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.

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.

Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain.

BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Like other cytotoxicity-mediating effectors, BteA uses its multifunctional N-terminal domain to target phosphatidylinositol (PI)-rich microdomains in the host membrane. Despite their structural similarity, T3SS effectors exhibit a variable range of membrane interaction modes, and currently only limited structural information is available for the BteA membrane-targeting domain and the molecular mechanisms underlying its function. Employing a synergistic combination of structural methods, here we determine the structure of this functional domain and uncover key molecular determinants mediating its interaction with membranes. Residues 29-121 of BteA form an elongated four-helix bundle packed against two shorter perpendicular helices, the second of which caps the domain in a critical 'tip motif'. A flexible region preceding the BteA helical bundle contains the characteristic ß-motif required for binding its cognate chaperone BtcA. We show that BteA targets PI(4,5)P2-containing lipoprotein nanodiscs and binds a soluble PI(4,5)P2 analog via an extensive positively charged surface spanning its first two helices, and that this interaction is weaker for PI(3,5)P2 and abolished for PI(4)P. We confirmed this model of membrane-targeting by observation of BteA-induced changes in the structure of PI(4,5)P2-containing phospholipid bilayers using small-angle X-ray scattering (SAXS). We also extended these results to a larger BteA domain (residues 1-287), confirming its interaction with bilayers using calorimetry, fluorescence and SAXS methods. This novel view of the structural underpinnings of membrane targeting by BteA is an important step towards a comprehensive understanding of cytotoxicity in Bordetella, as well as interactions of a broad range of pathogens with their respective hosts.

A role for the Salmonella Type III Secretion System 1 in bacterial adaptation to the cytosol of epithelial cells.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that invades the intestinal epithelium. Following invasion of epithelial cells, Salmonella survives and replicates within two distinct intracellular niches. While all of the bacteria are initially taken up into a membrane bound vacuole, the Salmonella-containing vacuole or SCV, a significant proportion of them promptly escape into the cytosol. Cytosolic Salmonella replicates more rapidly compared to the vacuolar population, although the reasons for this are not well understood. SipA, a multi-function effector protein, has been shown to affect intracellular replication and is secreted by cytosolic Salmonella via the invasion-associated Type III Secretion System 1 (T3SS1). Here, we have used a multipronged microscopy approach to show that SipA does not affect bacterial replication rates per se, but rather mediates intra-cytosolic survival and/or initiation of replication following bacterial egress from the SCV. Altogether, our findings reveal an important role for SipA in the early survival of cytosolic Salmonella.

Salmonella enterica Effectors SifA, SpvB, SseF, SseJ, and SteA Contribute to Type III Secretion System 1-Independent Inflammation in a Streptomycin-Pretreated Mouse Model of Colitis.

Salmonella enterica serovar Typhimurium (S. Typhimurium) induces inflammatory changes in the ceca of streptomycin-pretreated mice. In this mouse model of colitis, the type III secretion system 1 (T3SS-1) has been shown to induce rapid inflammatory change in the cecum at early points, 10 to 24 h after infection. Five proteins, SipA, SopA, SopB, SopD, and SopE2, have been identified as effectors involved in eliciting intestinal inflammation within this time range. In contrast, a T3SS-1-deficient strain was shown to exhibit inflammatory changes in the cecum at 72 to 120 h postinfection. However, the effectors eliciting T3SS-1-independent inflammation remain to be clarified. In this study, we focused on two T3SS-2 phenotypes, macrophage proliferation and cytotoxicity, to identify the T3SS-2 effectors involved in T3SS-1-independent inflammation. We identified a mutant strain that could not induce cytotoxicity in a macrophage-like cell line and that reduced intestinal inflammation in streptomycin-pretreated mice. We also identified five T3SS-2 effectors, SifA, SpvB, SseF, SseJ, and SteA, associated with T3SS-1-independent macrophage cytotoxicity. We then constructed a strain lacking T3SS-1 and all the five T3SS-2 effectors, termed T1S5. The S. Typhimurium T1S5 strain significantly reduced cytotoxicity in macrophages in the same manner as a mutant invA spiB strain (T1T2). Finally, the T1S5 strain elicited no inflammatory changes in the ceca of streptomycin-pretreated mice. We conclude that these five T3SS-2 effectors contribute to T3SS-1-independent inflammation.

Intestinal cell migration damage induced by enteropathogenic Escherichia coli strains.

Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.

Frequent pauses in Escherichia coli flagella elongation revealed by single cell real-time fluorescence imaging.

The bacterial flagellum is a large extracellular protein organelle that extrudes from the cell surface. The flagellar filament is assembled from tens of thousands of flagellin subunits that are exported through the flagellar type III secretion system. Here, we measure the growth of Escherichia coli flagella in real time and find that, although the growth rate displays large variations at similar lengths, it decays on average as flagella lengthen. By tracking single flagella, we show that the large variations in growth rate occur as a result of frequent pauses. Furthermore, different flagella on the same cell show variable growth rates with correlation. Our observations are consistent with an injection-diffusion model, and we propose that an insufficient cytoplasmic flagellin supply is responsible for the pauses in flagellar growth in E. coli.

Intestinal cell migration damage induced by enteropathogenic Escherichia coli strains

Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.

Controlled Activity of the Salmonella Invasion-Associated Injectisome Reveals Its Intracellular Role in the Cytosolic Population.

The Salmonella invasion-associated type III secretion system (T3SS1) is an essential virulence factor required for entry into nonphagocytic cells and consequent uptake into a Salmonella-containing vacuole (SCV). While Salmonella is typically regarded as a vacuolar pathogen, a subset of bacteria escape from the SCV in epithelial cells and eventually hyperreplicate in the cytosol. T3SS1 is downregulated following bacterial entry into mammalian cells, but cytosolic Salmonella cells are T3SS1 induced, suggesting prolonged or resurgent activity of T3SS1 in this population. In order to investigate the postinternalization contributions of T3SS1 to the Salmonella infectious cycle in epithelial cells, we bypassed its requirement for bacterial entry by tagging the T3SS1-energizing ATPase InvC at the C terminus with peptides that are recognized by bacterial tail-specific proteases. This caused a dramatic increase in InvC turnover which rendered even assembled injectisomes inactive. Bacterial strains conditionally expressing these unstable InvC variants were proficient for invasion but underwent rapid and sustained intracellular inactivation of T3SS1 activity when InvC expression ceased. This allowed us to directly implicate T3SS1 activity in cytosolic colonization and bacterial egress. We subsequently identified two T3SS1-delivered effectors, SopB and SipA, that are required for efficient colonization of the epithelial cell cytosol. Overall, our findings support a multifaceted, postinvasion role for T3SS1 and its effectors in defining the cytosolic population of intracellular SalmonellaIMPORTANCE A needle-like apparatus, the type III secretion system (T3SS) injectisome, is absolutely required for Salmonella enterica to enter epithelial cells; this requirement has hampered the analysis of its postentry contributions. To identify T3SS1-dependent intracellular activities, in this study we overcame this limitation by developing a conditional inactivation in the T3SS whereby T3SS activity is chemically induced during culture in liquid broth, permitting bacterial entry into epithelial cells, but is quickly and perpetually inactivated in the absence of inducer. In this sense, the mutant acts like wild-type bacteria when extracellular and as a T3SS mutant once it enters a host cell. This "conditional" mutant allowed us to directly link activity of this T3SS with nascent vacuole lysis, cytosolic proliferation, and cellular egress, demonstrating that the invasion-associated T3SS also contributes to essential intracellular stages of the S. enterica infectious cycle.

Supplemental invasion of Salmonella from the perspective of Salmonella enterica serovars Kentucky and Typhimurium.

BACKGROUND: Critical to the development of Salmonellosis in humans is the interaction of the bacterium with the epithelial lining of the gastrointestinal tract. Traditional scientific reasoning held type III secretion system (T3SS) as the virulence factor responsible for bacterial invasion. In this study, field-isolated Salmonella enterica serovar Kentucky and a known human pathogen Salmonella enterica serovar Typhimurium were mutated and evaluated for the invasion of human colorectal adenocarcinoma epithelial cells. RESULTS: S. enterica serovar Kentucky was shown to actively invade a eukaryotic monolayer, though at a rate that was significantly lower than Typhimurium. Additionally, strains mutated for T3SS formation were less invasive than the wild-type strains, but the decrease in invasion was not significant in Kentucky. CONCLUSIONS: Strains mutated for T3SS formation were able to initiate invasion of the eukaryotic monolayer to varying degrees based on strain, In the case of Kentucky, the mutated strain initiated invasion at a level that was not significantly different from the wild-type strain. A different result was observed for Typhimurium as the mutation significantly lowered the rate of invasion in comparison to the wild-type strain.

Type III secretion as a generalizable strategy for the production of full-length biopolymer-forming proteins.

Biopolymer-forming proteins are integral in the development of customizable biomaterials, but recombinant expression of these proteins is challenging. In particular, biopolymer-forming proteins have repetitive, glycine-rich domains and, like many heterologously expressed proteins, are prone to incomplete translation, aggregation, and proteolytic degradation in the production host. This necessitates tailored purification processes to isolate each full-length protein of interest from the truncated forms as well as other contaminating proteins; owing to the repetitive nature of these proteins, the truncated polypeptides can have very similar chemistry to the full-length form and are difficult to separate from the full-length protein. We hypothesized that bacterial expression and secretion would be a promising alternative option for biomaterials-forming proteins, simplifying isolation of the full-length target protein. By using a selective secretion system, truncated forms of the protein are not secreted and thus are not found in the culture harvest. We show that a synthetically upregulated type III secretion system leads to a general increase in secretion titer for each protein that we tested. Moreover, we observe a substantial enhancement in the homogeneity of full-length forms of pro-resilin, tropo-elastin crosslinking domains, and silk proteins produced in this manner, as compared with proteins purified from the cytosol. Secretion via the type III apparatus limits co-purification of truncated forms of the target protein and increases protein purity without extensive purification steps. Demonstrating the utility of such a system, we introduce several modifications to resilin-based peptides and use an un-optimized, single-column process to purify these proteins. The resulting materials are of sufficiently high quantity and yield for the production of antimicrobial hydrogels with highly reproducible rheological properties. The ease of this process and its applicability to an array of engineered biomaterial-forming peptides lend support for the application of bacterial expression and secretion for other proteins that are traditionally difficult to express and isolate from the bacterial cytoplasm. Biotechnol. Bioeng. 2016;113: 2313-2320. © 2015 Wiley Periodicals, Inc.

Nuclear Magnetic Resonance Characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa.

Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS), to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologues are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residue 16 to 41 and from residue 55 to 76. The helices of PcrG are partially formed, have similar backbone dynamics, and are flexible. NMR titrations show that the entire length of PcrG residues from position 9 to 76 is involved in binding to PcrV. PcrG adds to the growing list of partially folded or unstructured proteins with important roles in type III secretion.

Effect of FliG three amino acids deletion in Vibrio polar-flagellar rotation and formation.

Most of bacteria can swim by rotating flagella bidirectionally. The C ring, located at the bottom of the flagellum and in the cytoplasmic space, consists of FliG, FliM and FliN, and has an important function in flagellar protein secretion, torque generation and rotational switch of the motor. FliG is the most important part of the C ring that interacts directly with a stator subunit. Here, we introduced a three-amino acids in-frame deletion mutation (ΔPSA) into FliG from Vibrio alginolyticus, whose corresponding mutation in Salmonella confers a switch-locked phenotype, and examined its phenotype. We found that this FliG mutant could not produce flagellar filaments in a fliG null strain but the FliG(ΔPSA) protein could localize at the cell pole as does the wild-type protein. Unexpectedly, when this mutant was expressed in a wild-type strain, cells formed flagella efficiently but the motor could not rotate. We propose that this different phenotype in Vibrio and Salmonella might be due to distinct interactions between FliG mutant and FliM in the C ring between the bacterial species.

Fueling type III secretion.

Type III secretion systems (T3SSs) are complex nanomachines that export proteins from the bacterial cytoplasm across the cell envelope in a single step. They are at the core of the machinery used to assemble the bacterial flagellum, and the needle complex many Gram-negative pathogens use to inject effector proteins into host cells and cause disease. Several models have been put forward to explain how this export is energized, and the mechanism has been the subject of considerable debate. Here we present an overview of these models and discuss their relative merits. Recent evidence suggests that the proton motive force (pmf) is the primary energy source for type III secretion, although contribution from refolding of secreted proteins has not been ruled out. The mechanism by which the pmf is converted to protein export remains enigmatic.

Yersinia pseudotuberculosis†YopD mutants that genetically separate effector protein translocation from host membrane disruption.

The Yersinia type III secretion system (T3SS) translocates Yop effector proteins into host cells to manipulate immune defenses such as phagocytosis and reactive oxygen species (ROS) production. The T3SS translocator proteins YopB and YopD form pores in host membranes, facilitating Yop translocation. While the YopD amino and carboxy termini participate in pore formation, the role of the YopD central region between amino acids 150-227 remains unknown. We assessed the contribution of this region by generating Y. pseudotuberculosis yopD(Δ150-170) and yopD(Δ207-227) mutants and analyzing their T3SS functions. These strains exhibited wild-type levels of Yop secretion in vitro and enabled robust pore formation in macrophages. However, the yopDΔ150-170 and yopD(Δ207-227) mutants were defective in Yop translocation into CHO cells and splenocyte-derived neutrophils and macrophages. These data suggest that YopD-mediated host membrane disruption and effector Yop translocation are genetically separable activities requiring distinct protein domains. Importantly, the yopD(Δ150-170) and yopD(Δ207-227) mutants were defective in Yop-mediated inhibition of macrophage cell death and ROS production in neutrophil-like cells, and were attenuated in disseminated Yersinia infection. Therefore, the ability of the YopD central region to facilitate optimal effector protein delivery into phagocytes, and therefore robust effector Yop function, is important for Yersinia virulence.