully constructed to determine their virulence and proteolytic activity, which indicated that seven proteins are associated with bacterial virulence, and two proteins, AS87_RS04190 and AS87_RS07295, are protease-activity-associated virulence factors. In summary, we have identified at least 19 genes encoding T9SS-secreted proteins in the R. anatipestifer strain Yb2 genome, which encode multiple functions associated with the bacterium's virulence and proteolytic activity. IMPORTANCE Riemerella anatipestifer T9SS plays an important role in bacterial virulence. We have previously reported nine R. anatipestifer T9SS-secreted proteins and clarified the function of the metallophosphoesterase. In this study, we identified 10 more secreted proteins associated with the R. anatipestifer T9SS, in addition to the nine previously reported. Of these, 14 proteins showed significantly reduced secretion into the bacterial culture medium but increased expression in the bacterial cells of the T9SS mutant Yb2ΔgldM; seven proteins were shown to be associated with bacterial virulence; and two proteins, AS87_RS04190 and AS87_RS07295, were shown to be protease-activity-associated virulence factors. Thus, we have demonstrated that multiple R. anatipestifer T9SS-secreted proteins function in virulence and proteolytic activity.

Glycosyltransferase-Related Protein GtrA Is Essential for Localization of Type IX Secretion System Cargo Protein Cellulase Cel9A and Affects Cellulose Degradation in Cytophaga hutchinsonii.

The Gram-negative bacterium Cytophaga hutchinsonii digests cellulose through a novel cellulose degradation mechanism. It possesses the lately characterized type IX secretion system (T9SS). We recently discovered that N-glycosylation of the C-terminal domain (CTD) of a hypothetical T9SS substrate protein in the periplasmic space of C. hutchinsonii affects protein secretion and localization. In this study, green fluorescent protein (GFP)-CTDCel9A recombinant protein was found with increased molecular weight in the periplasm of C. hutchinsonii. Site-directed mutagenesis studies on the CTD of cellulase Cel9A demonstrated that asparagine residue 900 in the D-X-N-X-S motif is important for the processing of the recombinant protein. We found that the glycosyltransferase-related protein GtrA (CHU_0012) located in the cytoplasm of C. hutchinsonii is essential for outer membrane localization of the recombinant protein. The deletion of gtrA decreased the abundance of the outer membrane proteins and affected cellulose degradation by C. hutchinsonii. This study provided a link between the glycosylation system and cellulose degradation in C. hutchinsonii. IMPORTANCE N-Glycosylation systems are generally limited to some pathogenic bacteria in prokaryotes. The disruption of the N-glycosylation pathway is related to adherence, invasion, colonization, and other phenotypic characteristics. We recently found that the cellulolytic bacterium Cytophaga hutchinsonii also has an N-glycosylation system. The cellulose degradation mechanism of C. hutchinsonii is novel and mysterious; cellulases and other proteins on the cell surface are involved in utilizing cellulose. In this study, we identified an asparagine residue in the C-terminal domain of cellulase Cel9A that is necessary for the processing of the T9SS cargo protein. Moreover, the glycosyltransferase-related protein GtrA is essential for the localization of the GFP-CTDCel9A recombinant protein. Deletion of gtrA affected cellulose degradation and the abundance of outer membrane proteins. This study enriched the understanding of the N-glycosylation system in C. hutchinsonii and provided a link between N-glycosylation and cellulose degradation, which also expanded the role of the N-glycosylation system in bacteria.

Riemerella anatipestifer T9SS Effector SspA Functions in Bacterial Virulence and Defending Natural Host Immunity.

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Recent studies have shown that the R. anatipestifer type IX secretion system (T9SS) is a crucial factor in bacterial virulence. The AS87_RS04190 protein was obviously missing from the secreted proteins of the T9SS mutant strain Yb2ΔgldM. A bioinformatic analysis indicated that the AS87_RS04190 protein contains a T9SS C-terminal domain sequence and encodes a putative subtilisin-like serine protease (SspA). To determine the role of the putative SspA protein in R. anatipestifer pathogenesis and proteolysis, we constructed two strains with an sspA mutation and complementation, respectively, and determined their median lethal doses, their bacterial loads in infected duck blood, and their adherence to and invasion of cells. Our results demonstrate that the SspA protein functions in bacterial virulence. It is also associated with the bacterial protease activity and has a conserved catalytic triad structure (Asp126, His158, and Ser410), which is necessary for protein function. The optimal reactive pH and temperature were determined to be 7.0 and 50°C, respectively, and Km and Vmax were determined to be 10.15 mM and 246.96 U/mg, respectively. The enzymatic activity of SspA is activated by Ca2+, Mg2+, and Mn2+ and inhibited by Cu2+ and EDTA. SspA degrades gelatin, fibrinogen, and bacitracin LL-37. These results demonstrate that SspA is an effector protein of T9SS and functions in R. anatipestifer virulence and its proteolysis of gelatin, fibrinogen, and bacitracin LL-37. IMPORTANCE In recent years, Riemerella anatipestifer T9SS has been reported to act as a virulence factor. However, the functions of the proteins secreted by R. anatipestifer T9SS are not entirely clear. In this study, a secreted subtilisin-like serine protease SspA was shown to be associated with R. anatipestifer virulence, host complement evasion, and degradation of gelatin, fibrinogen, and LL-37. The enzymatic activity of recombinant SspA was determined, and its Km and Vmax were 10.15 mM and 246.96 U/mg, respectively. Three conserved sites (Asp126, His158, and Ser410) are necessary for the protein's function. The median lethal dose of the sspA-deleted mutant strain was reduced >10,000-fold, indicating that SspA is an important virulence factor. In summary, we demonstrate that the R. anatipestifer AS87_RS04190 gene encodes an important T9SS effector, SspA, which plays an important role in bacterial virulence.

A Type IX Secretion System Substrate Involved in Crystalline Cellulose Degradation by Affecting Crucial Cellulose Binding Proteins in Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an abundant soil cellulolytic bacterium that uses a unique cellulose degradation mechanism different from those that involve free cellulases or cellulosomes. Though several proteins have been identified as important for cellulose degradation, the mechanism used by C. hutchinsonii to digest crystalline cellulose remains a mystery. In this study, chu_0922 was identified by insertional mutation and gene deletion as an important gene locus indispensable for crystalline cellulose utilization. Deletion of chu_0922 resulted in defects in crystalline cellulose utilization. The Δ0922 mutant completely lost the ability to grow on crystalline cellulose, even with extended incubation, and selectively utilized the amorphous region of cellulose, leading to increased crystallinity. As a protein secreted by the type IX secretion system (T9SS), CHU_0922 was found to be located on the outer membrane, and the outer membrane localization of CHU_0922 relied on the T9SS. Comparative analysis of the outer membrane proteins revealed that the abundance of several cellulose-binding proteins, including CHU_1276, CHU_1277, and CHU_1279, was reduced in the Δ0922 mutant. Further study showed that CHU_0922 is crucial for the full expression of the gene cluster containing chu_1276, chu_1277, chu_1278, chu_1279, and chu_1280 (cel9C), which is essential for cellulose utilization. Moreover, CHU_0922 is required for the cell surface localization of CHU_3220, a cellulose-binding protein that is essential for crystalline cellulose utilization. Our study provides insights into the complex system that C. hutchinsonii uses to degrade crystalline cellulose. IMPORTANCE The widespread aerobic cellulolytic bacterium Cytophaga hutchinsonii, belonging to the phylum Bacteroidetes, utilizes a novel mechanism to degrade crystalline cellulose. No genes encoding proteins specialized in loosening or disruption the crystalline structure of cellulose were identified in the genome of C. hutchinsonii, except for chu_3220 and chu_1557. The crystalline cellulose degradation mechanism remains enigmatic. This study identified a new gene locus, chu_0922, encoding a typical T9SS substrate that is essential for crystalline cellulose degradation. Notably, CHU_0922 is crucial for the normal transcription of chu_1276, chu_1277, chu_1278, chu_1279, and chu_1280 (cel9C), which play important roles in the degradation of cellulose. Moreover, CHU_0922 participates in the cell surface localization of CHU_3220. These results demonstrated that CHU_0922 plays a key role in the crystalline cellulose degradation network. Our study will promote the uncovering of the novel cellulose utilization mechanism of C. hutchinsonii.

N-Glycosylation of a Cargo Protein C-Terminal Domain Recognized by the Type IX Secretion System in Cytophaga hutchinsonii Affects Protein Secretion and Localization.

Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the phylum Bacteroidetes. It digests crystalline cellulose with an unknown mechanism and possesses a type IX secretion system (T9SS) that can recognize the C-terminal domain (CTD) of the cargo protein as a signal. In this study, the functions of the CTD in the secretion and localization of T9SS substrates in C. hutchinsonii were studied by fusing the green fluorescent protein (GFP) with the CTD from CHU_2708. The CTD is necessary for the secretion of GFP by C. hutchinsonii T9SS. The GFP-CTDCHU_2708 fusion protein was found to be glycosylated in the periplasm, with a molecular mass about 5 kDa higher than that predicted from its sequence. The glycosylated protein was sensitive to peptide-N-glycosidase F, which can hydrolyze N-linked oligosaccharides. Analyses of mutants obtained by site-directed mutagenesis of asparagine residues in the N-X-S/T motif of CTDCHU_2708 suggested that N-glycosylation occurred on the CTD. CTD N-glycosylation is important for the secretion and localization of GFP-CTD recombinant proteins in C. hutchinsonii. Glycosyltransferase-encoding gene chu_3842, a homologous gene of Campylobacter jejuni pglA, was found to participate in the N-glycosylation of C. hutchinsonii. Deletion of chu_3842 affected cell motility, cellulose degradation, and cell resistance to some chemicals. Our study provided evidence that the CTD as the signal of T9SS was N-glycosylated in the periplasm of C. hutchinsonii. IMPORTANCE The bacterial N-glycosylation system has previously been found only in several species of Proteobacteria and Campylobacterota, and the role of N-linked glycans in bacteria is still not fully understood. C. hutchinsonii has a unique cell contact cellulose degradation mechanism, and many cell surface proteins, including cellulases, are secreted by the T9SS. In this study, we found that C. hutchinsonii, a member of the phylum Bacteroidetes, has an N-glycosylation system. Glycosyltransferase CHU_3842 was found to participate in the N-glycosylation of C. hutchinsonii proteins and had effects on cell resistance to some chemicals, cell motility, and cellulose degradation. Moreover, N-glycosylation occurs on the CTD translocation signal of T9SS. The glycosylation of the CTD appears to play an important role in affecting T9SS substrate transportation and localization. This study enriched our understanding of the widespread existence and multiple biological roles of N-glycosylation in bacteria.

Type B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage.

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 ß-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.

A Riemerella anatipestifer Metallophosphoesterase That Displays Phosphatase Activity and Is Associated with Virulence.

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our previous study, we demonstrated that bacterial virulence and secretion proteins of the type IX secretion system (T9SS) mutant strains Yb2ΔgldK and Yb2ΔgldM were significantly reduced, in comparison to those of wild-type strain Yb2. In this study, the T9SS secretion protein AS87_RS00980, which is absent from the secretion proteins of Yb2ΔgldK and Yb2ΔgldM, was investigated by construction of gene mutation and complementation strains. The virulence assessment showed >1,000-fold attenuated virulence and significantly reduced bacterial loads in the blood of ducks infected with Yb2Δ00980, the AS87_RS00980 gene deletion mutant strain. Bacterial virulence was recovered in complementation strain cYb2Δ00980 Further study indicated that the T9SS secretion protein AS87_RS00980 is a metallophosphoesterase (MPPE), which displayed phosphatase activity and was cytomembrane localized. Moreover, the optimal reactive pH and temperature were determined to be 7.0 and 60°C, respectively, and the Km and Vmax were determined to be 3.53 mM and 198.1 U/mg. The rMPPE activity was activated by Zn2+ and Cu2+ but inhibited by Fe3+, Fe2+, and EDTA. There are five conserved sites, namely, N267, H268 H351, H389, and H391, in the metallophosphatase domain. Mutant proteins Y267-rMPPE and Y268-rMPPE retained 29.30% and 19.81% relative activity, respectively, and mutant proteins Y351-rMPPE, Y389-rMPPE, and Y391-rMPPE lost almost all MPPE activity. Taken together, these results indicate that the R. anatipestiferAS87_RS00980 gene encodes an MPPE that is a secretion protein of T9SS that plays an important role in bacterial virulence.IMPORTANCERiemerella anatipestifer T9SS was recently discovered to be associated with bacterial gliding motility and secretion of virulence factors. Several T9SS genes have been identified, but no effector has been reported in R. anatipestifer to date. In this study, we identified the T9SS secretion protein AS87_RS00980 as an MPPE that displays phosphatase activity and is associated with bacterial virulence. The enzymatic activity of the rMPPE was determined, and the Km and Vmax were 3.53 mM and 198.1 U/mg, respectively. Five conserved sites were also identified. The AS87_RS00980 gene deletion mutant strain was attenuated >1,000-fold, indicating that MPPE is an important virulence factor. In summary, we identified that the R. anatipestiferAS87_RS00980 gene encodes an important T9SS effector, MPPE, which plays an important role in bacterial virulence.

PorM, a core component of bacterial type IX secretion system, forms a dimer with a unique kinked-rod shape.

Porphyromonas gingivalis, which is a major pathogen of the periodontal disease, secrets virulence factors such as gingipain proteases via the type IX secretion system (T9SS). T9SS consists of a trans-periplasmic core complex, the outer membrane translocon complex and the cell-surface complex attached on the outer membrane. PorM is a major component of the trans-periplasmic core complex and is believed to connect the outer membrane component with the inner membrane component. Recent structural studies have revealed that the periplasmic region of GldM, a PorM homolog of a gliding bacterium, consist of four domains and forms a dimer with a straight rod shape. However, only fragment structures are known for PorM. Moreover, one of the PorM fragment structure shows a kink. Here we show the structure of the entire structure of the periplasmic region of PorM (PorMp) at 3.7 Å resolution. PorMp is made up of four domains and forms a unique dimeric structure with an asymmetric, kinked-rod shape. The structure and the following mutational analysis revealed that R204 stabilizes the kink between the D1 and D2 domains and is essential for gingipains secretion, suggesting that the kinked structure of PorM is important for the functional T9SS formation.

Cytophaga hutchinsonii gldN, Encoding a Core Component of the Type IX Secretion System, Is Essential for Ion Assimilation, Cellulose Degradation, and Cell Motility.

The type IX secretion system (T9SS), which is involved in pathogenicity, motility, and utilization of complex biopolymers, is a novel protein secretion system confined to the phylum BacteroidetesCytophaga hutchinsonii, a common cellulolytic soil bacterium belonging to the phylum Bacteroidetes, can rapidly digest crystalline cellulose using a novel strategy. In this study, the deletion mutant of chu_0174 (gldN) was obtained using PY6 medium supplemented with Stanier salts. GldN was verified to be a core component of C. hutchinsonii T9SS, and is indispensable for cellulose degradation, motility, and secretion of C-terminal domain (CTD) proteins. Notably, the ΔgldN mutant showed significant growth defects in Ca2+- and Mg2+-deficient media. These growth defects could be relieved by the addition of Ca2+ or Mg2+ The intracellular concentrations of Ca2+ and Mg2+ were markedly reduced in ΔgldN These results demonstrated that GldN is essential for the acquisition of trace amounts of Ca2+ and Mg2+, especially for Ca2+ Moreover, an outer membrane efflux protein, CHU_2807, which was decreased in abundance on the outer membrane of ΔgldN, is essential for normal growth in PY6 medium. The reduced intracellular accumulation of Ca2+ and Mg2+ in the Δ2807 mutant indicated that CHU_2807 is involved in the uptake of trace amounts of Ca2+ and Mg2+ This study provides insights into the role of T9SS in metal ion assimilation in C. hutchinsoniiIMPORTANCE The widespread Gram-negative bacterium Cytophaga hutchinsonii uses a novel but poorly understood strategy to utilize crystalline cellulose. Recent studies showed that a T9SS exists in C. hutchinsonii and is involved in cellulose degradation and motility. However, the main components of the C. hutchinsonii T9SS and their functions are still unclear. Our study characterized the function of GldN, which is a core component of the T9SS. GldN was proved to play vital roles in cellulose degradation and cell motility. Notably, GldN is essential for the acquisition of Ca2+ and Mg2+ ions under Ca2+- and Mg2+-deficient conditions, revealing a link between the T9SS and the metal ion transport system. The outer membrane abundance of CHU_2807, which is essential for Ca2+ and Mg2+ uptake in PY6 medium, was affected by the deletion of GldN. This study demonstrated that the C. hutchinsonii T9SS has extensive functions, including cellulose degradation, motility, and metal ion assimilation, and contributes to further understanding of the function of the T9SS in the phylum Bacteroidetes.

Deletion of the Riemerella anatipestifer type IX secretion system gene sprA results in differential expression of outer membrane proteins and virulence.

Riemerella anatipestifer (RA), the causative agent of infectious serositis that targets ducklings and other poultry, secretes protein via the type IX secretion system (T9SS). The proteins transported by T9SS are located on the bacterial cell surface or secreted into the extracellular milieu. In this study, a sprA deletion mutant was constructed encoding a core protein of T9SS to investigate its influence on outer membrane protein expression and its role in virulence. Compared with the wild-type RA-YM strain, the deletion mutant ΔsprA failed to digest gelatin, showed the same growth rate in the logarithmic phase and exhibited greater sensitivity to the bactericidal activity of duck sera, whereas the complemented strain restored these phenotypes. The outer membrane proteome of RA-YM and the ΔsprA mutant were analyzed by Tandem Mass Tags, which revealed 198 proteins with predicted localization to the cell envelope. Sixty-three of these proteins were differentially expressed in the outer membrane, with 43 up-regulated and 20 down-regulated. Among the twelve outer membrane proteins which were secreted by T9SS, four proteins were up-regulated and one protein was down-regulated. Animal experiments demonstrated that the median lethal dose of the mutant strain ΔsprA was about 500 times higher than that of the wild-type RA-YM strain, and bacterial loads in blood, brain, heart, liver and spleen of the ΔsprA-infected ducks were significantly reduced. Our results indicate that the SprA is a virulence-associated factor of RA, and its absence results in altered abundance of outer membrane proteins, and secretion disorders associated with some of the T9SS effector proteins.

Structural and functional insights into the C-terminal signal domain of the Bacteroidetes type-IX secretion system.

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 ß-sandwich (ß1-ß7). 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 ß3 and ß4 ('motif Lß3ß4') 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 Lß3ß4 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.

Structural Modeling of T9SS Outer Membrane Proteins and Their Complexes.

The type 9 secretion system (T9SS) is a recently discovered machinery that both transports cargo proteins across the Gram-negative bacterial outer membrane and attaches them to lipopolysaccharides on the extracellular surface. Outer membrane proteins (OMPs) are key components of the T9SS and are involved in both steps. In this chapter, we describe a method for the in silico modeling of T9SS OMPs and their complexes, and model validation. This is useful when the production of recombinant OMPs is difficult, and these protocols can also be applied to OMP complexes outside of the T9SS.

Riemerella anatipestifer GldG is necessary for secretion of effectors by type IX secretion system.

Riemerella anatipestifer secretes proteins through the type IX secretion system (T9SS). Recent studies have shown that the R. anatipestifer T9SS component proteins GldM and GldK also act as crucial virulence factors. In our previous study, the disruption of AS87_RS00460 gene, which encodes the predicted protein GldG, significantly reduced the bacterial virulence of R. anatipestifer wild-type strain Yb2, but the mechanism was unclear. In this study, we investigated the function of the GldG in bacterial virulence and protein secretion using the mutant strain Yb2ΔgldG and complementation strain cYb2ΔgldG. Our results demonstrate that the gldG gene encodes a gliding-motility-associated ABC transporter substrate-binding protein GldG, which was localized to the bacterial membrane in an immunoblotting analysis, and functions in the bacterium's adherence to and invasion of host cells and its survival in host blood. The resistance of mutant strain Yb2ΔgldG to complement-dependent killing was significantly reduced. Yb2ΔgldG displayed reduced gliding motility and deficient protein secretion. Label-free quantification (LFQ) with liquid chromatography-mass spectrometry (LC-MS) showed that 10 proteins with a conserved T9SS C-terminal domain were differentially secreted by Yb2ΔgldG and Yb2. The secretion levels of those 10 proteins were determined with immunoblotting, and the results were consistent with the LFQ LC-MS data. All of these effects were rescued by complementation with a plasmid encoding Yb2 gldG. Our results demonstrate that the R. anatipestifer gldG gene encodes the protein GldG, which is involved in bacterial virulence and protein secretion.

Riemerella anatipestifer Endonuclease I displays enzymatic activity and is associated with bacterial virulence.

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. We previously reported that the R. anatipestifer AS87_RS02625 is a secretory protein of the type IX secretion system (T9SS). In this study, R. anatipestifer T9SS protein AS87_RS02625 was determined to be a functional Endonuclease I (EndoI), which has DNase and RNase activities. Optimal temperature and pH of the recombinant R. anatipestifer EndoI (rEndoI) to cleave λDNA were determined as 55-60 °C and 7.5 respectively. The DNase activity of the rEndoI was dependent on the presence of divalent metal ions. Presence of Mg2+ at a concentration range of 7.5-15 mM in the rEndoI reaction buffer displayed the highest DNase activity. In addition, the rEndoI displayed RNase activity to cleave MS2-RNA (ssRNA), either in the absence or presence of divalent cations Mg2+, Mn2+, Ca2+, Zn2+ and Cu2+. The DNase activity of the rEndoI was significantly enhanced by Mg2+, Mn2+ and Ca2+ but not Zn2+ and Cu2+. Moreover, we indicated that R. anatipestifer EndoI functioned on the bacterial adherence, invasion, in vivo survival and inducing inflammatory cytokines. These results indicate that the R. anatipestifer T9SS protein AS87_RS02625 is a novel EndoI, displays endonuclease activity and plays an important role in bacterial virulence.

A conditional gene expression system in Porphyromonas gingivalis for study of the secretion mechanisms of lipoproteins and T9SS cargo proteins.

The Gram-negative anaerobe, Porphyromonas gingivalis, is known to be a pathogen associated with chronic periodontitis. P. gingivalis possesses virulence factors such as fimbriae and gingipain proteinases. Fimbrial proteins are secreted to the cell surface as lipoproteins. In contrast, gingipain proteinases are secreted into the bacterial cell surface via the type IX secretion system (T9SS). The transport mechanisms of lipoproteins and T9SS cargo proteins are entirely different and remain unknown. Therefore, using the Tet-on system developed for the genus Bacteroides, we newly created a conditional gene expression system in P. gingivalis. We succeeded in establishing conditional expression of nanoluciferase and its derivatives for lipoprotein export, of FimA for a representative of lipoprotein export, and of T9SS cargo proteins such as Hbp35 and PorA for representatives of type 9 protein export. Using this system, we showed that the lipoprotein export signal, which has recently been found in other species in the phylum Bacteroidota, is also functional in FimA, and that a proton motive force inhibitor can affect type 9 protein export. Collectively, our conditional protein expression method is useful for screening inhibitors of virulence factors, and may be used to investigate the role of proteins essential to bacterial survival in vivo.

Secreted peptidases contribute to virulence of fish pathogen Flavobacterium columnare.

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 ΔgldN and ΔporV, 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.

Protein Interactome Analysis of the Type IX Secretion System Identifies PorW as the Missing Link between the PorK/N Ring Complex and the Sov Translocon.

The type IX secretion system (T9SS) transports cargo proteins through the outer membrane of Bacteroidetes and attaches them to the cell surface for functions including pathogenesis, gliding motility, and degradation of carbon sources. The T9SS comprises at least 20 different proteins and includes several modules: the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, the outer membrane Sov translocon, and the cell attachment complex. However, the spatial organization of these modules is unknown. We have characterized the protein interactome of the Sov translocon in Porphyromonas gingivalis and identified Sov-PorV-PorA as well as Sov-PorW-PorN-PorK to be novel networks. PorW also interacted with PGN_1783 (PorD), which was required for maximum secretion efficiency. The identification of PorW as the missing link completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore. IMPORTANCE The T9SS is a newly identified protein secretion system of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum used by pathogens associated with diseases of humans, fish, and poultry for the secretion and cell surface attachment of virulence factors. The T9SS comprises three known modules: (i) the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, (ii) the outer membrane Sov translocon, and (iii) the cell surface attachment complex. The spatial organization and interaction of these modules have been a mystery. Here, we describe the protein interactome of the Sov translocon in the human pathogen Porphyromonas gingivalis and have identified PorW as the missing link which bridges PorN with Sov and so completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing, for the first time, a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore.

Structural Model of a Porphyromonas gingivalis type IX Secretion System Shuttle Complex.

Porphyromonas gingivalis is a gram-negative oral anaerobic pathogen and is one of the key causative agents of periodontitis. P. gingivalis utilises a range of virulence factors, including the cysteine protease RgpB, to drive pathogenesis and these are exported and attached to the cell surface via the type IX secretion system (T9SS). All cargo proteins possess a conserved C-terminal signal domain (CTD) which is recognised by the T9SS, and the outer membrane ß-barrel protein PorV (PG0027/LptO) can interact with cargo proteins as they are exported to the bacterial surface. Using a combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical analyses, machine-learning-based modelling and molecular dynamics (MD) simulations, we present a structural model of a PorV:RgpB-CTD complex from P. gingivalis. This is the first structural insight into CTD recognition by the T9SS and shows how the conserved motifs in the CTD are the primary sites that mediate binding. In PorV, interactions with extracellular surface loops are important for binding the CTD, and together these appear to cradle and lock RgpB-CTD in place. This work provides insight into cargo recognition by PorV but may also have important implications for understanding other aspects of type-IX dependent secretion.