scholarly journals Lack of a surface layer in Tannerella forsythia mutants deficient in the type IX secretion system

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2295-2303 ◽  
Author(s):  
Yuka Narita ◽  
Keiko Sato ◽  
Hideharu Yukitake ◽  
Mikio Shoji ◽  
Daisuke Nakane ◽  
...  
Keyword(s):  
Surface Layer ◽  
Biofilm Formation ◽  
Anaerobic Bacterium ◽  
Secretion System ◽  
Tannerella Forsythia ◽  
Gram Negative ◽  
Extracellular Milieu ◽  
Genes Encoding ◽  
Type Ix Secretion System ◽  
Encoding Genes

Tannerella forsythia, a Gram-negative anaerobic bacterium, is an important pathogen in periodontal disease. This bacterium possesses genes encoding all known components of the type IX secretion system (T9SS). T. forsythia mutants deficient in genes orthologous to the T9SS-encoding genes porK, porT and sov were constructed. All porK, porT and sov single mutants lacked the surface layer (S-layer) and expressed less-glycosylated versions of the S-layer glycoproteins TfsA and TfsB. In addition, these mutants exhibited decreased haemagglutination and increased biofilm formation. Comparison of the proteins secreted by the porK and WT strains revealed that the secretion of several proteins containing C-terminal domain (CTD)-like sequences is dependent on the porK gene. These results indicate that the T9SS is functional in T. forsythia and contributes to the translocation of CTD proteins to the cell surface or into the extracellular milieu.

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.

scholarly journals Identification of a NovelN-Acetylmuramic Acid Transporter in Tannerella forsythia

2016 ◽  
Vol 198 (22) ◽  
pp. 3119-3125 ◽  
Author(s):  
Angela Ruscitto ◽  
Isabel Hottmann ◽  
Graham P. Stafford ◽  
Christina Schäffer ◽  
Christoph Mayer ◽  
...  
Keyword(s):  
De Novo ◽  
Amino Sugar ◽  
Periodontal Pathogen ◽  
Tannerella Forsythia ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
Sugar Transporters ◽  
E Coli ◽  
Genes Encoding

ABSTRACTTannerella forsythiais a Gram-negative periodontal pathogen lacking the ability to undergode novosynthesis of amino sugarsN-acetylmuramic acid (MurNAc) andN-acetylglucosamine (GlcNAc) that form the disaccharide repeating unit of the peptidoglycan backbone.T. forsythiarelies on the uptake of these sugars from the environment, which is so far unexplored. Here, we identified a novel transporter system ofT. forsythiainvolved in the uptake of MurNAc across the inner membrane and characterized a homolog of theEscherichia coliMurQ etherase involved in the conversion of MurNAc-6-phosphate (MurNAc-6-P) to GlcNAc-6-P. The genes encoding these components were identified on a three-gene cluster spanning Tanf_08375 to Tanf_08385 located downstream from a putative peptidoglycan recycling locus. We show that the three genes, Tanf_08375, Tanf_08380, and Tanf_08385, encoding a MurNAc transporter, a putative sugar kinase, and a MurQ etherase, respectively, are transcriptionally linked. Complementation of the Tanf_08375 and Tanf_08380 genes together intrans, but not individually, rescued the inability of anE. colimutant deficient in the phosphotransferase (PTS) system-dependent MurNAc transporter MurP as well as that of a double mutant deficient in MurP and components of the PTS system to grow on MurNAc. In addition, complementation with this two-gene construct inE. colicaused depletion of MurNAc in the medium, further confirming this observation. Our results show that the products of Tanf_08375 and Tanf_08380 constitute a novel non-PTS MurNAc transporter system that seems to be widespread among bacteria of theBacteroidetesphylum. To the best of our knowledge, this is the first identification of a PTS-independent MurNAc transporter in bacteria.IMPORTANCEIn this study, we report the identification of a novel transporter for peptidoglycan amino sugarN-acetylmuramic acid (MurNAc) in the periodontal pathogenT. forsythia. It has been known since the late 1980s thatT. forsythiais a MurNAc auxotroph relying on environmental sources for this essential sugar. Most sugar transporters, and the MurNAc transporter MurP in particular, require a PTS phosphorelay to drive the uptake and concurrent phosphorylation of the sugar through the inner membrane in Gram-negative bacteria. Our study uncovered a novel type of PTS-independent MurNAc transporter, and although so far, it seems to be unique toT. forsythia, it may be present in a range of bacteria both of the oral cavity and gut, especially of the phylumBacteroidetes.

scholarly journals A novel class of polymorphic toxins in Bacteroidetes

2020 ◽  
Vol 3 (4) ◽  
pp. e201900631
Author(s):  
Biswanath Jana ◽  
Dor Salomon ◽  
Eran Bosis
Keyword(s):  
Gut Microbiota ◽  
Unknown Function ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
New Class ◽  
Terminal Domain ◽  
Domain Of Unknown Function ◽  
Type Ix Secretion System ◽  
Encoding Gene

Bacteroidetes are Gram-negative bacteria that are abundant in the environment as well as in the gut microbiota of animals. Many bacteroidetes encode large proteins containing an N-terminal domain of unknown function, named TANFOR. In this work, we show that TANFOR-containing proteins carry polymorphic C-terminal toxin domains with predicted antibacterial and anti-eukaryotic activities. We also show that a C-terminal domain that is prevalent in TANFOR-containing proteins represents a novel family of antibacterial DNase toxins, which we named BaCT (Bacteroidetes C-terminal Toxin). Finally, we discover that TANFOR-encoding gene neighborhoods are enriched with genes that encode substrates of the type IX secretion system (T9SS), which is involved in exporting proteins from the periplasm across the outer membrane. Based on these findings, we conclude that TANFOR-containing proteins are a new class of polymorphic toxins, and we hypothesize that they are T9SS substrates.

Type IX secretion system effectors and virulence of the model Flavobacterium columnare strain MS-FC-4

2021 ◽  
Author(s):  
Nicole C. Thunes ◽  
Rachel A. Conrad ◽  
Haitham H. Mohammed ◽  
Yongtao Zhu ◽  
Paul Barbier ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Virulence Factors ◽  
Genetic Manipulation ◽  
Secretion System ◽  
Gliding Motility ◽  
Secreted Proteins ◽  
Flavobacterium Columnare ◽  
Genes Encoding ◽  
Columnaris Disease ◽  
Type Ix Secretion System

Flavobacterium columnare causes columnaris disease in wild and cultured freshwater fish and is a major problem for sustainable aquaculture worldwide. The F. columnare type IX secretion system (T9SS) secretes many proteins and is required for virulence. The T9SS component GldN is required for secretion and for gliding motility over surfaces. Genetic manipulation of F. columnare is inefficient, which has impeded identification of secreted proteins that are critical for virulence. Here we identified a virulent wild-type F. columnare strain (MS-FC-4) that is highly amenable to genetic manipulation. This facilitated isolation and characterization of two deletion mutants lacking core components of the T9SS. Deletion of gldN disrupted protein secretion and gliding motility and eliminated virulence in zebrafish and rainbow trout. Deletion of porV disrupted secretion and virulence but not motility. Both mutants exhibited decreased extracellular proteolytic, hemolytic, and chondroitin sulfate lyase activities. They also exhibited decreased biofilm formation and decreased attachment to fish fins and to other surfaces. Using genomic and proteomic approaches, we identified proteins secreted by the T9SS. We deleted ten genes encoding secreted proteins and characterized the virulence of mutants lacking individual or multiple secreted proteins. A mutant lacking two genes encoding predicted peptidases exhibited reduced virulence in rainbow trout, and mutants lacking a predicted cytolysin showed reduced virulence in zebrafish and rainbow trout. The results establish F. columnare strain MS-FC-4 as a genetically amenable model to identify virulence factors. This may aid development of measures to control columnaris disease and impact fish health and sustainable aquaculture. IMPORTANCE: Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish and is a major problem for aquaculture. Little is known regarding the virulence factors involved in this disease and control measures are inadequate. The type IX secretion system (T9SS) secretes many proteins and is required for virulence, but the secreted virulence factors are not known. We identified a strain of F. columnare (MS-FC-4) that is well suited for genetic manipulation. The components of the T9SS and the proteins secreted by this system were identified. Deletion of core T9SS genes eliminated virulence. Genes encoding ten secreted proteins were deleted. Deletion of two peptidase-encoding genes resulted in decreased virulence in rainbow trout, and deletion of a cytolysin-encoding gene resulted in decreased virulence in rainbow trout and zebrafish. Secreted peptidases and cytolysins are likely virulence factors and are targets for the development of control measures.

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 On the path to uncover the bacterial type II secretion system

2012 ◽  
Vol 367 (1592) ◽  
pp. 1059-1072 ◽  
Author(s):  
Badreddine Douzi ◽  
Alain Filloux ◽  
Romé Voulhoux
Keyword(s):  
Protein Interactions ◽  
Secretion System ◽  
Target Cells ◽  
Type Ii Secretion ◽  
Gram Negative Bacteria ◽  
Type Ii ◽  
Protein Protein Interactions ◽  
Gram Negative ◽  
Extracellular Milieu ◽  
Type Ii Secretion System

Gram-negative bacteria have evolved several secretory pathways to release enzymes or toxins into the surrounding environment or into the target cells. The type II secretion system (T2SS) is conserved in Gram-negative bacteria and involves a set of 12 to 16 different proteins. Components of the T2SS are located in both the inner and outer membranes where they assemble into a supramolecular complex spanning the bacterial envelope, also called the secreton. The T2SS substrates transiently go through the periplasm before they are translocated across the outer membrane and exposed to the extracellular milieu. The T2SS is unique in its ability to promote secretion of large and sometimes multimeric proteins that are folded in the periplasm. The present review describes recently identified protein–protein interactions together with structural and functional advances in the field that have contributed to improve our understanding on how the type II secretion apparatus assembles and on the role played by individual proteins of this highly sophisticated system.

scholarly journals The Type VI Secretion System: A Multipurpose Delivery System with a Phage-Like Machinery

2011 ◽  
Vol 24 (7) ◽  
pp. 751-757 ◽  
Author(s):  
Angela R. Records
Keyword(s):  
Plant Growth Promoting Rhizobacteria ◽  
Secretion System ◽  
Host Cells ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Gram Negative ◽  
Extracellular Milieu ◽  
Type Vi Secretion ◽  
Plant Growth Promoting ◽  
And Function

Whether they live in the soil, drift in the ocean, survive in the lungs of human hosts or reside on the surfaces of leaves, all bacteria must cope with an array of environmental stressors. Bacteria have evolved an impressive suite of protein secretion systems that enable their survival in hostile environments and facilitate colonization of eukaryotic hosts. Collectively, gram-negative bacteria produce six distinct secretion systems that deliver proteins to the extracellular milieu or directly into the cytosol of host cells. The type VI secretion system (T6SS) was discovered recently and is encoded in at least one fourth of all sequenced gram-negative bacterial genomes. T6SS proteins are evolutionarily and structurally related to phage proteins, and it is likely that the T6SS apparatus is reminiscent of phage injection machinery. Most studies of T6SS function have been conducted in the context of host-pathogen interactions. However, the totality of data suggests that the T6SS is a versatile tool with roles in virulence, symbiosis, interbacterial interactions, and antipathogenesis. This review gives a brief history of T6SS discovery and an overview of the pathway's predicted structure and function. Special attention is paid to research addressing the T6SS of plant-associated bacteria, including pathogens, symbionts and plant growth–promoting rhizobacteria.

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 The Type 9 Secretion System Is Required for Flavobacterium johnsoniae Biofilm Formation

2021 ◽  
Vol 12 ◽  
Author(s):  
Todd J. Eckroat ◽  
Camillus Greguske ◽  
David W. Hunnicutt
Keyword(s):  
Biofilm Formation ◽  
Deletion Mutant ◽  
Secretion System ◽  
Gliding Motility ◽  
Microplate Assay ◽  
Partial Reduction ◽  
Wild Type ◽  
Flavobacterium Johnsoniae ◽  
Biofilm Production ◽  
Type Ix Secretion System

Flavobacterium johnsoniae forms biofilms in low nutrient conditions. Protein secretion and cell motility may have roles in biofilm formation. The F. johnsoniae type IX secretion system (T9SS) is important for both secretion and motility. To determine the roles of each process in biofilm formation, mutants defective in secretion, in motility, or in both processes were tested for their effects on biofilm production using a crystal violet microplate assay. All mutants that lacked both motility and T9SS-mediated secretion failed to produce biofilms. A porV deletion mutant, which was severely defective for secretion, but was competent for motility, also produced negligible biofilm. In contrast, mutants that retained secretion but had defects in gliding formed biofilms. An sprB mutant that is severely but incompletely defective in gliding motility but retains a fully functional T9SS was similar to the wild type in biofilm formation. Mutants with truncations of the gldJ gene that compromise motility but not secretion showed partial reduction in biofilm formation compared to wild type. Unlike the sprB mutant, these gldJ truncation mutants were essentially nonmotile. The results show that a functional T9SS is required for biofilm formation. Gliding motility, while not required for biofilm formation, also appears to contribute to formation of a robust biofilm.

scholarly journals Peptidoglycan Acetylation of Campylobacter jejuni Is Essential for Maintaining Cell Wall Integrity and Colonization in Chicken Intestines

2016 ◽  
Vol 82 (20) ◽  
pp. 6284-6290 ◽  
Author(s):  
Taketoshi Iwata ◽  
Ayako Watanabe ◽  
Masahiro Kusumoto ◽  
Masato Akiba
Keyword(s):  
Cell Wall ◽  
Campylobacter Jejuni ◽  
Biofilm Formation ◽  
Cell Wall Integrity ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Genes Encoding ◽  
Isogenic Mutants ◽  
Bacterial Diarrhea

ABSTRACTPeptidoglycan (PG) acetylation of Gram-positive bacteria confers lysozyme resistance and contributes to survival in the host. However, the importance of PG acetylation in Gram-negative bacteria has not been fully elucidated. The genes encoding putative PG acetyltransferase A (PatA) and B (PatB) are highly conserved inCampylobacter jejuni, the predominant cause of bacterial diarrhea worldwide. To evaluate the importance of PatA and PatB ofC. jejuni, we constructedpatAandpatBisogenic mutants and compared their phenotypes with those of the parental strains. Although transmission electron microscopy did not reveal morphological changes, both mutants exhibited decreased motility and biofilm formationin vitro. The extent of acetylation of the PG purified from thepatAandpatBmutants was significantly lower than the PG acetylation in the parental strains. Both mutants exhibited decreased lysozyme resistance and intracellular survival in macrophage cells. In a chick colonization experiment, significant colonization deficiency was observed for both mutants. These results suggest that PatA and PatB ofC. jejuniplay important roles in maintaining cell wall integrity by catalyzing PGO-acetylation and that the loss of these enzymes causes decreased motility and biofilm formation, thus leading to colonization deficiency in chicken infection.IMPORTANCEThe importance of peptidoglycan (PG) acetylation in Gram-negative bacteria has not been fully elucidated. The genes encoding putative PG acetyltransferase A (PatA) and B (PatB) are highly conserved inCampylobacter jejuni, the predominant cause of bacterial diarrhea worldwide. We evaluated the importance of these enzymes using isogenic mutants. The results of this study suggest that PatA and PatB ofC. jejuniplay important roles in maintaining cell wall integrity. The loss of these factors caused multiple phenotypic changes, leading to colonization deficiency in chicken infection. These data should be useful in developing novel control measures to prevent chicken colonization byC. jejuni. Inhibitors of the PG acetylation enzymes PatA and PatB might serve as potent anti-C. jejuniagents.

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