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.

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.

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.

Structure-function analysis of PorXFj, the PorX homolog from Flavobacterium johnsioniae, suggests a role of the CheY-like domain in type IX secretion motor activity.

The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.

A type VI secretion system effector TseG of Pantoea ananatis is involved in virulence and antibacterial activity.

The type VI secretion system (T6SS) of many gram-negative bacteria injects toxic effectors into adjacent cells to manipulate host cells during pathogenesis or to kill competing bacteria. However, the identification and function of the T6SS effectors remains only partly known. Pantoea ananatis, a gram-negative bacterium, is commonly found in various plants and natural environments, including water and soil. In the current study, genomic analysis of P. ananatis DZ-12 causing brown stalk rot on maize demonstrated that it carries three T6SS gene clusters, namely, T6SS-1, T6SS-2, and T6SS-3. Interestingly, only T6SS-1 secretion systems are involved in pathogenicity and bacterial competition. The study also investigated the T6SS-1 system in detail and identified an unknown T6SS-1-secreted effector TseG by using the upstream T6SS effector chaperone TecG containing a conserved domain of DUF2169. TseG can directly interact with the chaperone TecG for delivery and with a downstream immunity protein TsiG for protection from its toxicity. TseG, highly conserved in the Pantoea genus, is involved in virulence in maize, potato, and onion. Additionally, P. ananatis uses TseG to target Escherichia coli, gaining a competitive advantage. This study provides the first report on the T6SS-1-secreted effector from P. ananatis, thereby enriching our understanding of the various types and functions of type VI effector proteins.

Structural insights into the mechanism of protein transport by the Type 9 Secretion System translocon.

Secretion systems are protein export machines that enable bacteria to exploit their environment through the release of protein effectors. The Type 9 Secretion System (T9SS) is responsible for protein export across the outer membrane (OM) of bacteria of the phylum Bacteroidota. Here we trap the T9SS of Flavobacterium johnsoniae in the process of substrate transport by disrupting the T9SS motor complex. Cryo-EM analysis of purified substrate-bound T9SS translocons reveals an extended translocon structure in which the previously described translocon core is augmented by a periplasmic structure incorporating the proteins SprE, PorD and a homologue of the canonical periplasmic chaperone Skp. Substrate proteins bind to the extracellular loops of a carrier protein within the translocon pore. As transport intermediates accumulate on the translocon when energetic input is removed, we deduce that release of the substrate-carrier protein complex from the translocon is the energy-requiring step in T9SS transport.

Caught in the act: In situ visualization of bacterial secretion systems by cryo-electron tomography.

Bacterial secretion systems, such as the type 3, 4, and 6 are multiprotein nanomachines expressed at the surface of pathogens with Gram-negative like envelopes. They are known to be crucial for virulence and to translocate bacteria-encoded effector proteins into host cells to manipulate cellular functions. This facilitates either pathogen attachment or invasion of the targeted cell. Effector proteins also promote evasion of host immune recognition. Imaging by cryo-electron microscopy in combination with structure determination has become a powerful approach to understand how these nanomachines work. Still, questions on their assembly, the precise secretion mechanisms, and their direct involvement in pathogenicity remain unsolved. Here, we present an overview of the recent developments in in situ cryo-electron microscopy. We discuss its potential for the investigation of the role of bacterial secretion systems during the host-bacterial crosstalk at the molecular level. These in situ studies open new perspectives for our understanding of secretion system structure and function.

Assembly mechanism of a Tad secretion system secretin-pilotin complex.

The bacterial Tight adherence Secretion System (TadSS) assembles surface pili that drive cell adherence, biofilm formation and bacterial predation. The structure and mechanism of the TadSS is mostly unknown. This includes characterisation of the outer membrane secretin through which the pilus is channelled and recruitment of its pilotin. Here we investigate RcpA and TadD lipoprotein from Pseudomonas aeruginosa. Light microscopy reveals RcpA colocalising with TadD in P. aeruginosa and when heterologously expressed in Escherichia coli. We use cryogenic electron microscopy to determine how RcpA and TadD assemble a secretin channel with C13 and C14 symmetries. Despite low sequence homology, we show that TadD shares a similar fold to the type 4 pilus system pilotin PilF. We establish that the C-terminal four residues of RcpA bind TadD - an interaction essential for secretin formation. The binding mechanism between RcpA and TadD appears distinct from known secretin-pilotin pairings in other secretion systems.

Bacterial secretion systems contribute to rapid tissue decay in button mushroom soft rot disease.

The soft rot pathogen Janthinobacterium agaricidamnosum causes devastating damage to button mushrooms (Agaricus bisporus), one of the most cultivated and commercially relevant mushrooms. We previously discovered that this pathogen releases the membrane-disrupting lipopeptide jagaricin. This bacterial toxin, however, could not solely explain the rapid decay of mushroom fruiting bodies, indicating that J. agaricidamnosum implements a more sophisticated infection strategy. In this study, we show that secretion systems play a crucial role in soft rot disease. By mining the genome of J. agaricidamnosum, we identified gene clusters encoding a type I (T1SS), a type II (T2SS), a type III (T3SS), and two type VI secretion systems (T6SSs). We targeted the T2SS and T3SS for gene inactivation studies, and subsequent bioassays implicated both in soft rot disease. Furthermore, through a combination of comparative secretome analysis and activity-guided fractionation, we identified a number of secreted lytic enzymes responsible for mushroom damage. Our findings regarding the contribution of secretion systems to the disease process expand the current knowledge of bacterial soft rot pathogens and represent a significant stride toward identifying targets for their disarmament with secretion system inhibitors. IMPORTANCE The button mushroom (Agaricus bisporus) is the most popular edible mushroom in the Western world. However, mushroom crops can fall victim to serious bacterial diseases that are a major threat to the mushroom industry, among them being soft rot disease caused by Janthinobacterium agaricidamnosum. Here, we show that the rapid dissolution of mushroom fruiting bodies after bacterial invasion is due to degradative enzymes and putative effector proteins secreted via the type II secretion system (T2SS) and the type III secretion system (T3SS), respectively. The ability to degrade mushroom tissue is significantly attenuated in secretion-deficient mutants, which establishes that secretion systems are key factors in mushroom soft rot disease. This insight is of both ecological and agricultural relevance by shedding light on the disease processes behind a pathogenic bacterial-fungal interaction which, in turn, serves as a starting point for the development of secretion system inhibitors to control disease progression.

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.

A conditional gene expression system in Porphyromonas gingivalis for study of the secretion mechanisms of lipoproteins and T9SS cargo proteins.

The Gram-negative anaerobe, Porphyromonas gingivalis, is known to be a pathogen associated with chronic periodontitis. P. gingivalis possesses virulence factors such as fimbriae and gingipain proteinases. Fimbrial proteins are secreted to the cell surface as lipoproteins. In contrast, gingipain proteinases are secreted into the bacterial cell surface via the type IX secretion system (T9SS). The transport mechanisms of lipoproteins and T9SS cargo proteins are entirely different and remain unknown. Therefore, using the Tet-on system developed for the genus Bacteroides, we newly created a conditional gene expression system in P. gingivalis. We succeeded in establishing conditional expression of nanoluciferase and its derivatives for lipoprotein export, of FimA for a representative of lipoprotein export, and of T9SS cargo proteins such as Hbp35 and PorA for representatives of type 9 protein export. Using this system, we showed that the lipoprotein export signal, which has recently been found in other species in the phylum Bacteroidota, is also functional in FimA, and that a proton motive force inhibitor can affect type 9 protein export. Collectively, our conditional protein expression method is useful for screening inhibitors of virulence factors, and may be used to investigate the role of proteins essential to bacterial survival in vivo.

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.

Bacterial secretion system functions: evidence of interactions and downstream implications.

Unprecedented insights into the biology and functions of bacteria have been and continue to be gained through studying bacterial secretion systems in isolation. This method, however, results in our understanding of the systems being primarily based on the idea that they operate independently, ignoring the subtleties of downstream interconnections. Gram-negative bacteria are naturally able to adapt to and navigate their frequently varied and dynamic surroundings, mostly because of the covert connections between secretion systems. Therefore, to comprehend some of the linked downstream repercussions for organisms that follow this discourse, it is vital to have mechanistic insights into how the intersecretion system functions in bacterial rivalry, virulence, and survival, among other things. To that purpose, this paper discusses a few key instances of molecular antagonistic and interdependent relationships between bacterial secretion systems and their produced functional products.

Chitinase A, a tightly regulated virulence factor of Salmonella enterica serovar Typhimurium, is actively secreted by a Type 10 Secretion System.

As a facultative intracellular pathogen, Salmonella enterica serovar Typhimurium is one of the leading causes of food-borne diseases in humans. With the ingestion of fecal contaminated food or water, S. Typhimurium reaches the intestine. Here, the pathogen efficiently invades intestinal epithelial cells of the mucosal epithelium by the use of multiple virulence factors. Recently, chitinases have been described as emerging virulence factors of S. Typhimurium that contribute to the attachment and invasion of the intestinal epithelium, prevent immune activation, and modulate the host glycome. Here we find that the deletion of chiA leads to diminished adhesion and invasion of polarized intestinal epithelial cells (IEC) compared to wild-type S. Typhimurium. Interestingly, no apparent impact on interaction was detected when using non-polarized IEC or HeLa epithelial cells. In concordance, we demonstrate that chiA gene and ChiA protein expression was solely induced when bacteria gain contact with polarized IEC. The induction of chiA transcripts needs the specific activity of transcriptional regulator ChiR, which is co-localized with chiA in the chitinase operon. Moreover, we established that after chiA is induced, a major portion of the bacterial population expresses chiA, analyzed by flow cytometry. Once expressed, we found ChiA in the bacterial supernatants using Western blot analyses. ChiA secretion was completely abolished when accessory genes within the chitinase operon encoding for a holin and a peptidoglycan hydrolase were deleted. Holins, peptidoglycan hydrolases, and large extracellular enzymes in close proximity have been described as components of the bacterial holin/peptidoglycan hydrolase-dependent protein secretion system or Type 10 Secretion System. Overall, our results confirm that chitinase A is an important virulence factor, tightly regulated by ChiR, that promotes adhesion and invasion upon contact with polarized IEC and is likely secreted by a Type 10 Secretion System (T10SS).

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.

Recruitment of heterologous substrates by bacterial secretion systems for transkingdom translocation.

Bacterial secretion systems mediate the selective exchange of macromolecules between bacteria and their environment, playing a pivotal role in processes such as horizontal gene transfer or virulence. Among the different families of secretion systems, Type III, IV and VI (T3SS, T4SS and T6SS) share the ability to inject their substrates into human cells, opening up the possibility of using them as customized injectors. For this to happen, it is necessary to understand how substrates are recruited and to be able to engineer secretion signals, so that the transmembrane machineries can recognize and translocate the desired substrates in place of their own. Other factors, such as recruiting proteins, chaperones, and the degree of unfolding required to cross through the secretion channel, may also affect transport. Advances in the knowledge of the secretion mechanism have allowed heterologous substrate engineering to accomplish translocation by T3SS, and to a lesser extent, T4SS and T6SS into human cells. In the case of T4SS, transport of nucleoprotein complexes adds a bonus to its biotechnological potential. Here, we review the current knowledge on substrate recognition by these secretion systems, the many examples of heterologous substrate translocation by engineering of secretion signals, and the current and future biotechnological and biomedical applications derived from this approach.

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 structure of a tautomerase superfamily member linked to the type VI secretion system of Acinetobacter baumannii.

Bacteria exploit specialized secretion systems to assist in competition for resources, in collaboration and in communication. Here, a protocol for the recombinant production, purification and crystallization of a protein linked to the Acinetobacter baumannii type VI secretion system is provided. A high-resolution structure of this trimeric protein is reported, revealing the characteristic dual ß-α-ß subunit fold typical of longer subunit members of the tautomerase superfamily. The protein does not appear to be toxic to bacteria or yeast under the conditions tested. The possible biological role of this protein is discussed.