Et tu, Vibrio cholerae? Kin-cannibalism and a bacterial secretion system.

Type VI secretion systems are molecular syringes used by Gram-negative bacteria to kill heterospecific (non-kin) niche competitors. In this issue of Cell, Mashruwala et al. show that colonies of the pathogen Vibrio cholera can also exhibit T6SS-mediated cell killing of kin cells and that this process benefits emerging resistant mutants, thereby increasing genetic diversity.

Endocannabinoids Inhibit the Induction of Virulence in Enteric Pathogens.

Endocannabinoids are host-derived lipid hormones that fundamentally impact gastrointestinal (GI) biology. The use of cannabis and other exocannabinoids as anecdotal treatments for various GI disorders inspired the search for mechanisms by which these compounds mediate their effects, which led to the discovery of the mammalian endocannabinoid system. Dysregulated endocannabinoid signaling was linked to inflammation and the gut microbiota. However, the effects of endocannabinoids on host susceptibility to infection has not been explored. Here, we show that mice with elevated levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG) are protected from enteric infection by Enterobacteriaceae pathogens. 2-AG directly modulates pathogen function by inhibiting virulence programs essential for successful infection. Furthermore, 2-AG antagonizes the bacterial receptor QseC, a histidine kinase encoded within the core Enterobacteriaceae genome that promotes the activation of pathogen-associated type three secretion systems. Taken together, our findings establish that endocannabinoids are directly sensed by bacteria and can modulate bacterial function.

Protein-Injection Machines in Bacteria.

Many bacteria have evolved specialized nanomachines with the remarkable ability to inject multiple bacterially encoded effector proteins into eukaryotic or prokaryotic cells. Known as type III, type IV, and type VI secretion systems, these machines play a central role in the pathogenic or symbiotic interactions between multiple bacteria and their eukaryotic hosts, or in the establishment of bacterial communities in a diversity of environments. Here we focus on recent progress elucidating the structure and assembly pathways of these machines. As many of the interactions shaped by these machines are of medical importance, they provide an opportunity to develop novel therapeutic approaches to combat important human diseases.

Cellular Electron Cryotomography: Toward Structural Biology In Situ.

Electron cryotomography (ECT) provides three-dimensional views of macromolecular complexes inside cells in a native frozen-hydrated state. Over the last two decades, ECT has revealed the ultrastructure of cells in unprecedented detail. It has also allowed us to visualize the structures of macromolecular machines in their native context inside intact cells. In many cases, such machines cannot be purified intact for in vitro study. In other cases, the function of a structure is lost outside the cell, so that the mechanism can be understood only by observation in situ. In this review, we describe the technique and its history and provide examples of its power when applied to cell biology. We also discuss the integration of ECT with other techniques, including lower-resolution fluorescence imaging and higher-resolution atomic structure determination, to cover the full scale of cellular processes.

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine.

Type III protein secretion systems have specifically evolved to deliver bacterially encoded proteins into target eukaryotic cells. The core elements of this multi-protein machine are the envelope-associated needle complex, the inner membrane export apparatus, and a large cytoplasmic sorting platform. Here, we report a high-resolution in situ structure of the Salmonella Typhimurium type III secretion machine obtained by high-throughput cryo-electron tomography and sub-tomogram averaging. Through molecular modeling and comparative analysis of machines assembled with protein-tagged components or from different deletion mutants, we determined the molecular architecture of the secretion machine in situ and localized its structural components. We also show that docking of the sorting platform results in significant conformational changes in the needle complex to provide the symmetry adaptation required for the assembly of the entire secretion machine. These studies provide major insight into the structure and assembly of a broadly distributed protein secretion machine.

Atomic structure of T6SS reveals interlaced array essential to function.

Type VI secretion systems (T6SSs) are newly identified contractile nanomachines that translocate effector proteins across bacterial membranes. The Francisella pathogenicity island, required for bacterial phagosome escape, intracellular replication, and virulence, was presumed to encode a T6SS-like apparatus. Here, we experimentally confirm the identity of this T6SS and, by cryo electron microscopy (cryoEM), show the structure of its post-contraction sheath at 3.7 Å resolution. We demonstrate the assembly of this T6SS by IglA/IglB and secretion of its putative effector proteins in response to environmental stimuli. The sheath has a quaternary structure with handedness opposite that of contracted sheath of T4 phage tail and is organized in an interlaced two-dimensional array by means of ß sheet augmentation. By structure-based mutagenesis, we show that this interlacing is essential to secretion, phagosomal escape, and intracellular replication. Our atomic model of the T6SS will facilitate design of drugs targeting this highly prevalent secretion apparatus.

Structure of the type VI secretion system contractile sheath.

Bacteria use rapid contraction of a long sheath of the type VI secretion system (T6SS) to deliver effectors into a target cell. Here, we present an atomic-resolution structure of a native contracted Vibrio cholerae sheath determined by cryo-electron microscopy. The sheath subunits, composed of tightly interacting proteins VipA and VipB, assemble into a six-start helix. The helix is stabilized by a core domain assembled from four ß strands donated by one VipA and two VipB molecules. The fold of inner and middle layers is conserved between T6SS and phage sheaths. However, the structure of the outer layer is distinct and suggests a mechanism of interaction of the bacterial sheath with an accessory ATPase, ClpV, that facilitates multiple rounds of effector delivery. Our results provide a mechanistic insight into assembly of contractile nanomachines that bacteria and phages use to translocate macromolecules across membranes.

Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion.

Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increase in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.

Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions.

The bacterial type VI secretion system (T6SS) is a dynamic organelle that bacteria use to target prey cells for inhibition via translocation of effector proteins. Time-lapse fluorescence microscopy has documented striking dynamics of opposed T6SS organelles in adjacent sister cells of Pseudomonas aeruginosa. Such cell-cell interactions have been termed "T6SS dueling" and likely reflect a biological process that is driven by T6SS antibacterial attack. Here, we show that T6SS dueling behavior strongly influences the ability of P. aeruginosa to prey upon heterologous bacterial species. We show that, in the case of P. aeruginosa, T6SS-dependent killing of either Vibrio cholerae or Acinetobacter baylyi is greatly stimulated by T6SS activity occurring in those prey species. Our data suggest that, in P. aeruginosa, T6SS organelle assembly and lethal counterattack are regulated by a signal that corresponds to the point of attack of the T6SS apparatus elaborated by a second aggressive T6SS(+) bacterial cell. PAPERFLICK:

Identification of trypsin-degrading commensals in the large intestine.

Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions1-3. However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells4,5. Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.

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.

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.

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).

Dynamic proton-dependent motors power type IX secretion and gliding motility in Flavobacterium.

Motile bacteria usually rely on external apparatus like flagella for swimming or pili for twitching. By contrast, gliding bacteria do not rely on obvious surface appendages to move on solid surfaces. Flavobacterium johnsoniae and other bacteria in the Bacteroidetes phylum use adhesins whose movement on the cell surface supports motility. In F. johnsoniae, secretion and helicoidal motion of the main adhesin SprB are intimately linked and depend on the type IX secretion system (T9SS). Both processes necessitate the proton motive force (PMF), which is thought to fuel a molecular motor that comprises the GldL and GldM cytoplasmic membrane proteins. Here, we show that F. johnsoniae gliding motility is powered by the pH gradient component of the PMF. We further delineate the interaction network between the GldLM transmembrane helices (TMHs) and show that conserved glutamate residues in GldL TMH2 are essential for gliding motility, although having distinct roles in SprB secretion and motion. We then demonstrate that the PMF and GldL trigger conformational changes in the GldM periplasmic domain. We finally show that multiple GldLM complexes are distributed in the membrane, suggesting that a network of motors may be present to move SprB along a helical path on the cell surface. Altogether, our results provide evidence that GldL and GldM assemble dynamic membrane channels that use the proton gradient to power both T9SS-dependent secretion of SprB and its motion at the cell surface.

A unique bacterial secretion machinery with multiple secretion centers.

The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron tomography, we report that the P. gingivalis T9SS consists of 18 PorM dimers arranged as a large, caged ring in the periplasm. Near the outer membrane, PorM dimers interact with a PorKN ring complex of ∼52 nm in diameter. PorMKN translocation complexes of a given T9SS adopt distinct conformations energized by the proton motive force, suggestive of different activation states. At the inner membrane, PorM associates with a cytoplasmic complex that exhibits 12-fold symmetry and requires both PorM and PorL for assembly. Activated motors deliver substrates across the outer membrane via one of eight Sov translocons arranged in a ring. The T9SSs are unique among known secretion systems in bacteria and eukaryotes in their assembly as supramolecular machines composed of apparently independently functioning translocation motors and export pores.

Multiple genetic paths including massive gene amplification allow Mycobacterium tuberculosis to overcome loss of ESX-3 secretion system substrates.

Mycobacterium tuberculosis (Mtb) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. Mtb strains deleted for the genes encoding substrates of the ESX-3 T7SS, esxG or esxH, require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of esxG or esxH deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of Mtb chromosome evolution involving "accordion-type" amplification.

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.

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.

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.

Assembly and Subcellular Localization of Bacterial Type VI Secretion Systems.

Bacteria need to deliver large molecules out of the cytosol to the extracellular space or even across membranes of neighboring cells to influence their environment, prevent predation, defeat competitors, or communicate. A variety of protein-secretion systems have evolved to make this process highly regulated and efficient. The type VI secretion system (T6SS) is one of the largest dynamic assemblies in gram-negative bacteria and allows for delivery of toxins into both bacterial and eukaryotic cells. The recent progress in structural biology and live-cell imaging shows the T6SS as a long contractile sheath assembled around a rigid tube with associated toxins anchored to a cell envelope by a baseplate and membrane complex. Rapid sheath contraction releases a large amount of energy used to push the tube and toxins through the membranes of neighboring target cells. Because reach of the T6SS is limited, some bacteria dynamically regulate its subcellular localization to precisely aim at their targets and thus increase efficiency of toxin translocation.