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 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 Type IX Secretion System and Its Role in Bacterial Function and Pathogenesis

Porphyromonas, Tannerella, and Prevotella species found in severe periodontitis use the Type IX Secretion System (T9SS) to load their outer membrane surface with an array of virulence factors. These virulence factors are then released on outer membrane vesicles (OMVs), which penetrate the host to dysregulate the immune response to establish a positive feedback loop of chronic, inflammatory destruction of the tooth’s supporting tissues. In this review, we present the latest information on the molecular architecture of the T9SS and provide mechanistic insight into its role in secretion and attachment of cargo proteins to produce a virulence coat on cells and OMVs. The recent molecular structures of the T9SS motor comprising PorL and PorM as well as the secretion pore Sov, together with advances in the overall interactome, have provided insight into the possible mechanisms of secretion. We propose the presence of PorL/M motors arranged in a circle at the inner membrane with bent periplasmic rotors interacting with the PorN protein. At the outer membrane, we envisage a slide carousel model where the PorN protein is driven around a circular track composed of PorK. Cargo proteins are transported by PorN to PorW and the Sov translocon just as slides are rotated to the projection window. Secreted proteins are proposed to then be shuttled along highways consisting of the PorV shuttle protein to an array of attachment complexes distributed around the cell. The cell surface attachment of cargo is a hallmark of the T9SS, and in Porphyromonas gingivalis and Tannerella forsythia, this attachment is achieved via covalent bonding to a linking sugar synthesized by the Wbp/Vim pathway. The cell-surface attached cargo are enriched on OMVs, which are then released from the cell.

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

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.

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 CTD in the secretion and localization of T9SS substrates in C. hutchinsonii were studied by fusing the green fluorescent protein (GFP) with CTD from CHU_2708. CTD is necessary for the secretion of GFP by C. hutchinsonii T9SS. The GFP-CTD CHU_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 CTD CHU_2708 suggest 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 the evidence that CTD as the signal of T9SS was N -glycosylated in the periplasm of C. hutchinsonii . IMPORTANCE The bacterial N -glycosylation system has previously only been found 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. Here, 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 CTD apears to play an important role in affecting T9SS substrates transportation and localization. This study enriched our understanding of the widespread existence and multiple biological roles of N -glycosylation in bacteria.

Intermolecular latency regulates the essential C-terminal signal peptidase and sortase of the Porphyromonas gingivalis 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.

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

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.

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

Increasing problems with antibiotic resistance has directed interest towards phage therapy in the aquaculture industry. However, phage resistance evolving in target bacteria is considered a challenge. To investigate how phage resistance influences the fish pathogen Flavobacterium columnare , two wild-type bacterial isolates, FCO-F2 and FCO-F9, were exposed to phages (FCO-F2 to FCOV-F2, FCOV-F5 and FCOV-F25, and FCO-F9 to FCL-2, FCOV-F13 and FCOV-F45), and resulting phenotypic and genetic changes in bacteria were analyzed. Bacterial viability first decreased in the exposure cultures, but started to increase after 1-2 days, along with a change in colony morphology from original rhizoid to rough, leading to 98% prevalence of the rough morphotype. Twenty-four isolates (including four isolates from no-phage treatments) were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion and biofilm formation, protease activity, whole genome sequencing and virulence in rainbow trout fry. The rough isolates arising in phage exposure were phage-resistant with low virulence, whereas rhizoid isolates maintained phage susceptibility and high virulence. Gliding motility and protease activity were also related to the phage susceptibility. Observed mutations in phage-resistant isolates were mostly located in genes coding for type IX secretion system, a component of the Bacteroidetes gliding motility machinery. However, not all phage-resistant isolates had mutations, indicating that phage resistance in F. columnare 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. Importance Phage 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-susceptible 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.

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

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.

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

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.

Complementation in trans of Porphyromonas gingivalis lipopolysaccharide biosynthetic mutants demonstrates lipopolysaccharide exchange

Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to A-lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled “in situ” for correct secretion and A-LPS modification of cargo proteins, combinations of non-pigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Re-acquisition of pigmentation only occurred between an A-LPS mutant and a T9SS mutant which coincided with A-LPS modification of cargo-proteins detected by Western blot and co-immunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two strain crosses involving only the five T9SS OM component mutants produced black pigmentation implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation and hence a more ordered temporal in situ assembly of T9SS components maybe required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host. Importance Porphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.