scholarly journals Involvement of an Skp-Like Protein, PGN_0300, in the Type IX Secretion System of Porphyromonas gingivalis

2015 ◽  
Vol 84 (1) ◽  
pp. 230-240 ◽  
Author(s):  
Yuko Taguchi ◽  
Keiko Sato ◽  
Hideharu Yukitake ◽  
Tetsuyoshi Inoue ◽  
Masaaki Nakayama ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Virulence Factors ◽  
Membrane Fraction ◽  
Secretion System ◽  
Signal Peptidase ◽  
Blood Agar ◽  
Bacterial Cells ◽  
Loss Of Function ◽  
Content Type ◽  
Type Ix Secretion System

The oral Gram-negative anaerobic bacteriumPorphyromonas gingivalisis an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in theomp17mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in theomp17mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of theomp17mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, theomp17mutant was less virulent than the wild type. These results suggested that Omp17 is involved inP. gingivalisvirulence.

scholarly journals Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Paul D. Veith ◽  
Mikio Shoji ◽  
Richard A. J. O’Hair ◽  
Michael G. Leeming ◽  
Shuai Nie ◽  
...  
Keyword(s):  
Cell Surface ◽  
Porphyromonas Gingivalis ◽  
Biosynthetic Pathway ◽  
Secretion System ◽  
Tannerella Forsythia ◽  
Diverse Range ◽  
Content Type ◽  
C Terminus ◽  
Cargo Proteins ◽  
Type Ix Secretion System

ABSTRACT Porphyromonas gingivalis and Tannerella forsythia use the type IX secretion system to secrete cargo proteins to the cell surface where they are anchored via glycolipids. In P. gingivalis, the glycolipid is anionic lipopolysaccharide (A-LPS), of partially known structure. Modified cargo proteins were deglycosylated using trifluoromethanesulfonic acid and digested with trypsin or proteinase K. The residual modifications were then extensively analyzed by tandem mass spectrometry. The C terminus of each cargo protein was amide-bonded to a linking sugar whose structure was deduced to be 2-N-seryl, 3-N-acetylglucuronamide in P. gingivalis and 2-N-glycyl, 3-N-acetylmannuronic acid in T. forsythia. The structures indicated the involvement of the Wbp pathway to produce 2,3-di-N-acetylglucuronic acid and a WbpS amidotransferase to produce the uronamide form of this sugar in P. gingivalis. The wbpS gene was identified as PGN_1234 as its deletion resulted in the inability to produce the uronamide. In addition, the P. gingivalis vimA mutant which lacks A-LPS was successfully complemented by the T. forsythia vimA gene; however, the linking sugar was altered to include glycine rather than serine. After removal of the acetyl group at C-2 by the putative deacetylase, VimE, VimA presumably transfers the amino acid to complete the biosynthesis. The data explain all the enzyme activities required for the biosynthesis of the linking sugar accounting for six A-LPS-specific genes. The linking sugar is therefore the key compound that enables the attachment of cargo proteins in P. gingivalis and T. forsythia. We propose to designate this novel linking sugar biosynthetic pathway the Wbp/Vim pathway. IMPORTANCE Porphyromonas gingivalis and Tannerella forsythia, two pathogens associated with severe gum disease, use the type IX secretion system (T9SS) to secrete and attach toxic arrays of virulence factor proteins to their cell surfaces. The proteins are tethered to the outer membrane via glycolipid anchors that have remained unidentified for more than 2 decades. In this study, the first sugar molecules (linking sugars) in these anchors are identified and found to be novel compounds. The novel biosynthetic pathway of these linking sugars is also elucidated. A diverse range of bacteria that do not have the T9SS were found to have the genes for this pathway, suggesting that they may synthesize similar linking sugars for utilization in different systems. Since the cell surface attachment of virulence factors is essential for virulence, these findings reveal new targets for the development of novel therapies.

Intermolecular latency regulates the essential C-terminal signal peptidase and sortase of the Porphyromonas gingivalis type-IX secretion system

2021 ◽  
Vol 118 (40) ◽  
pp. e2103573118
Author(s):  
Danuta Mizgalska ◽  
Theodoros Goulas ◽  
Arturo Rodríguez-Banqueri ◽  
Florian Veillard ◽  
Mariusz Madej ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Catalytic Domain ◽  
Secretion System ◽  
Oral Microbiome ◽  
Signal Peptidase ◽  
Dual Function ◽  
Secretion Signal ◽  
Long Latency ◽  
Type Ix Secretion System

Porphyromonas gingivalis is a keystone pathogen of the human dysbiotic oral microbiome that causes severe periodontitis. It employs a type-IX secretion system (T9SS) to shuttle proteins across the outer membrane (OM) for virulence. Uniquely, T9SS cargoes carry a C-terminal domain (CTD) as a secretion signal, which is cleaved and replaced with anionic lipopolysaccharide by transpeptidation for extracellular anchorage to the OM. Both reactions are carried out by PorU, the only known dual-function, C-terminal signal peptidase and sortase. PorU is itself secreted by the T9SS, but its CTD is not removed; instead, intact PorU combines with PorQ, PorV, and PorZ in the OM-inserted “attachment complex.” Herein, we revealed that PorU transits between active monomers and latent dimers and solved the crystal structure of the ∼260-kDa dimer. PorU has an elongated shape ∼130 Å in length and consists of seven domains. The first three form an intertwined N-terminal cluster likely engaged in substrate binding. They are followed by a gingipain-type catalytic domain (CD), two immunoglobulin-like domains (IGL), and the CTD. In the first IGL, a long “latency β-hairpin” protrudes ∼30 Å from the surface to form an intermolecular β-barrel with β-strands from the symmetric CD, which is in a latent conformation. Homology modeling of the competent CD followed by in vivo validation through a cohort of mutant strains revealed that PorU is transported and functions as a monomer through a C690/H657 catalytic dyad. Thus, dimerization is an intermolecular mechanism for PorU regulation to prevent untimely activity until joining the attachment complex.

scholarly journals Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

2016 ◽  
Vol 82 (6) ◽  
pp. 1756-1766 ◽  
Author(s):  
Daichi Kita ◽  
Satoshi Shibata ◽  
Yuichiro Kikuchi ◽  
Eitoyo Kokubu ◽  
Koji Nakayama ◽  
...  
Keyword(s):  
Cell Surface ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Secretion System ◽  
Gliding Motility ◽  
Confocal Laser ◽  
Bacterial Cells ◽  
Wild Type ◽  
Content Type ◽  
Type Ix Secretion System

ABSTRACTCapnocytophaga ochraceais a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces.C. ochraceapossesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) inFlavobacterium johnsoniaewas shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins inF. johnsoniaehave been identified in the genome ofC. ochracea; therefore, the T9SS may be involved in biofilm formation byC. ochracea. Here we constructed three ortholog-deficientC. ochraceamutants lackingsprB(which encodes a gliding motility adhesin) orgldKorsprT(which encode T9SS proteins inF. johnsoniae). Gliding motility was lost in each mutant, suggesting that, inC. ochracea, the proteins encoded bysprB,gldK, andsprTare necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprBstrains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-typeC. ochraceawere denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation ofC. ochracea.

scholarly journals PorZ, an Essential Component of the Type IX Secretion System of Porphyromonas gingivalis, Delivers Anionic Lipopolysaccharide to the PorU Sortase for Transpeptidase Processing of T9SS Cargo Proteins

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mariusz Madej ◽  
Zuzanna Nowakowska ◽  
Miroslaw Ksiazek ◽  
Anna M. Lasica ◽  
Danuta Mizgalska ◽  
...  
Keyword(s):  
Cell Surface ◽  
Porphyromonas Gingivalis ◽  
Secretion System ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Content Type ◽  
Essential Component ◽  
Wide Range ◽  
Cargo Proteins ◽  
Type Ix Secretion System

ABSTRACT 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.

scholarly journals A PorX/PorY and σ P Feedforward Regulatory Loop Controls Gene Expression Essential for Porphyromonas gingivalis Virulence

mSphere ◽  
2021 ◽  
Author(s):  
Chizhou Jiang ◽  
Dezhi Yang ◽  
Tangsiyuan Hua ◽  
Zichun Hua ◽  
Wei Kong ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Secretion System ◽  
Etiologic Agent ◽  
Noncommunicable Diseases ◽  
Two Component System ◽  
Content Type ◽  
Two Component ◽  
Genes Encoding ◽  
Type Ix Secretion System ◽  
Regulatory Loop

The anaerobic bacterium Porphyromonas gingivalis is not only the major etiologic agent for chronic periodontitis, but also prevalent in some common noncommunicable diseases such as cardiovascular disease, Alzheimer's disease, and rheumatoid arthritis. We present genetic, biochemical, and biological results to demonstrate that the PorX/PorY two-component system and sigma factor σ P build a specific regulatory network to coordinately control transcription of the genes encoding the type IX secretion system, and perhaps also other virulence factors.

scholarly journals Author Correction: Crystal structure of Type IX secretion system PorE C-terminal domain from Porphyromonas gingivalis in complex with a peptidoglycan fragment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nhung Thi Trang Trinh ◽  
Hieu Quang Tran ◽  
Quyen Van Dong ◽  
Christian Cambillau ◽  
Alain Roussel ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Porphyromonas Gingivalis ◽  
Secretion System ◽  
Terminal Domain ◽  
Type Ix Secretion System

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

scholarly journals The Carboxy-Terminal Region ofFlavobacterium johnsoniaeSprB Facilitates Its Secretion by the Type IX Secretion System and Propulsion by the Gliding Motility Machinery

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Surashree S. Kulkarni ◽  
Joseph J. Johnston ◽  
Yongtao Zhu ◽  
Zachary T. Hying ◽  
Mark J. McBride
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Surface ◽  
Type A ◽  
Secretion System ◽  
Gliding Motility ◽  
Foreign Protein ◽  
Type B ◽  
Content Type ◽  
Type Ix Secretion System

ABSTRACTFlavobacterium johnsoniaeSprB moves rapidly along the cell surface, resulting in gliding motility. SprB secretion requires the type IX secretion system (T9SS). Proteins secreted by the T9SS typically have conserved C-terminal domains (CTDs) belonging to the type A CTD or type B CTD family. Attachment of 70- to 100-amino-acid type A CTDs to a foreign protein allows its secretion. Type B CTDs are common but have received little attention. Secretion of the foreign protein superfolder green fluorescent protein (sfGFP) fused to regions spanning the SprB type B CTD (sfGFP-CTDSprB) was analyzed. CTDs of 218 amino acids or longer resulted in secretion of sfGFP, whereas a 149-amino-acid region did not. Some sfGFP was secreted in soluble form, whereas the rest was attached on the cell surface. Surface-attached sfGFP was rapidly propelled along the cell, suggesting productive interaction with the motility machinery. This did not result in rapid cell movement, which apparently requires additional regions of SprB. Secretion of sfGFP-CTDSprBrequired coexpression withsprF, which lies downstream ofsprB. SprF is similar in sequence toPorphyromonas gingivalisPorP. MostF. johnsoniaegenes encoding proteins with type B CTDs lie immediately upstream ofporP/sprF-like genes. sfGFP was fused to the type B CTD from one such protein (Fjoh_3952). This resulted in secretion of sfGFP only when it was coexpressed with its cognate PorP/SprF-like protein. These results highlight the need for extended regions of type B CTDs and for coexpression with the appropriate PorP/SprF-like protein for efficient secretion and cell surface localization of cargo proteins.IMPORTANCETheF. johnsoniaegliding motility adhesin SprB is delivered to the cell surface by the type IX secretion system (T9SS) and is rapidly propelled along the cell by the motility machinery. How this 6,497-amino-acid protein interacts with the secretion and motility machines is not known. Fusion of the C-terminal 218 amino acids of SprB to a foreign cargo protein resulted in its secretion, attachment to the cell surface, and rapid movement by the motility machinery. Efficient secretion of SprB required coexpression with the outer membrane protein SprF. Secreted proteins that have sequence similarity to SprB in their C-terminal regions are common in the phylumBacteroidetesand may have roles in adhesion, motility, and virulence.

scholarly journals Non-O1/Non-O139 Vibrio cholerae Carrying Multiple Virulence Factors and V. cholerae O1 in the Chesapeake Bay, Maryland

2015 ◽  
Vol 81 (6) ◽  
pp. 1909-1918 ◽  
Author(s):  
Daniela Ceccarelli ◽  
Arlene Chen ◽  
Nur A. Hasan ◽  
Shah M. Rashed ◽  
Anwar Huq ◽  
...  
Keyword(s):  
Chesapeake Bay ◽  
Vibrio Cholerae ◽  
Virulence Factors ◽  
Fluorescent Antibody ◽  
Secretion System ◽  
Surveillance Program ◽  
Toxin Gene ◽  
Protease Gene ◽  
Content Type ◽  
Pathogenic Variants

ABSTRACTNon-O1/non-O139Vibrio choleraeinhabits estuarine and coastal waters globally, but its clinical significance has not been sufficiently investigated, despite the fact that it has been associated with septicemia and gastroenteritis. The emergence of virulent non-O1/non-O139V. choleraeis consistent with the recognition of new pathogenic variants worldwide. Oyster, sediment, and water samples were collected during a vibrio surveillance program carried out from 2009 to 2012 in the Chesapeake Bay, Maryland.V. choleraeO1 was detected by a direct fluorescent-antibody (DFA) assay but was not successfully cultured, whereas 395 isolates of non-O1/non-O139V. choleraewere confirmed by multiplex PCR and serology. Only a few of the non-O1/non-O139V. choleraeisolates were resistant to ampicillin and/or penicillin. Most of the isolates were sensitive to all antibiotics tested, and 77 to 90% carried the El Tor variant hemolysin genehlyAET, the actin cross-linking repeats in toxin genertxA, the hemagglutinin protease genehap, and the type 6 secretion system. About 19 to 21% of the isolates carried the neuraminidase-encoding genenanHand/or the heat-stable toxin (NAG-ST), and only 5% contained a type 3 secretion system. None of the non-O1/non-O139V. choleraeisolates containedVibriopathogenicity island-associated genes. However,ctxA,ace, orzotwas present in nine isolates. Fifty-five different genotypes showed up to 12 virulence factors, independent of the source of isolation, and represent the first report of both antibiotic susceptibility and virulence associated with non-O1/non-O139V. choleraefrom the Chesapeake Bay. Since these results confirm the presence of potentially pathogenic non-O1/non-O139V. cholerae, monitoring for totalV. cholerae, regardless of serotype, should be done within the context of public health.

scholarly journals Genomic Avenue to Avian Colisepticemia

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Sagi Huja ◽  
Yaara Oren ◽  
Eva Trost ◽  
Elzbieta Brzuszkiewicz ◽  
Dvora Biran ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Analysis ◽  
Virulence Factors ◽  
Iron Uptake ◽  
Secretion System ◽  
Economic Losses ◽  
Content Type ◽  
E Coli ◽  
Type 3 Secretion System ◽  
Type 3

ABSTRACTHere we present an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78 that represent the major cause of avian colisepticemia, an invasive infection caused by avian pathogenicEscherichia coli(APEC) strains. It is associated with high mortality and morbidity, resulting in significant economic consequences for the poultry industry. To understand the genetic basis of the virulence of avian septicemic E. coli, we sequenced the entire genome of a clinical isolate of serotype O78—O78:H19 ST88 isolate 789 (O78-9)—and compared it with three publicly available APEC O78 sequences and one complete genome of APEC serotype O1 strain. Although there was a large variability in genome content between the APEC strains, several genes were conserved, which are potentially critical for colisepticemia. Some of these genes are present in multiple copies per genome or code for gene products with overlapping function, signifying their importance. A systematic deletion of each of these virulence-related genes identified three systems that are conserved in all septicemic strains examined and are critical for serum survival, a prerequisite for septicemia. These are the plasmid-encoded protein, the defective ETT2 (E. colitype 3 secretion system 2) type 3 secretion system ETT2sepsis, and iron uptake systems. Strain O78-9 is the only APEC O78 strain that also carried the regulon coding for yersiniabactin, the iron binding system of theYersiniahigh-pathogenicity island. Interestingly, this system is the only one that cannot be complemented by other iron uptake systems under iron limitation and in serum.IMPORTANCEAvian colisepticemia is a severe systemic disease of birds causing high morbidity and mortality and resulting in severe economic losses. The bacteria associated with avian colisepticemia are highly antibiotic resistant, making antibiotic treatment ineffective, and there is no effective vaccine due to the multitude of serotypes involved. To understand the disease and work out strategies to combat it, we performed an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78, the major cause of the disease. We identified several potential virulence factors, conserved in all the colisepticemic strains examined, and determined their contribution to growth in serum, an absolute requirement for septicemia. These findings raise the possibility that specific vaccines or drugs can be developed against these critical virulence factors to help combat this economically important disease.

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