Bacterial type III effector-induced plant C8 volatiles elicit antibacterial immunity in heterospecific neighbouring plants via airborne signalling.

Upon sensing attack by pathogens and insect herbivores, plants release complex mixtures of volatile compounds. Here, we show that the infection of lima bean (Phaseolus lunatus L.) plants with the non-host bacterial pathogen Pseudomonas syringae pv. tomato led to the production of microbe-induced plant volatiles (MIPVs). Surprisingly, the bacterial type III secretion system, which injects effector proteins directly into the plant cytosol to subvert host functions, was found to prime both intra- and inter-specific defense responses in neighbouring wild tobacco (Nicotiana benthamiana) plants. Screening of each of 16 effectors using the Pseudomonas fluorescens effector-to-host analyser revealed that an effector, HopP1, was responsible for immune activation in receiver tobacco plants. Further study demonstrated that 1-octen-3-ol, 3-octanone and 3-octanol are novel MIPVs emitted by the lima bean plant in a HopP1-dependent manner. Exposure to synthetic 1-octen-3-ol activated immunity in tobacco plants against a virulent pathogen Pseudomonas syringae pv. tabaci. Our results show for the first time that a bacterial type III effector can trigger the emission of C8 plant volatiles that mediate defense priming via plant-plant interactions. These results provide novel insights into the role of airborne chemicals in bacterial pathogen-induced inter-specific plant-plant interactions.

Structural and Functional Analysis of SsaV Cytoplasmic Domain and Variable Linker States in the Context of the InvA-SsaV Chimeric Protein.

The type III secretion (T3S) injectisome is a syringe-like protein-delivery nanomachine widely utilized by Gram-negative bacteria. It can deliver effector proteins directly from bacteria into eukaryotic host cells, which is crucial for the bacterial-host interaction. Intracellular pathogen Salmonella enterica serovar Typhimurium encodes two sets of T3S injectisomes from Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), which are critical for its host invasion and intracellular survival, respectively. The inner membrane export gate protein, SctV (InvA in SPI-1 and SsaV in SPI-2), is the largest component of the injectisome and is essential for assembly and function of T3SS. Here, we report the 2.11 Å cryo-EM structure of the SsaV cytoplasmic domain (SsaVC) in the context of a full-length SctV chimera consisting of the transmembrane region of InvA, the linker of SsaV (SsaVL) and SsaVC. The structural analysis shows that SsaVC exists in a semi-open state and SsaVL exhibits two major orientations, implying a highly dynamic process of SsaV for the substrate selection and secretion in a full-length context. A biochemical assay indicates that SsaVL plays an essential role in maintaining the nonameric state of SsaV. This study offers near atomic-level insights into how SsaVC and SsaVL facilitate the assembly and function of SsaV and may lead to the development of potential anti-virulence therapeutics against T3SS-mediated bacterial infection. IMPORTANCE Type III secretion system (T3SS) is a multicomponent nanomachine and a critical virulence factor for a wide range of Gram-negative bacterial pathogens. It can deliver numbers of effectors into the host cell to facilitate the bacterial host infection. Export gate protein SctV, as one of the engines of T3SS, is at the center of T3SS assembly and function. In this study, we show the high-resolution atomic structure of the cytosolic domain of SctV in the nonameric state with variable linker conformations. Our first observation of conformational changes of the linker region of SctV and the semi-open state of the cytosolic domain of SctV in the full-length context further support that the substrate selection and secretion process of SctV is highly dynamic. These findings have important implications for the development of therapeutic strategies targeting SctV to combat T3SS-mediated bacterial infection.

Cryo-EM structure of the needle filament tip complex of the Salmonella type III secretion injectisome.

Type III secretion systems are multiprotein molecular machines required for the virulence of several important bacterial pathogens. The central element of these machines is the injectisome, a ∼5-Md multiprotein structure that mediates the delivery of bacterially encoded proteins into eukaryotic target cells. The injectisome is composed of a cytoplasmic sorting platform, and a membrane-embedded needle complex, which is made up of a multiring base and a needle-like filament that extends several nanometers from the bacterial surface. The needle filament is capped at its distal end by another substructure known as the tip complex, which is crucial for the translocation of effector proteins through the eukaryotic cell plasma membrane. Here we report the cryo-EM structure of the Salmonella Typhimurium needle tip complex docked onto the needle filament tip. Combined with a detailed analysis of structurally guided mutants, this study provides major insight into the assembly and function of this essential component of the type III secretion protein injection machine.

α-Helices in the Type III Secretion Effectors: A Prevalent Feature with Versatile Roles.

Type III Secretion Systems (T3SSs) are multicomponent nanomachines located at the cell envelope of Gram-negative bacteria. Their main function is to transport bacterial proteins either extracellularly or directly into the eukaryotic host cell cytoplasm. Type III Secretion effectors (T3SEs), latest to be secreted T3S substrates, are destined to act at the eukaryotic host cell cytoplasm and occasionally at the nucleus, hijacking cellular processes through mimicking eukaryotic proteins. A broad range of functions is attributed to T3SEs, ranging from the manipulation of the host cell's metabolism for the benefit of the bacterium to bypassing the host's defense mechanisms. To perform this broad range of manipulations, T3SEs have evolved numerous novel folds that are compatible with some basic requirements: they should be able to easily unfold, pass through the narrow T3SS channel, and refold to an active form when on the other side. In this review, the various folds of T3SEs are presented with the emphasis placed on the functional and structural importance of α-helices and helical domains.

Type 3 secretion system of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen, mainly affecting severe patients, such as those in intensive care units (ICUs). High levels of antibiotic resistance and a long battery of virulence factors characterise this pathogen. Among virulence factors, the T3SS (Type 3 Secretion Systems) are especially relevant, being one of the most important virulence factors in P. aeruginosa. T3SS are a complex "molecular syringe" able to inject different effectors in host cells, subverting cell machinery influencing immune responses, and increasing bacterial survival rates. While T3SS have been largely studied and the molecular structure and main effector functions have been established, a series of questions and further points remain to be clarified or established. The key role of T3SS in P. aeruginosa virulence has resulted in the search for T3SS-targeting molecules able to impair their functions and subsequently improve patient outcomes. This review aims to summarise the most relevant features of the P. aeruginosa T3SS.

Got mutants? How advances in chlamydial genetics have furthered the study of effector proteins.

Chlamydia trachomatis is the leading cause of infectious blindness and a sexually transmitted infection. All chlamydiae are obligate intracellular bacteria that replicate within a membrane-bound vacuole termed the inclusion. From the confines of the inclusion, the bacteria must interact with many host organelles to acquire key nutrients necessary for replication, all while promoting host cell viability and subverting host defense mechanisms. To achieve these feats, C. trachomatis delivers an arsenal of virulence factors into the eukaryotic cell via a type 3 secretion system (T3SS) that facilitates invasion, manipulation of host vesicular trafficking, subversion of host defense mechanisms and promotes bacteria egress at the conclusion of the developmental cycle. A subset of these proteins intercalate into the inclusion and are thus referred to as inclusion membrane proteins. Whereas others, referred to as conventional T3SS effectors, are released into the host cell where they localize to various eukaryotic organelles or remain in the cytosol. Here, we discuss the functions of T3SS effector proteins with a focus on how advances in chlamydial genetics have facilitated the identification and molecular characterization of these important factors.

Cryoelectron-microscopy structure of the enteropathogenic Escherichia coli type III secretion system EspA filament.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) utilize a macromolecular type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. This apparatus spans the inner and outer bacterial membranes and includes a helical needle protruding into the extracellular space. Thus far observed only in EPEC and EHEC and not found in other pathogenic Gram-negative bacteria that have a T3SS is an additional helical filament made by the EspA protein that forms a long extension to the needle, mediating both attachment to eukaryotic cells and transport of effector proteins through the intestinal mucus layer. Here, we present the structure of the EspA filament from EPEC at 3.4 Å resolution. The structure reveals that the EspA filament is a right-handed 1-start helical assembly with a conserved lumen architecture with respect to the needle to ensure the seamless transport of unfolded cargos en route to the target cell. This functional conservation is despite the fact that there is little apparent overall conservation at the level of sequence or structure with the needle. We also unveil the molecular details of the immunodominant EspA epitope that can now be exploited for the rational design of epitope display systems.

Cell-type-specific hypertranslocation of effectors by the Pseudomonas aeruginosa type III secretion system.

Many Gram-negative pathogens use a type III secretion system (T3SS) to promote disease by injecting effector proteins into host cells. Common to many T3SSs is that injection of effector proteins is feedback inhibited. The mechanism of feedback inhibition and its role in pathogenesis are unclear. In the case of P. aeruginosa, the effector protein ExoS is central to limiting effector injection. ExoS is bifunctional, with an amino-terminal RhoGAP and a carboxy-terminal ADP-ribosyltransferase domain. We demonstrate that both domains are required to fully feedback inhibit effector injection. The RhoGAP-, but not the ADP-ribosyltransferase domain of the related effector protein ExoT also participates. Feedback inhibition does not involve translocator insertion nor pore-formation. Instead, feedback inhibition is due, in part, to a loss of the activating trigger for effector injection, and likely also decreased translocon stability. Surprisingly, feedback inhibition is abrogated in phagocytic cells. The lack of feedback inhibition in these cells requires phagocytic uptake of the bacteria, but cannot be explained through acidification of the phagosome or calcium limitation. Given that phagocytes are crucial for controlling P. aeruginosa infections, our data suggest that feedback inhibition allows P. aeruginosa to direct its effector arsenal against the cell types most damaging to its survival.

Adaptivity and dynamics in type III secretion systems.

The type III secretion system is the common core of two bacterial molecular machines: the flagellum and the injectisome. The flagellum is the most widely distributed prokaryotic locomotion device, whereas the injectisome is a syringe-like apparatus for inter-kingdom protein translocation, which is essential for virulence in important human pathogens. The successful concept of the type III secretion system has been modified for different bacterial needs. It can be adapted to changing conditions, and was found to be a dynamic complex constantly exchanging components. In this review, we highlight the flexibility, adaptivity, and dynamic nature of the type III secretion system.

Anaerobic Respiration of NOX1-Derived Hydrogen Peroxide Licenses Bacterial Growth at the Colonic Surface.

The colonic microbiota exhibits cross-sectional heterogeneity, but the mechanisms that govern its spatial organization remain incompletely understood. Here we used Citrobacter rodentium, a pathogen that colonizes the colonic surface, to identify microbial traits that license growth and survival in this spatial niche. Previous work showed that during colonic crypt hyperplasia, type III secretion system (T3SS)-mediated intimate epithelial attachment provides C. rodentium with oxygen for aerobic respiration. However, we find that prior to the development of colonic crypt hyperplasia, T3SS-mediated intimate attachment is not required for aerobic respiration but for hydrogen peroxide (H2O2) respiration using cytochrome c peroxidase (Ccp). The epithelial NADPH oxidase NOX1 is the primary source of luminal H2O2 early after C. rodentium infection and is required for Ccp-dependent growth. Our results suggest that NOX1-derived H2O2 is a resource that governs bacterial growth and survival in close proximity to the mucosal surface during gut homeostasis.

Ancient co-option of an amino acid ABC transporter locus in Pseudomonas syringae for host signal-dependent virulence gene regulation.

Pathogenic bacteria frequently acquire virulence traits via horizontal gene transfer, yet additional evolutionary innovations may be necessary to integrate newly acquired genes into existing regulatory pathways. The plant bacterial pathogen Pseudomonas syringae relies on a horizontally acquired type III secretion system (T3SS) to cause disease. T3SS-encoding genes are induced by plant-derived metabolites, yet how this regulation occurs, and how it evolved, is poorly understood. Here we report that the two-component system AauS-AauR and substrate-binding protein AatJ, proteins encoded by an acidic amino acid-transport (aat) and -utilization (aau) locus in P. syringae, directly regulate T3SS-encoding genes in response to host aspartate and glutamate signals. Mutants of P. syringae strain DC3000 lacking aauS, aauR or aatJ expressed lower levels of T3SS genes in response to aspartate and glutamate, and had decreased T3SS deployment and virulence during infection of Arabidopsis. We identified an AauR-binding motif (Rbm) upstream of genes encoding T3SS regulators HrpR and HrpS, and demonstrated that this Rbm is required for maximal T3SS deployment and virulence of DC3000. The Rbm upstream of hrpRS is conserved in all P. syringae strains with a canonical T3SS, suggesting AauR regulation of hrpRS is ancient. Consistent with a model of conserved function, an aauR deletion mutant of P. syringae strain B728a, a bean pathogen, had decreased T3SS expression and growth in host plants. Together, our data suggest that, upon acquisition of T3SS-encoding genes, a strain ancestral to P. syringae co-opted an existing AatJ-AauS-AauR pathway to regulate T3SS deployment in response to specific host metabolite signals.

Envelope Stress and Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System.

Salmonella enterica serovar Typhimurium uses a type three secretion system (T3SS) encoded on the Salmonella pathogenicity island 1 (SPI1) to invade intestinal epithelial cells and induce inflammatory diarrhea. The SPI1 T3SS is regulated by numerous environmental and physiological signals, integrated to either activate or repress invasion. Transcription of hilA, encoding the transcriptional activator of the SPI1 structural genes, is activated by three AraC-like regulators, HilD, HilC, and RtsA, that act in a complex feed-forward loop. Deletion of bamB, encoding a component of the ß-barrel assembly machinery, causes a dramatic repression of SPI1, but the mechanism was unknown. Here, we show that partially defective ß-barrel assembly activates the RcsCDB regulon, leading to decreased hilA transcription. This regulation is independent of RpoE activation. Though Rcs has been previously shown to repress SPI1 when disulfide bond formation is impaired, we show that activation of Rcs in a bamB background is dependent on the sensor protein RcsF, whereas disulfide bond status is sensed independently. Rcs decreases transcription of the flagellar regulon, including fliZ, the product of which indirectly activates HilD protein activity. Rcs also represses hilD, hilC, and rtsA promoters by an unknown mechanism. Both dsbA and bamB mutants have motility defects, though this is simply regulatory in a bamB background; motility is restored in the absence of Rcs. Effector secretion assays show that repression of SPI1 in a bamB background is also regulatory; if expressed, the SPI1 T3SS is functional in a bamB background. This emphasizes the sensitivity of SPI1 regulation to overall envelope homeostasis.IMPORTANCESalmonella causes worldwide foodborne illness, leading to massive disease burden and an estimated 600,000 deaths per year. Salmonella infects orally and invades intestinal epithelial cells using a type 3 secretion system that directly injects effector proteins into host cells. This first step in invasion is tightly regulated by a variety of inputs. In this work, we demonstrate that Salmonella senses the functionality of outer membrane assembly in determining regulation of invasion machinery, and we show that Salmonella uses distinct mechanisms to detect specific perturbations in envelope assembly.

ACNNT3: Attention-CNN Framework for Prediction of Sequence-Based Bacterial Type III Secreted Effectors.

The type III secretion system (T3SS) is a special protein delivery system in Gram-negative bacteria which delivers T3SS-secreted effectors (T3SEs) to host cells causing pathological changes. Numerous experiments have verified that T3SEs play important roles in many biological activities and in host-pathogen interactions. Accurate identification of T3SEs is therefore essential to help understand the pathogenic mechanism of bacteria; however, many existing biological experimental methods are time-consuming and expensive. New deep-learning methods have recently been successfully applied to T3SE recognition, but improving the recognition accuracy of T3SEs is still a challenge. In this study, we developed a new deep-learning framework, ACNNT3, based on the attention mechanism. We converted 100 residues of the N-terminal of the protein sequence into a fusion feature vector of protein primary structure information (one-hot encoding) and position-specific scoring matrix (PSSM) which are used as the feature input of the network model. We then embedded the attention layer into CNN to learn the characteristic preferences of type III effector proteins, which can accurately classify any protein directly as either T3SEs or non-T3SEs. We found that the introduction of new protein features can improve the recognition accuracy of the model. Our method combines the advantages of CNN and the attention mechanism and is superior in many indicators when compared to other popular methods. Using the common independent dataset, our method is more accurate than the previous method, showing an improvement of 4.1-20.0%.

Extracellular DNA controls expression of Pseudomonas aeruginosa genes involved in nutrient utilization, metal homeostasis, acid pH tolerance and virulence.

Introduction. Pseudomonas aeruginosa grows in extracellular DNA (eDNA)-enriched biofilms and infection sites. eDNA is generally considered to be a structural biofilm polymer required for aggregation and biofilm maturation. In addition, eDNA can sequester divalent metal cations, acidify growth media and serve as a nutrient source.Aim. We wanted to determine the genome-wide influence on the transcriptome of planktonic P. aeruginosa PAO1 grown in the presence of eDNA.Methodology. RNA-seq analysis was performed to determine the genome-wide effects on gene expression of PAO1 grown with eDNA. Transcriptional lux fusions were used to confirm eDNA regulation and to validate phenotypes associated with growth in eDNA.Results. The transcriptome of eDNA-regulated genes included 89 induced and 76 repressed genes (FDR<0.05). A large number of eDNA-induced genes appear to be involved in utilizing DNA as a nutrient. Several eDNA-induced genes are also induced by acidic pH 5.5, and eDNA/acidic pH promoted an acid tolerance response in P. aeruginosa. The cyoABCDE terminal oxidase is induced by both eDNA and pH 5.5, and contributed to the acid tolerance phenotype. Quantitative metal analysis confirmed that DNA binds to diverse metals, which helps explain why many genes involved in a general uptake of metals were controlled by eDNA. Growth in the presence of eDNA also promoted intracellular bacterial survival and influenced virulence in the acute infection model of fruit flies.Conclusion. The diverse functions of the eDNA-regulated genes underscore the important role of this extracellular polymer in promoting antibiotic resistance, virulence, acid tolerance and nutrient utilization; phenotypes that contribute to long-term survival.

A revised model for the role of GacS/GacA in regulating type III secretion by Pseudomonas syringae pv. tomato DC3000.

GacS/GacA is a conserved two-component system that functions as a master regulator of virulence-associated traits in many bacterial pathogens, including Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/GacA-regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogen Pseudomonas aeruginosa, GacS/GacA negatively regulates the expression of type III secretion system (T3SS)-encoding genes. However, in the plant pathogenic bacterium Pseudomonas syringae, strain-to-strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we re-evaluated the function of GacA in P. syringae pv. tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence inside Arabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface-inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulating P. syringae virulence.

Mapping of Shigella flexneri's tissue distribution and type III secretion apparatus activity during infection of the large intestine of guinea pigs.

Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo.

Multiple proteins arising from a single gene: The role of the Spa33 variants in Shigella T3SS regulation.

Shigella invasion and dissemination in intestinal epithelial cells relies on a type 3 secretion system (T3SS), which mediates translocation of virulence proteins into host cells. T3SSs are composed of three major parts: an extracellular needle, a basal body, and a cytoplasmic complex. Three categories of proteins are hierarchically secreted: (a) the needle components, (b) the translocator proteins which form a pore (translocon) inside the host cell membrane and (c) the effectors interfering with the host cell signaling pathways. In the absence of host cell contact, the T3SS is maintained in an "off" state by the presence of a tip complex. Secretion is activated by host cell contact which allows the release of a gatekeeper protein called MxiC. In this work, we have investigated the role of Spa33, a component of the cytoplasmic complex, in the regulation of secretion. The spa33 gene encodes a 33-kDa protein and a smaller fragment of 12 kDa (Spa33C ) which are both essential components of the cytoplasmic complex. We have shown that the spa33 gene gives rise to 5 fragments of various sizes. Among them, three are necessary for T3SS. Interestingly, we have shown that Spa33 is implicated in the regulation of secretion. Indeed, the mutation of a single residue in Spa33 induces an effector mutant phenotype, in which MxiC is sequestered. Moreover, we have shown a direct interaction between Spa33 and MxiC.

Analysis of the capacity of Salmonella enterica Typhimurium to infect the human Placenta.

INTRODUCTION: Salmonella species are gram-negative facultative intracellular bacteria that are common causes of foodborne illness in North America. Infections by Salmonella during pregnancy are a significant cause of fetal loss in domestic livestock, and fetal and maternal mortality in mice. Furthermore, Salmonella infection is associated with miscarriage, stillbirth and preterm birth in pregnant women. Despite these collective associations, the extent to which Salmonella can infect the human placenta has not been investigated. METHODS: Human placental villous explants from several gestational ages were exposed to Salmonella enterica serovar Typhimurium (STm) ex vivo. Infection was assessed by colony forming unit assay and whole mount immunofluorescence (WMIF). RESULTS: Viable bacteria were recovered from placental villous explants of all gestational ages tested, but the bacterial burden was highest in 1st trimester explants. Bacterial numbers did not change appreciably with time post-infection in explants from any gestational age examined, suggesting that STm does not proliferate in placental villi. Exposure of villous explants to STm strains defective for the type III secretion systems revealed that Salmonella pathogenicity island 1 is essential for optimal invasion. In contrast to placental explants, STm infected and proliferated within villous cytotrophoblast cells isolated from term placentas. WMIF demonstrated that STm was restricted primarily to the syncytiotrophoblast layer in infected placentas. DISCUSSION: Our study demonstrates that STm can invade into the syncytiotrophoblast but does not subsequently proliferate. Thus, the syncytiotrophoblast may function as a barrier to STm infection of the fetus.