Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system.

Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification, and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE2R2Q2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits.

Cytolysin A is an intracellularly induced and secreted cytotoxin of typhoidal Salmonella.

Typhoidal Salmonella enterica serovars, such as Typhi and Paratyphi A, cause severe systemic infections, thereby posing a significant threat as human-adapted pathogens. This study focuses on cytolysin A (ClyA), a virulence factor essential for bacterial dissemination within the human body. We show that ClyA is exclusively expressed by intracellular S. Paratyphi A within the Salmonella-containing vacuole (SCV), regulated by the PhoP/Q system and SlyA. ClyA localizes in the bacterial periplasm, suggesting potential secretion. Deletion of TtsA, an essential Type 10 Secretion System component, completely abolishes intracellular ClyA detection and its presence in host cell supernatants. Host cells infected with wild-type S. Paratyphi A contain substantial ClyA, with supernatants capable of lysing neighboring cells. Notably, ClyA selectively lyses macrophages and erythrocytes while sparing epithelial cells. These findings identify ClyA as an intracellularly induced cytolysin, dependent on the SCV environment and secreted via a Type 10 Secretion System, with specific cytolytic activity.

A coordinated attack by a bacterial secretion system and a small molecule drives prey specificity.

Vibrio species are recognized for their role in food- and water-borne diseases in humans, fish, and aquatic invertebrates. We screened bacterial strains isolated from raw food shrimp for those that are bactericidal to Vibrio strains. Here we identify and characterize Aeromonas dhakensis strain A603 which shows robust bactericidal activity specifically towards Vibrio and related taxa but less potency toward other Gram-negative species. Using the A603 genome and genetic analysis, we show that two antibacterial mechanisms account for its vibriocidal activity -- a highly potent Type Six Secretion System (T6SS) and biosynthesis of a vibriocidal phenazine-like small molecule, named here as Ad-Phen. Further analysis indicates coregulation between Ad-Phen and a pore-forming T6SS effector TseC, which potentiates V. cholerae to killing by Ad-Phen.

Transcriptome Analysis Reveals Cross-Talk between the Flagellar Transcriptional Hierarchy and Secretion System in Plesiomonas shigelloides.

Plesiomonas shigelloides, a Gram-negative bacillus, is the only member of the Enterobacteriaceae family able to produce polar and lateral flagella and cause gastrointestinal and extraintestinal illnesses in humans. The flagellar transcriptional hierarchy of P. shigelloides is currently unknown. In this study, we identified FlaK, FlaM, FliA, and FliAL as the four regulators responsible for polar and lateral flagellar regulation in P. shigelloides. To determine the flagellar transcription hierarchy of P. shigelloides, the transcriptomes of the WT and ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were carried out for comparison in this study. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and luminescence screening assays were used to validate the RNA-seq results, and the Electrophoretic Mobility Shift Assay (EMSA) results revealed that FlaK can directly bind to the promoters of fliK, fliE, flhA, and cheY, while the FlaM protein can bind directly to the promoters of flgO, flgT, and flgA. Meanwhile, we also observed type VI secretion system (T6SS) and type II secretion system 2 (T2SS-2) genes downregulated in the transcriptome profiles, and the killing assay revealed lower killing abilities for ΔflaK, ΔflaM, ΔfliA, and ΔfliAL compared to the WT, indicating that there was a cross-talk between the flagellar hierarchy system and bacterial secretion system. Invasion assays also showed that ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were less effective in infecting Caco-2 cells than the WT. Additionally, we also found that the loss of flagellar regulators causes the differential expression of some of the physiological metabolic genes of P. shigelloides. Overall, this study aims to reveal the transcriptional hierarchy that controls flagellar gene expression in P. shigelloides, as well as the cross-talk between motility, virulence, and physiological and metabolic activity, laying the groundwork for future research into P. shigelloides' coordinated survival in the natural environment and the mechanisms that infect the host.

Ecological, beneficial, and pathogenic functions of the Type 9 Secretion System.

The recently discovered Type 9 Secretion System (T9SS) is present in bacteria of the Fibrobacteres-Bacteroidetes-Chlorobi superphylum, which are key constituents of diverse microbiomes. T9SS is instrumental in the extracellular secretion of over 270,000 proteins, including peptidases, sugar hydrolases, metal ion-binding proteins, and metalloenzymes. These proteins are essential for the interaction of bacteria with their environment. This mini-review explores the extensive array of proteins secreted by the T9SS. It highlights the diverse functions of these proteins, emphasizing their roles in pathogenesis, bacterial interactions, host colonization, and the overall health of the ecosystems inhabited by T9SS-containing bacteria.

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.

Mycobacterial type VII secretion systems.

Mycobacteria, such as the pathogen M. tuberculosis, utilize up to five paralogous type VII secretion systems to transport proteins across their cell envelope. Since these proteins associate in pairs that depend on each other for transport to a different extent, the secretion pathway to the bacterial surface remained challenging to address. Structural characterization of the inner-membrane embedded secretion machineries along with recent advances on the substrates' co-dependencies for transport allow for the first time more detailed and testable models for secretion.

The regulation of bacterial two-partner secretion systems.

Two-partner secretion (TPS) systems, also known as Type Vb secretion systems, allow the translocation of effector proteins across the outer membrane of Gram-negative bacteria. By secreting different classes of effectors, including cytolysins and adhesins, TPS systems play important roles in bacterial pathogenesis and host interactions. Here, we review the current knowledge on TPS systems regulation and highlight specific and common regulatory mechanisms across TPS functional classes. We discuss in detail the specific regulatory networks identified in various bacterial species and emphasize the importance of understanding the context-dependent regulation of TPS systems. Several regulatory cues reflecting host environment during infection, such as temperature and iron availability, are common determinants of expression for TPS systems, even across relatively distant species. These common regulatory pathways often affect TPS systems across subfamilies with different effector functions, representing conserved global infection-related regulatory mechanisms.

Bacterial Type II Secretion System and Its Mitochondrial Counterpart.

Over the billions of years that bacteria have been around, they have evolved several sophisticated protein secretion nanomachines to deliver toxins, hydrolytic enzymes, and effector proteins into their environments. Of these, the type II secretion system (T2SS) is used by Gram-negative bacteria to export a wide range of folded proteins from the periplasm across the outer membrane. Recent findings have demonstrated that components of the T2SS are localized in mitochondria of some eukaryotic lineages, and their behavior is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). This review focuses on recent advances in the field and discusses open questions concerning the function and evolution of miT2SSs.

The Coxiella burnetii effector EmcB is a deubiquitinase that inhibits RIG-I signaling.

Eukaryotes have cytosolic surveillance systems to detect invading microorganisms and initiate protective immune responses. In turn, host-adapted pathogens have evolved strategies to modulate these surveillance systems, which can promote dissemination and persistence in the host. The obligate intracellular pathogen Coxiella burnetii infects mammalian hosts without activating many innate immune sensors. The Defect in Organelle Trafficking/Intracellular Multiplication (Dot/Icm) protein secretion system is necessary for C. burnetii to establish a vacuolar niche inside of host cells, which sequesters these bacteria in a specialized organelle that could evade host surveillance systems. However, bacterial secretion systems often introduce agonists of immune sensors into the host cytosol during infection. For instance, nucleic acids are introduced to the host cytosol by the Dot/Icm system of Legionella pneumophila, which results in type I interferon production. Despite host infection requiring a homologous Dot/Icm system, C. burnetii does not induce type I interferon production during infection. Here, it was found that type I interferons are detrimental to C. burnetii infection and that C. burnetii blocks type I interferon production mediated by retionic acid inducible gene I (RIG-I) signaling. Two Dot/Icm effector proteins, EmcA and EmcB, are required for C. burnetii inhibition of RIG-I signaling. EmcB is sufficient to block RIG-I signaling and is a ubiquitin-specific cysteine protease capable of deconjugating ubiquitin chains from RIG-I that are necessary for signaling. EmcB preferentially cleaves K63-linked ubiquitin chains of three or more monomers, which represent ubiquitin chains that potently activate RIG-I signaling. Identification of a deubiquitinase encoded by C. burnetii provides insights into how a host-adapted pathogen antagonizes immune surveillance.

Biochemical analysis of protein-protein interfaces underlying the regulation of bacterial secretion systems.

Bacterial pathogens such as Pseudomonas aeruginosa use complex regulatory networks to tailor gene expression patterns to meet complex environmental challenges. P. aeruginosa is capable of causing both acute and chronic persistent infections, each type being characterized by distinct symptoms brought about by distinct sets of virulence mechanisms. The GacS/GacA phosphorelay system sits at the heart of a complex regulatory network that reciprocally governs the expression of virulence factors associated with either acute or chronic infections. A second non-enzymatic signaling cascade involving four proteins, ExsA, ExsC, ExsD, and ExsE is a key player in regulating the expression of the type three secretion system, an essential facilitator of acute infections. Both signaling pathways involve a remarkable array of non-canonical interactions that we sought to characterize. In the following section, we will outline several strategies, we adapted to map protein-protein interfaces and quantify the strength of biomolecular interactions by pairing complex mutational analyses with FRET binding assays and Bacterial-Two-Hybrid assays with appropriate functional assays. In the process, protocols were developed for disrupting large hydrophobic interfaces, deleting entire domains within a protein, and for mapping protein-protein interfaces formed primarily through backbone interactions.

Lack of evidence that Pseudomonas aeruginosa AmpDh3-PA0808 constitute a type VI secretion system effector-immunity pair.

Type VI secretion systems (T6SSs) are cell envelope-spanning protein complexes that Gram-negative bacteria use to inject a diverse arsenal of antibacterial toxins into competitor cells. Recently, Wang et al. reported that the H2-T6SS of Pseudomonas aeruginosa delivers the peptidoglycan recycling amidase, AmpDh3, into the periplasm of recipient cells where it is proposed to act as a peptidoglycan degrading toxin. They further reported that PA0808, the open reading frame downstream of AmpDh3, encodes an immunity protein that localizes to the periplasm where it binds to and inactivates intercellularly delivered AmpDh3, thus protecting against its toxic activity. Given that AmpDh3 has an established role in cell wall homeostasis and that no precedent exists for cytosolic enzymes moonlighting as T6SS effectors, we attempted to replicate these findings. We found that cells lacking PA0808 are not susceptible to bacterial killing by AmpDh3 and that PA0808 and AmpDh3 do not physically interact in vitro or in vivo. Additionally, we found no evidence that AmpDh3 is exported from cells, including by strains with a constitutively active H2-T6SS. Finally, subcellular fractionation experiments and a 1.97 Å crystal structure reveal that PA0808 does not contain a canonical signal peptide or localize to the correct cellular compartment to confer protection against a cell wall targeting toxin. Taken together, these results cast doubt on the assertion that AmpDh3-PA0808 constitutes an H2-T6SS effector-immunity pair.

Protein interactome mapping of Porphyromonas gingivalis provides insights into the formation of the PorQ-Z complex of the type IX secretion system.

Porphyromonas gingivalis is an anaerobic Gram-negative human oral pathogen highly associated with the more severe forms of periodontal disease. Porphyromonas gingivalis utilises the type IX secretion system (T9SS) to transport ∼30 cargo proteins, including multiple virulence factors, to the cell surface. The T9SS is a multiprotein system consisting of at least 20 proteins, and recently, we characterised the protein interactome of these components. Similar to the T9SS, almost all biological processes are mediated through protein-protein interactions (PPIs). Therefore, mapping PPIs is important to understand the biological functions of many proteins in P. gingivalis. Herein, we provide native migration profiles of over 1000 P. gingivalis proteins. Using the T9SS, we demonstrate that our dataset is a useful resource for identifying novel protein interactions. Using this dataset and further analysis of T9SS P. gingivalis mutants, we discover new mechanistic insights into the formation of the PorQ-Z complex of the T9SS. This dataset is a valuable resource for studies of P. gingivalis.

Purification, crystallization and crystallographic analysis of the PorX response regulator associated with the type IX secretion system.

Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane(s) and manipulate their infected host. To date, 11 secretion systems have been identified, including the type IX secretion system (T9SS) associated with human, avian and farmed-fish diseases. As a bacterial secretion system, the T9SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria. PorX is a highly conserved protein that regulates the transcription of essential T9SS components and additionally mediates the function of T9SS via direct interaction with PorL, the rotary motor protein of the T9SS. PorX is also a member of a two-component system regulatory cascade, where it serves as the response regulator that relays a signal transduced from a conserved sensor histidine kinase, PorY, to a designated sigma factor. Here, the recombinant expression and purification of PorX homologous proteins from the pathogenic bacterium Porphyromonas gingivalis and the nonpathogenic bacterium Flavobacterium johnsoniae are reported. A bioinformatical characterization of the different domains comprising the PorX protein is also provided, and the crystallization and X-ray analysis of PorX from F. johnsoniae are reported.

Molecular architecture of bacterial type IV secretion systems.

Bacterial type IV secretion systems (T4SSs) are a versatile group of nanomachines that can horizontally transfer DNA through conjugation and deliver effector proteins into a wide range of target cells. The components of T4SSs in gram-negative bacteria are organized into several large subassemblies: an inner membrane complex, an outer membrane core complex, and, in some species, an extracellular pilus. Cryo-electron tomography has been used to define the structures of T4SSs in intact bacteria, and high-resolution structural models are now available for isolated core complexes from conjugation systems, the Xanthomonas citri T4SS, the Helicobacter pylori Cag T4SS, and the Legionella pneumophila Dot/Icm T4SS. In this review, we compare the molecular architectures of these T4SSs, focusing especially on the structures of core complexes. We discuss structural features that are shared by multiple T4SSs as well as evolutionary strategies used for T4SS diversification. Finally, we discuss how structural variations among T4SSs may confer specialized functional properties.

Insertional Inactivation and Gene Complementation of Prevotella intermedia Type IX Secretion System Reveals Its Indispensable Roles in Black Pigmentation, Hemagglutination, Protease Activity of Interpain A, and Biofilm Formation.

Prevotella intermedia, a Gram-negative oral anaerobic bacterium, is frequently isolated from the periodontal pockets of patients with chronic periodontitis. In recent years, the involvement of the bacterium in respiratory tract infections as well as in oral infections has been revealed. P. intermedia possesses several potent virulence factors, such as cysteine proteinase interpain A encoded by the inpA gene. The genome of P. intermedia carries genes of the type IX secretion system (T9SS), which enables the translocation of virulence factors across the outer membrane in several pathogens belonging to the phylum Bacteroidetes; however, it is still unclear whether the T9SS is functional in this microorganism. Recently, we performed targeted mutagenesis in the strain OMA14 of P. intermedia. Here, we successfully obtained mutants deficient in inpA and the T9SS component genes porK and porT. None of the mutants exhibited protease activity of interpain A. The porK and porT mutants, but not the inpA mutant, showed defects in colony pigmentation, hemagglutination, and biofilm formation. We also obtained a complemented strain for the porK gene that recovered all the above abilities. These results indicate that T9SS functions in P. intermedia and that interpain A is one of the T9SS cargo proteins. IMPORTANCE The virulence factors of periodontal pathogens such as Prevotella intermedia have not been elucidated. Using our established procedure, we succeeded in generating type IX secretion system mutants and gene complementation strains that might transfer virulence factors to the bacterial surface. The generated strains clearly indicate that T9SS in P. intermedia is essential for colonial pigmentation, hemagglutination, and biofilm formation. These results indicated that interpain A is a T9SS cargo protein.

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.

Bacterial protein secretion systems: Game of types.

Protein trafficking across the bacterial envelope is a process that contributes to the organisation and integrity of the cell. It is the foundation for establishing contact and exchange between the environment and the cytosol. It helps cells to communicate with one another, whether they establish symbiotic or competitive behaviours. It is instrumental for pathogenesis and for bacteria to subvert the host immune response. Understanding the formation of envelope conduits and the manifold strategies employed for moving macromolecules across these channels is a fascinating playground. The diversity of the nanomachines involved in this process logically resulted in an attempt to classify them, which is where the protein secretion system types emerged. As our knowledge grew, so did the number of types, and their rightful nomenclature started to be questioned. While this may seem a semantic or philosophical issue, it also reflects scientific rigour when it comes to assimilating findings into textbooks and science history. Here I give an overview on bacterial protein secretion systems, their history, their nomenclature and why it can be misleading for newcomers in the field. Note that I do not try to suggest a new nomenclature. Instead, I explore the reasons why naming could have escaped our control and I try to reiterate basic concepts that underlie protein trafficking cross membranes.

Structures of the Type IX Secretion/Gliding Motility Motor from across the Phylum Bacteroidetes.

Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are energized by the GldLM motor complex, which transduces the proton motive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9-Å resolution (R. Hennell James, J. C. Deme, A. Kjaer, F. Alcock, et al., Nat Microbiol 6:221-233, 2021, https://dx.doi.org/10.1038/s41564-020-00823-6). Here, we present structures of homologous complexes from a range of pathogenic and environmental Bacteroidetes species at up to 3.0-Å resolution. These structures show that the architecture of the GldLM motor core is conserved across the Bacteroidetes phylum, although there are species-specific differences at the N terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility. IMPORTANCE Many bacteria in the Bacteroidetes phylum use the type IX secretion system to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex, which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species, including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organization of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the type IX secretion system, which is a virulence determinant in human and animal diseases.