scholarly journals Crystal structures of two camelid nanobodies raised against GldL, a component of the type IX secretion system from Flavobacterium johnsoniae

2021 ◽  
Vol 77 (6) ◽  
Author(s):  
Thi Trang Nhung Trinh ◽  
Anaïs Gaubert ◽  
Pauline Melani ◽  
Christian Cambillau ◽  
Alain Roussel ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Cytoplasmic Domain ◽  
Secretion System ◽  
Gliding Motility ◽  
Molecular Replacement ◽  
Flavobacterium Johnsoniae ◽  
Type Ix Secretion System ◽  
Inner Membrane Protein ◽  
And Function ◽  
Rotary Motor

GldL is an inner-membrane protein that is essential for the function of the type IX secretion system (T9SS) in Flavobacterium johnsoniae. The complex that it forms with GldM is supposed to act as a new rotary motor involved in the gliding motility of the bacterium. In the context of structural studies of GldL to gain information on the assembly and function of the T9SS, two camelid nanobodies were selected, produced and purified. Their interaction with the cytoplasmic domain of GldL was characterized and their crystal structures were solved. These nanobodies will be used as crystallization chaperones to help in the crystallization of the cytoplasmic domain of GldL and could also help to solve the structure of the complex using molecular replacement.

scholarly journals The Type IX Secretion System: Advances in Structure, Function and Organisation

2020 ◽  
Vol 8 (8) ◽  
pp. 1173 ◽  
Author(s):  
Dhana G. Gorasia ◽  
Paul D. Veith ◽  
Eric C. Reynolds
Keyword(s):  
Cell Surface ◽  
Structure Function ◽  
Secretion System ◽  
Gliding Motility ◽  
Flavobacterium Johnsoniae ◽  
Bacteroidetes Phylum ◽  
Transcription Regulators ◽  
Type Ix Secretion System ◽  
Insight Into ◽  
Made In

The type IX secretion system (T9SS) is specific to the Bacteroidetes phylum. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilises the T9SS to transport many proteins—including its gingipain virulence factors—across the outer membrane and attach them to the cell surface. Additionally, the T9SS is also required for gliding motility in motile organisms, such as Flavobacterium johnsoniae. At least nineteen proteins have been identified as components of the T9SS, including the three transcription regulators, PorX, PorY and SigP. Although the components are known, the overall organisation and the molecular mechanism of how the T9SS operates is largely unknown. This review focusses on the recent advances made in the structure, function, and organisation of the T9SS machinery to provide further insight into this highly novel secretion system.

scholarly journals Flavobacterium johnsoniae PorV Is Required for Secretion of a Subset of Proteins Targeted to the Type IX Secretion System

2014 ◽  
Vol 197 (1) ◽  
pp. 147-158 ◽  
Author(s):  
Sampada S. Kharade ◽  
Mark J. McBride
Keyword(s):  
Cell Surface ◽  
Secretion System ◽  
Gliding Motility ◽  
Content Type ◽  
Flavobacterium Johnsoniae ◽  
Protein Secretion System ◽  
Surface Motility ◽  
Type Ix Secretion System ◽  
Carboxy Terminal ◽  
Novel Protein

Flavobacterium johnsoniaeexhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT.Porphyromonas gingivalishas orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS.P. gingivalisporUandporVhave also been linked to T9SS-mediated secretion, andF. johnsoniaehas orthologs of these. Mutations inF. johnsoniaeporUandporVwere constructed to determine if they function in secretion. Cells of aporVdeletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and theporVmutant exhibited gliding motility and formed spreading colonies. However, theporVmutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. TheporVmutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in theF. johnsoniaeT9SS.

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 In situ structure and organisation of the type IX secretion system

2020 ◽  
Author(s):  
DG Gorasia ◽  
G Chreifi ◽  
CA Seers ◽  
CA Butler ◽  
JE Heath ◽  
...  
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Electron Tomography ◽  
Secretion System ◽  
Gliding Motility ◽  
Type B ◽  
Surface Attachment ◽  
Flavobacterium Johnsoniae ◽  
Close Proximity ◽  
Type Ix Secretion System

AbstractThe Bacteroidetes type IX secretion system (T9SS) consists of at least 19 components that translocate proteins with a type A or type B C-terminal domain (CTD) signal across the outer membrane. The overall organisation and architecture of this system including how the secretion pore (Sov) interacts with the other components is unknown. We used cryo-electron tomography to obtain the first images of the T9SS including PorK/N rings inside intact Porphyromonas gingivalis cells. Using proteomics, we identified a novel complex between Sov, PorV and PorA and showed that Sov interacts with the PorK/N rings via PorW and a new component PGN_1783. A separate complex comprising the outer membrane β-barrel protein PorP, PorE, and the type B CTD protein PG1035 was also identified. Similarly, the Flavobacterium johnsoniae PorP-like protein, SprF was found bound to the major gliding motility adhesin, SprB. Based on these data, we propose cell surface anchorage for type B CTD proteins to PorP-like proteins and a unique model where the PorK/N rings function as an outer membrane barrier to maintain the close proximity of the translocon to the shuttle and attachment complexes inside the rings, ensuring the harmonized secretion and cell surface attachment of the T9SS substrates.

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 A molecular rack and pinion actuates a cell-surface adhesin and enables bacterial gliding motility

2020 ◽  
Vol 6 (10) ◽  
pp. eaay6616 ◽  
Author(s):  
Abhishek Shrivastava ◽  
Howard C. Berg
Keyword(s):  
Cell Surface ◽  
Rotation Axis ◽  
Gliding Motility ◽  
Flavobacterium Johnsoniae ◽  
Viscous Shear ◽  
Shear Cells ◽  
A Cell ◽  
Solid Substratum ◽  
Gliding Bacterium ◽  
Rotary Motor

The gliding bacterium Flavobacterium johnsoniae is known to have an adhesin, SprB, that moves along the cell surface on a spiral track. Following viscous shear, cells can be tethered by the addition of an anti-SprB antibody, causing spinning at 3 Hz. Labeling the type 9 secretion system (T9SS) with a YFP fusion of GldL showed a yellow fluorescent spot near the rotation axis, indicating that the motor driving the motion is associated with the T9SS. The distance between the rotation axis and the track (90 nm) was determined after adding a Cy3 label for SprB. A rotary motor spinning a pinion of radius 90 nm at 3 Hz would cause a spot on its periphery to move at 1.5 μm/s, the gliding speed. We suggest the pinion drives a flexible tread that carries SprB along a track fixed to the cell surface. Cells glide when this adhesin adheres to the solid substratum.

scholarly journals Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and Type IX Secretion System

2020 ◽  
Author(s):  
Heidi M. T. Kunttu ◽  
Anniina Runtuvuori-Salmela ◽  
Krister Sundell ◽  
Tom Wiklund ◽  
Mathias Middelboe ◽  
...  
Keyword(s):  
Phage Therapy ◽  
Secretion System ◽  
Gliding Motility ◽  
Genetic Mutations ◽  
Phage Resistance ◽  
Flavobacterium Columnare ◽  
Aquaculture Industry ◽  
Columnaris Disease ◽  
Phage Sensitivity ◽  
Type Ix Secretion System

AbstractIncreasing problems with antibiotic resistance has directed interest towards phages as tools to treat bacterial infections in the aquaculture industry. However, phage resistance evolves rapidly in bacteria posing a challenge for successful phage therapy. To investigate phage resistance in the fish pathogenic bacterium Flavobacterium columnare, two phage-sensitive, virulent wild-type isolates, FCO-F2 and FCO-F9, were exposed to phages and subsequently analyzed for bacterial viability and colony morphology. Twenty-four phage-exposed isolates were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion and biofilm formation on polystyrene surface, protease activity, whole genome sequencing and virulence against rainbow trout fry. Bacterial viability first decreased in the exposure cultures, subsequently increasing after 1-2 days. Simultaneously, the colony morphology of the phage-exposed isolates changed from original rhizoid to rough. The rough isolates arising in phage exposure were phage-resistant with low virulence, whereas rhizoid isolates maintained phage sensitivity, though reduced, and high virulence. Gliding motility and protease activity were also related to the phage sensitivity. Observed genetic mutations in phage-resistant isolates were mostly located in genes coding for type IX secretion system, a component of the flavobacterial gliding motility machinery. However, there were mutational differences between individual isolates, and not all phage-resistant isolates had genetic mutations. This indicates that development of phage resistance in F. columnare probably is a multifactorial process including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections, since phage resistance is associated with decrease in bacterial virulence.ImportancePhage resistance of infectious bacteria is a common phenomenon posing challenges for development of phage therapy. Along with growing World population and need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by F. columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce a development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-sensitive isolates and thus not a challenge for phage therapy against columnaris disease. This is a valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

scholarly journals Untangling Flavobacterium johnsoniae Gliding Motility and Protein Secretion

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
Joseph J. Johnston ◽  
Abhishek Shrivastava ◽  
Mark J. McBride
Keyword(s):  
Amino Acids ◽  
Protein Secretion ◽  
Secretion System ◽  
Gliding Motility ◽  
Content Type ◽  
Flavobacterium Johnsoniae ◽  
C Terminus ◽  
Genes Encoding ◽  
Surface Localization ◽  
Protein Secretion System

ABSTRACTFlavobacterium johnsoniaeexhibits rapid gliding motility over surfaces. At least 20 genes are involved in this process. Seven of these,gldK,gldL,gldM,gldN,sprA,sprE, andsprT, encode proteins of the type IX protein secretion system (T9SS). The T9SS is required for surface localization of the motility adhesins SprB and RemA, and for secretion of the soluble chitinase ChiA. Here, we demonstrate that the gliding motility proteins GldA, GldB, GldD, GldF, GldH, GldI, and GldJ are also essential for secretion. Cells with mutations in the genes encoding any of these seven proteins had normal levels ofgldKmRNA but dramatically reduced levels of the GldK protein, which may explain the secretion defects of the motility mutants. GldJ is necessary for stable accumulation of GldK, and each mutant lacked the GldJ protein.F. johnsoniaecells that produced truncated GldJ, lacking eight to 13 amino acids from the C terminus, accumulated GldK but were deficient in gliding motility. SprB was secreted by these cells but was not propelled along their surfaces. This C-terminal region of GldJ is thus required for gliding motility but not for secretion. The identification of mutants that are defective for motility but competent for secretion begins to untangle theF. johnsoniaegliding motility machinery from the T9SS.IMPORTANCEMany members of the phylumBacteroidetessecrete proteins using T9SSs. T9SSs appear to be confined to members of this phylum. Many of these bacteria also glide rapidly over surfaces using a motility machine that is also confined to theBacteroidetesand appears to be intertwined with the T9SS. This study identifiesF. johnsoniaeproteins that are required for both T9SS function and gliding motility. It also provides an explanation for the link between secretion and gliding and identifies mutants with defects in motility but not secretion.

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