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Bordetella bronchiseptica injects virulence proteins called effectors into host cells via a type III secretion system (T3SS) conserved among many Gram-negative bacteria. Small proteins called chaperones are required to stabilize some T3SS components or localize them to the T3SS machinery. In a previous study, we identified a chaperone-like protein named Bcr4 that regulates T3SS activity in B. bronchiseptica. Bcr4 does not show strong sequence similarity to well-studied T3SS proteins of other bacteria, and its function remains to be elucidated. Here, we investigated the mechanism by which Bcr4 controls T3SS activity. A pulldown assay revealed that Bcr4 interacts with BscI, based on its homology to other bacterial proteins, to be an inner rod protein of the T3SS machinery. An additional pulldown assay using truncated Bcr4 derivatives and secretion profiles of B. bronchiseptica producing truncated Bcr4 derivatives showed that the Bcr4 C-terminal region is necessary for the interaction with BscI and activation of the T3SS. Moreover, the deletion of BscI abolished the secretion of type III secreted proteins from B. bronchiseptica and the translocation of a cytotoxic effector into cultured mammalian cells. Finally, we show that BscI is unstable in the absence of Bcr4. These results suggest that Bcr4 supports the construction of the T3SS machinery by stabilizing BscI. This is the first demonstration of a chaperone for the T3SS inner rod protein among the virulence bacteria possessing the T3SS.

The blaNDM-1 gene encodes a carbapenemase, New Delhi metallo-β-lactamase (NDM-1), and the ability to produce NDM-1 is spread among Enterobacteriaceae via horizontal gene transfer of plasmids. It has been widely accepted that blasubNDM-1/subis regulated by a hybrid promoter (PsubISAba125/sub) consisting of a -10 box from the original blasubNDM-1/suband a -35 box from ISAba125. However, the conservation of this promoter and the vertical transmission of blasubNDM/subgenes by chromosomal integration have not been comprehensively analyzed. We retrieved the region containing the ORF of blasubNDM-1/sub(gt;95% translated protein identity) and a region 120 bp upstream of the blasubNDM-1/substart codon from the complete sequence data of Enterobacteriaceae plasmids (n = 10,914) and chromosomes (n = 4908) deposited in GenBank, and the 310 extracted blasubNDM/subgenes were analyzed by an in-silico approach. The results showed that most blasubNDM/subgenes (99.0%) utilized the promoter, PsubISAba125/sub. Interestingly, two blasubNDM-1/subgenes from the genus Citrobacter utilized the ISCR1-derived outward-oriented promoters PsubOUT/sub(PsubISCR1/sub). Furthermore, the insertion of ISAba125 and ISCR1 occurred upstream of the CCATATTT sequence, which is located upstream of the -10 box. We also confirmed that most of the blasubNDM/subgenes were disseminated by horizontal gene transfer of the plasmid, but 10 cases of the blasubNDM/subgenes were integrated into the chromosome via mobile genetic elements such as IS26, IS150, ISCR1, ICE, and Tn7-like elements. Thus, plasmid-mediated transmission of the PsubISAba125/sub-blasubNDM/subgenes is predominant in Enterobacteriaceae. However, the spread of blasubNDM/subgenes with new promoters and vertical dissemination via chromosomal integrations may pose additional serious clinical problems.

Accumulating evidence has revealed that lymphoid-tissue-resident commensal bacteria (e.g., Alcaligenes spp.) survive within dendritic cells. We extended our previous study by investigating microbes that persistently colonize colonic macrophages. 16S rRNA-based metagenome analysis using DNA purified from murine colonic macrophages revealed the presence of Stenotrophomonas maltophilia. The in situ intracellular colonization by S. maltophilia was recapitulated in vitro by using bone marrow-derived macrophages (BMDMs). Co-culture of BMDMs with clinically isolated S. maltophilia led to increased mitochondrial respiration and robust IL-10 production. We further identified a 25-kDa protein encoded by the gene assigned as smlt2713 (recently renamed as SMLT_RS12935) and secreted by S. maltophilia as the factor responsible for enhanced IL-10 production by BMDMs. IL-10 production is critical for maintenance of the symbiotic condition, because intracellular colonization by S. maltophilia was impaired in IL-10-deficient BMDMs, and smlt2713-deficient S. maltophilia failed to persistently colonize IL-10-competent BMDMs. These findings indicate a novel commensal network between colonic macrophages and S. maltophilia that is mediated by IL-10 and smlt2713.

Bordetella bronchiseptica injects virulence proteins called effectors into host cells via a type III secretion system (T3SS) conserved among many Gram-negative bacteria. Small proteins called chaperones are required for stabilizing some T3SS components or localizing them to the T3SS machinery. In a previous study, we identified a chaperone-like protein named Bcr4 that regulates T3SS activity in B. bronchiseptica. Bcr4 does not show strong sequence similarity to well-studied T3SS proteins of other bacteria, and its function remains to be elucidated. Here, we investigated the mechanism by which Bcr4 controls T3SS activity. A pull-down assay revealed that Bcr4 interacts with BscI, based on its homology to other bacterial proteins, to be an inner rod protein of the T3SS machinery. An additional pull-down assay using truncated Bcr4 derivatives and secretion profiles of B. bronchiseptica producing truncated Bcr4 derivatives showed that the Bcr4 C-terminal region is necessary for the interaction with BscI and activation of the T3SS. Moreover, the deletion of BscI abolished the secretion of type III secreted proteins from B. bronchiseptica and the translocation of a cytotoxic effector into cultured mammalian cells. Finally, we showed that BscI is unstable in the absence of Bcr4. These results suggest that Bcr4 supports the construction of the T3SS machinery by stabilizing BscI. This is the first demonstration of a chaperone for the T3SS inner rod protein among the virulence bacteria possessing the T3SS.ImportanceThe type III secretion system (T3SS) is a needle-like complex that projects outward from bacterial cells. Bordetella bronchiseptica uses the T3SS to inject virulence proteins into host cells. Our previous study reported that a protein named Bcr4 is essential for the secretion of virulence proteins from B. bronchiseptica bacterial cells and delivery through the T3SS. Because other bacteria lack a Bcr4 homologue, the function of Bcr4 has not been elucidated. In this study, we discovered that Bcr4 interacts with BscI, a component of the T3SS machinery. We showed that a B. bronchiseptica BscI-deficient strain was unable to secrete type III secreted proteins. Furthermore, in a B. bronchiseptica strain that overproduces T3SS component proteins, Bcr4 is required to maintain BscI in bacterial cells. These results suggest that Bcr4 stabilizes BscI to allow construction of the T3SS in B. bronchiseptica.

Aurodox was originally isolated in 1972 as a linear polyketide compound exhibiting antibacterial activity against Gram-positive bacteria. We have since identified aurodox as a specific inhibitor of the bacterial type III secretion system (T3SS) using our original screening system for inhibition of T3SS-mediated hemolysis in enteropathogenic Escherichia coli (EPEC). In this research, we synthesized 15 derivatives of aurodox and evaluated EPEC T3SS inhibitory activity as well as antibacterial activity against EPEC. One of the derivatives was highly selective for T3SS inhibition, equivalent to that of aurodox, but without exhibiting antibacterial activity (69-fold selectivity). This work revealed the structure-activity relationship for the inhibition of T3SS by aurodox and suggests that the target of T3SS is distinct from the target for antibacterial activity.

SummaryTranslocated intimin receptor (Tir) is an essential bacterial factor for enteropathogenic Escherichia coli (EPEC) to establish Tir-intimin interaction-mediated adherence to the epithelial cell and to form actin pedestal structures beneath the adherent bacteria. However, it remains unclear how the host cells eliminate Tir protein after infection. Here we show that intracellular translocated Tir is degraded via the host ubiquitin- proteasome system. We found that host CBL-C, an E3 ubiquitin-protein ligase, bound to and ubiquitinated the 454 tyrosine-phosphorylated Tir protein. Tir ubiquitination leads to proteasome-dependent degradation and attenuated EPEC colonization of the epithelial cell. Using Citrobacter rodentium, a mouse model for EPEC, we demonstrated that infection with C. rodentium mutant expressing a tyrosine- phenylalanine-substituted Tir (CBL-C resistant) showed increased bacterial loads in the colon and lethality compared with infection with C. rodentium expressing wild-type Tir. These results indicate that CBL-C is a critical host defense factor that determines the fate of cytosolic Tir and terminates bacterial colonization.Graphical Abstracts

Bordetella pertussisuses a type III secretion system (T3SS) to inject virulence proteins into host cells. Although theB. pertussisT3SS was presumed to be involved in host colonization, efficient secretion of type III secreted proteins fromB. pertussishas not been observed. To investigate the roles of type III secreted proteins during infection, we attempted to optimize culture conditions for the production and secretion of a type III secreted protein, BteA, inB. pertussis. We observed thatB. pertussisefficiently secretes BteA in ascorbic acid-depleted (AsA−) medium. When L2 cells, a rat lung epithelial cell line, were infected withB. pertussiscultured in the AsA−medium, BteA-dependent cytotoxicity was observed. We also performed an immunofluorescence assay of L2 cells infected withB. pertussis. Clear fluorescence signals of Bsp22, a needle structure of T3SS, were detected on the bacterial surface ofB. pertussiscultured in the AsA−medium. Since ascorbic acid is known as a reducing agent, we culturedB. pertussisin liquid medium containing other reducing agents such as 2-mercaptoethanol and dithioerythritol. Under these reducing conditions, the production of type III secreted proteins was repressed. These results suggest that inB. pertussis, the production and secretion of type III secreted proteins are downregulated under reducing conditions.IMPORTANCEThe type III secretion system (T3SS) ofBordetella pertussisforms a needlelike structure that protrudes from the bacterial cell surface.B. pertussisuses a T3SS to translocate virulence proteins called effectors into host cells. The culture conditions for effector production inB. pertussishave not been investigated. We attempted to optimize culture medium compositions for producing and secreting type III secreted proteins. We found thatB. pertussissecretes type III secreted proteins in reducing agent-deprived liquid medium and that BteA-secretingB. pertussisprovokes cytotoxicity against cultured mammalian cells. These results suggest that redox signaling is involved in the regulation ofB. pertussisT3SS.

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.

Bordetella bronchiseptica infects a wide variety of mammals, and the type III secretion system (T3SS) is involved in the long-term colonization of Bordetella in the trachea and lung. The T3SS translocates virulence factors (commonly referred to as effectors) into host cells, leading to alteration of the host physiological function. The Bordetella effectors BopN and BteA are known to have roles in the up-regulation of IL-10 and the cytotoxicity, respectively. Nevertheless, the mechanism by which BopN is translocated into host cells has not been examined in sufficient detail. Therefore, to determine the precise mechanisms of the BopN translocation into host cells, we built truncated derivatives of BopN and evaluated the derivatives' ability of translocation into host cells by adenylate cyclase-mediated translocation assay. N-terminal amino acid (aa) residues 1–200 of BopN are sufficient for its translocation in host cells. Interestingly, the BopN translocation was completely blocked by deletion of the N-terminal aa residues 6–50, indicating that the N-terminal region is critical for the BopN translocation. Furthermore, BopN appears to play an auxiliary role in the BteA-mediated cytotoxicity. Thus, BopN apparently has the ability to translocate into host cells and is suspected to facilitate the activity of BteA.

Four large laminar-box shaking table experiments are conducted to document pile response due to the mechanism of liquefaction-induced lateral spreading. Single-pile and pile-group configura...

Bordetella species, including B. pertussis, have a type III secretion system that is highly conserved among gram-negative pathogenic bacteria. Genes encoding the component proteins of the type III secretion system are localized at the bsc locus in the Bordetella genome. Here, the function of a hypothetical protein Bcr4 encoded at the bsc locus in the B. bronchiseptica genome was investigated. A Bcr4-deficient mutant was created and the amounts of type III secreted proteins (e.g., BopB, BopN and Bsp22) in both the supernatant fraction and whole-cell lysates of the Bcr4-deficient mutant were determined. It was found that the amounts of these proteins were significantly lower than in the wild-type strain. The amounts of type III secreted proteins in the supernatant fraction and whole-cell lysates were much greater in a Bcr4-overproducing strain than in the wild-type strain. The type III secreted protein BspR reportedly negatively regulates the type III secretion system. Here, it was observed that a Bcr4 + BspR double-knockout mutant did not secrete type III secreted proteins, whereas the amounts of these proteins in whole-cell lysates of this mutant were nearly equal to those in whole-cell lysates of the BspR-deficient mutant. Bcr4 thus appears to play an essential role in the extracellular secretion of type III secreted proteins. Our data also suggest that Bcr4 antagonizes the negative regulatory function of BspR.

This study presents a new guidance and control system using a constrained adaptive backstepping method for a space transportation system. In this method, the effects of input saturations by actuator dynamics (e.g. magnitude, rate and bandwidth) are considered to introduce the compensators on the basis of pseudo control hedging. The stability of the proposed entire system is guaranteed by the Lyapunov’ stability theorem. To confirm the realization and robustness of the proposed system, Monte Carlo simulations (MCSs) were performed. In addition, to obtain optimized control performance, a parameter optimization algorithm combined with the MCSs was introduced. Finally, automatic landing simulations using the six degrees-of-freedom nonlinear flight simulation model of the NASA’s Space Shuttle Orbiter were performed to verify the effectiveness of the proposed technique.

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.

Enteropathogenic Escherichia coli (EPEC) are worldwide human enteric pathogens inflicting significant morbidity and causing large economic losses. A type 3 secretion system (T3SS) is critical for EPEC intestinal colonization, and injection of effectors into host cells contributes to cellular subversion and innate immune evasion. Here, we demonstrate that two strictly conserved C-terminal tyrosine residues, Y152 and Y153, within the multicargo T3SS chaperone CesT serve differential roles in regulating effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine attenuated EPEC type 3 effector injection into host cells, and significantly limited Tir effector mediated intimate adherence, a key feature of attaching and effacing pathogenesis. Whereas CesT Y153 supported normal levels of Tir translocation, CesT Y152 was strictly required for the effector NleA to be expressed and subsequently translocated into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y152, Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating Y152 in CesT functionality. A mouse infection model of EPEC intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for strictly conserved tandem tyrosine residues within CesT. Tyrosine 152 of CesT is implicated in NleA expression, providing functional relevance for localized amino acid conservation. Therefore, CesT via its novel C-terminal domain, has relevant roles beyond typical T3SC that interact and stabilize effector proteins.

Bordetella bronchiseptica infects a wide variety of mammals, the type III secretion system (T3SS) being involved in long-term colonization by Bordetella of the trachea and lung. T3SS translocates virulence factors (commonly referred to as effectors) into host cells, leading to alterations in the host's physiological function. The Bordetella effectors BopN and BteA are known to have roles in up-regulation of IL-10 and cytotoxicity, respectively. Nevertheless, the mechanism by which BopN is translocated into host cells has not been examined in sufficient detail. Therefore, to determine the precise mechanisms of translocation of BopN into host cells, truncated derivatives of BopN were built and the derivatives' ability to translocate into host cells evaluated by adenylate cyclase-mediated translocation assay. It was found that N-terminal amino acid (aa) residues 1-200 of BopN are sufficient for its translocation into host cells. Interestingly, BopN translocation was completely blocked by deletion of the N-terminal aa residues 6-50, indicating that the N-terminal region is critical for BopN translocation. Furthermore, BopN appears to play an auxiliary role in BteA-mediated cytotoxicity. Thus, BopN can apparently translocate into host cells and may facilitate activity of BteA.

This paper presents the results of a large-scale shake table test at E-Defense facility on a pile group located adjacent to a gravity-type quay wall and were subjected to liquefaction-induced large ground displacements. Extensive liquefaction-induced large ground lateral spreading displaced the quay wall about 2.2 m and damaged the pile foundation. The pile foundation consisted of a six-pile group which supported a footing and a superstructure model. Large lateral soil displacements were measured by several sensors such as inclinometers and the results favorably agreed with the directly observed deformations. Soil lateral displacement decreased as the distance from the quay wall increased landward. The piles were densely instrumented and the measured bending strain records were able to explain the damage to the piles. Lateral pressures of the liquefied soil exerted on the piles were measured using earth pressure (EP) sensors. The application of two design guidelines (JRA [1] and JSWA [2] ) for estimation of liquefaction-induced lateral pressure on piles is discussed and their advantages and shortcomings are addressed. Furthermore, two simplified methods (Shamoto et al. [3] and Valsamis et al. [4] ) are employed to predict the extent of liquefaction-induced large ground displacements and they are compared to the measured deformations. Finally, their accuracy for predicting the liquefaction-induced lateral displacements is evaluated and practical recommendations are made.

The 2011 MW = 9.0 Tohoku-oki earthquake generated a large number of unique soil liquefaction case histories, including cases with strong ground motion recordings on liquefiable or potentially liquefiable soils. We have compiled a list of 22 strong motion stations (SMS) where surface evidence of liquefaction was observed and 16 SMS underlain by geologically recent sediments or fills where surface evidence of liquefaction was not observed. Pre-earthquake standard penetration test data and borehole shear wave velocity ( Vs) profiles are available for some stations, but critical information, such as grain size distribution and fines plasticity, are often lacking. In the heavily damaged city of Urayasu, we performed post-earthquake cone penetration testing at seven SMS and Vs profiles, using surface wave methods at 28 additional locations to supplement existing geotechnical data. We describe the liquefaction effects in Urayasu, the available site characterization data, and our initial data interpretations.

BteA is one of the effectors secreted from the Bordetella bronchiseptica type III secretion system. It has been reported that BteA induces necrosis in mammalian cells; however, the roles of BteA during the infection process are largely unknown. In order to investigate the BteA functions, morphological changes of the cells infected with the wild-type B. bronchiseptica were examined by time-lapse microscopy. L2 cells, a rat lung epithelial cell line, spread at 1.6 hours after B. bronchiseptica infection. Membrane ruffles were observed at peripheral parts of infected cells during the cell spreading. BteA-dependent cytotoxicity and cell detachment were inhibited by addition of cytochalasin D, an actin polymerization inhibitor. Domain analyses of BteA suggested that two separate amino acid regions, 200-312 and 400-658, were required for the necrosis induction. In order to examine the intra/intermolecular interactions of BteA, the amino- and the carboxyl-terminal moieties were purified as recombinant proteins from Escherichia coli. The amino-terminal moiety of BteA appeared to interact with the carboxyl-terminal moiety in the pull-down assay in vitro. When we measured the amounts of bacteria phagocytosed by J774A.1, a macrophage-like cell line, the phagocytosed amounts of B. bronchiseptica strains that deliver BteA into the host cell cytoplasm were significantly lower than those of strains that lost the ability to translocate BteA into the host cell cytoplasm. These results suggest that B. bronchiseptica induce necrosis by exploiting the actin polymerization signaling pathway and inhibit macrophage phagocytosis.

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.

Genetic evidence indicates that only the β-anomer of galactose is transported to Kluyveromyces lactis cells by galactose/glucose transporter Hgt1p, and that aldose-1-epimerase encoded by GAL10 is a prerequisite for growth on galactose. Minor aldose-1-epimerases other than Gal10p also exist in K. lactis. Using a mutant defective in both aldose-1-epimerases, we show by solid-state nuclear magnetic resonance spectroscopy that only β-anomer is transported in the cell and stays without or with a slow rate of conversion to α-anomer. Signals due to intracellular β-galactose appeared at two positions, both of which were shifted towards higher magnetic fields than that of β-galactose in aqueous solution, suggesting that incorporated galactose binds to cellular components, probably proteins.

The Bordetella pertussis pertussis toxin and pertactin (Prn) are protective antigens and are contained in acellular pertussis vaccines. Polymorphisms in the A subunit of pertussis toxin (PtxA) and pertactin have been proposed to mediate vaccine resistance and contribute to pertussis reemergence. To test this hypothesis, previous studies compared clinical isolates expressing different alleles for the proteins. However, other virulence factors or virulence factor expression levels also may vary, confounding the analysis. To overcome these limitations, we constructed isogenic mutants of B. pertussis Tohama expressing the alleles ptxA1 or ptxA2 and prn1 or prn2 and compared the efficacies of an acellular pertussis vaccine against the mutants in a mouse model. While the vaccine was effective against all of the B. pertussis strains regardless of the allele expression pattern, the strain expressing ptxA1 and prn2 displayed a survival advantage over the other strains. These results suggest that an allele shift to the ptxA1 prn2 genotype may play a role in the emergence of pertussis in vaccinated populations.

The inflammatory response is one of several host alert mechanisms that recruit neutrophils from the circulation to the area of infection. We demonstrate that Bordetella, a bacterial pathogen, exploits an antiinflammatory cytokine, interleukin-10 (IL-10), to evade the host immune system. We identified a Bordetella effector, BopN, that is translocated into the host cell via the type III secretion system, where it induces enhanced production of IL-10. Interestingly, the BopN effector translocates itself into the nucleus and is involved in the down-regulation of mitogen-activated protein kinases. Using pharmacological blockade, we demonstrated that BopN-induced IL-10 production is mediated, at least in part, by its ability to block the extracellular signal-regulated kinase pathway. We also showed that BopN blocks nuclear translocation of nuclear factor κB p65 (NF-κBp65) but, in contrast, promotes nuclear translocation of NF-κBp50. A BopN-deficient strain was unable to induce IL-10 production in mice, resulting in the elimination of bacteria via neutrophil infiltration into the pulmonary alveoli. Furthermore, IL-10–deficient mice effectively eliminated wild-type as well as BopN mutant bacteria. Thus, Bordetella exploits BopN as a stealth strategy to shut off the host inflammatory reaction. These results explain the ability of Bordetella species to avoid induction of the inflammatory response.

B. pertussis is a causative agent of whooping cough (pertussis) in humans. Despite wide-scale vaccination in many countries, there is serious concern about pertussis as a re-emerging disease. Re-emergence of pertussis may be explained by several factors: the short duration of protection by the currently available acellular pertussis vaccine, an increase in asymptomatic adult carriers and expansion of strains with certain antigenic variations which are not covered by currently available vaccines. To develop safer and more efficacious vaccines which confer more prolonged protection, researchers are focusing on identification and characterization of new virulence factors. One candidate for protective antigens is the type III secretion system and its secreted proteins.

Streptolysin O (SLO) is a membrane-damaging toxic protein produced by group A streptococci. We performed an ultrastructural analysis of pore formation and the mechanism of hemolysis by SLO, using a mutant form of SLO [SLO(C/A)-SS] and native SLO. SLO(C/A)-SS was unable to penetrate the erythrocyte membrane as a consequence of immobilization that was due to a disulfide bond between domains. The SLO(C/A)-SS molecules that bound to membranes formed numerous single-layered ring-shaped structures that did not result in pores on the membranes. These structures were similar to the structures formed by native SLO at 0 degrees C. After treatment with dithiothreitol, SLO(C/A)-SS that had bound to membranes formed double-layered rings with pores on the membranes, as does native SLO at room temperature. Our morphological evidence demonstrates that an increase in temperature is necessary for the occurrence of conformational changes and for the formation of double-layered rings after the insertion of domain 3 into the host cell membrane. On the basis of a model of the oligomeric structure of SLO, we propose some new details of the mechanism of hemolysis by SLO.

In Chile, which is a mining country, based on our company’s original technology, we have opened the Atacama Mine, a copper mine, in which our company owns a 60% majority share, and are conducting operations based on our own initiative.The Atacama Mine is located about 16 kilometers SSE of Copiapo, the capital city of the Third Region in the Republic of Chile, at an elevation above sea level of approx. 500 meters. We began investigations in 1990, starting basically from scratch with almost no existing data, and found and confirmed ore deposits that could be economically mined. In 1999 we founded a joint venture company and began mine construction in 2001. At the time, the price of copper was low, so our main propositions were to reduce development costs, and shorten the development period, which we successfully completed by the end of 2002, achieving a short development period of a year and a half.Our development of the Atacama Mine had the following technological features:i) Because the ore body was deep underground (approx. 400 meters from the surface), we placed the primary ore reduction equipment underground, and used three long belt conveyors (3,500 m total) to transport the ore to the surface. This allowed us to reduce operation costs, and realize a safe and comfortable underground environment.ii) Slime is transported to a mine residue dam 16 km from the dressing plant via a fluid transport system using a pipeline, which has allowed us to reduce transport costs.iii) By using a new type of flotation machine we have been able to maximize the extraction rate of shipping ore (recovery rate of 93% or more).Four years after beginning operations the Atacama Mine continues to operate stably.

Bordetella bronchiseptica is genetically related to B. pertussis and B. parapertussis, which cause respiratory tract infections in humans. These pathogens possess a large number of virulence factors, including the type III secretion system (T3SS), which is required for the delivery of effectors into the host cells. In a previous study, we identified a transcriptional regulator, BspR, that is involved in the regulation of the T3SS-related genes in response to iron-starved conditions. A unique feature of BspR is that this regulator is secreted into the extracellular milieu via the T3SS. To further characterize the role of BspR in extracellular localization, we constructed various truncated derivatives of BspR and investigated their translocation into the host cells using conventional translocation assays. In this study, the effector translocation was evaluated by the T3SS of enteropathogenic E. coli (EPEC), since the exogenous expression of BspR triggers severe repression of the Bordetella T3SS expression. The results of the translocation assays using the EPEC T3SS showed that the N-terminal 150 amino acid (aa) residues of BspR are sufficient for translocation into the host cells in a T3SS-dependent manner. In addition, exogenous expression of BspR in HeLa cells demonstrated that the N-terminal 100 aa residues are involved in the nuclear localization. In contrast, the N-terminal 54 aa residues are sufficient for the extracellular secretion into the bacterial culture supernatant via the EPEC T3SS. Thus, BspR is not only a transcriptional regulator in bacteria cytosol, but also functions as an effector that translocates into the nuclei of infected host cells.

Some pathogenic bacteria actually invade the cytoplasm of their target host cells. Invasive bacteria acquire the propulsive force to move by recruiting actin and inducing its polymerization. Here we show thatShigellamovement within the cytoplasm was severely hindered by microtubules and that the bacteria destroyed surrounding microtubules by secreting VirA by means of the type III secretion system. Degradation of microtubules by VirA was dependent on its α-tubulin–specific cysteine protease–like activity.virAmutants did not move within the host cytoplasm and failed to move into adjacent cells.

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in young children (12). This pathogen induces a characteristic histopathological lesion referred to as an attaching/effacing lesion, which is defined by the intimate attachment of bacteria to the epithelial surface and the effacement of host cell microvilli (25). Factors responsible for the formation of attaching/effacing lesions are encoded by a 35-kbp locus designated LEE (24), which encodes the following components: (i) the type III secretion (TTS) apparatus (15), (ii) E. coli-secreted proteins (EspA, EspB, and EspD) and several effectors, including Tir, (iii) type III-specific chaperones, and (iv) regulators (9). LEE is highly conserved among enterohemorrhagic E. coli, rabbit EPEC, and Citrobacter rodentium. TTS systems are found in many other gram-negative bacterial species, and they constitute a strategy for the delivery of effectors into host cells, a process which in turn disrupts host physiology and thereby contributes to the disease process (14). Unlike the sec-dependent secreted proteins, the effector does not have a typical signal sequence at its N terminus, and it is directly delivered via the TTS system without N-terminal processing in the bacterial periplasm. Analysis by transmission electron microscopy (TEM) revealed that the supermolecular structures of the TTS apparatus in EPEC (8, 31), Salmonella enterica serovar Typhimurium (18, 20-22), and Shigella flexneri (2, 32) are highly conserved with respect to each other and that their shapes are similar to that of the flagellar basal body complex. The core TTS apparatus, which is referred to as a needle complex (NC), is composed of two distinct portions: (i) a needle structure that extrudes from the bacterial outer membrane and functions as an injector of effectors into host cells and (ii) a cylindrical basal body that is similar to the flagellar basal body and functions as a channel that spans the outer and inner membranes of the bacterium as well as the periplasmic region. The basal body is further divided into three major portions: (i) an outer ring, (ii) an inner ring, and (iii) a presumed central rod (22) that can connect the outer and inner rings to build a channel. Although the supermolecular structure of the EPEC NC is similar in shape to that of the NCs of both Salmonella and Shigella, a unique extracellular appendage in the needle structure of the EPEC NC was discovered previously (31). The EPEC needle structure is composed of a thin needle (neck portion) and an expandable sheath-like structure (31). EPEC EscF is predicted to be an 8-kDa protein and may polymerize to form the thin needle in the needle structure. EscF shows homology to PrgI (24% identity) in the Salmonella pathogenicity island 1, Shigella MxiH (25% identity), and Yersinia enterocolitica YscF (20% identity), which are major components of the thin and stiff needle structures of the Salmonella, Shigella, and Yersinia NCs, respectively (21, 26, 32). EscF is required for NC formation and the secretion of the Esp proteins (31); this observation agrees with findings of secretion-defective phenotypes of Salmonella prgI and Shigella mxiH mutant strains (21, 32). EspA is predicted to be a 20-kDa protein and is secreted via the EPEC TTS system (17). We previously demonstrated that EspA is directly associated with the tip of the putative EscF needle and polymerizes into an expandable filamentous structure, referred to as the sheath-like structure (31). The EPEC needle structure, including the EspA sheath-like structure, extended to a length of more than 600 nm and was 10 times longer than the Shigella needle (45 nm) (31). Three-dimensional structure analysis of the EspA filament revealed that the structure consists of a helical tube with a diameter of 12 nm enclosing a central channel with a diameter of 2.5 nm (7). Recent TEM analysis of purified EspA revealed that EspA alone is able to polymerize into irregular short filaments (33). On the other hand, the widths of the outer and inner membrane rings in the basal body of the EPEC NC are estimated to be 17 and 18 nm, respectively, and the height of the basal body is 31 nm (31). However, the molecular composition of the EPEC NC basal body remains unclear. From the results of a membrane fractionation study (13), yeast two-hybrid analysis (5), whole mutation analyses of LEE (10), and computer modeling predictions, several proteins are thought to be components for the EPEC TTS apparatus. The outer ring protein of the basal body has been suggested to be EscC, according to a membrane fractionation study (13), and this hypothesis received further support from a demonstration of its similarity to the YscC protein (31.1% identity) in the Yersinia TTS system, which forms a ring-shaped oligomeric complex with a diameter of 20 nm in the outer membrane (4, 19). EscC belongs to a member of the secretin superfamily, which participates in the delivery of large molecules through the outer membrane by the formation of a channel. EscC is predicted to be synthesized as a 56-kDa preprotein possessing a signal sequence that is cleavable with type I signal peptidase after amino acid residue 19. Then, the EscC preprotein most likely undergoes signal peptide cleavage after its export across the inner membrane by a sec-dependent secretion pathway, thus generating a mature 54-kDa protein. Indeed, a mature form of Salmonella InvG, an EscC orthologue, starts at amino acid residue 25, suggesting that its preprotein is cleaved by type I signal peptidase in order to reach maturation (20). Recently, the association of EscC with EscD was suggested by use of a yeast two-hybrid system (5). EscD is predicted to be a 45-kDa protein and shows amino acid sequence similarity to Yersinia YscD, a bacterial inner membrane protein (27). In Salmonella, PrgK and PrgH together form the inner membrane ring of the TTS apparatus (18). Although a PrgH orthologue has not been found in EPEC, a PrgK orthologue, EscJ, is thought to form a portion of the inner ring. Molecular modeling using the 1.8-A crystal structure of EscJ suggests that EscJ oligomerizes to form a large 24-subunit ring structure (34). Furthermore, a membrane fraction study indicates that EscJ localizes mainly to the inner membrane (34). These findings suggest that the inner ring of the EPEC TTS apparatus contains the EscJ multimeric complex. EscJ is predicted to be produced as a 21-kDa preprotein with a lipoprotein signal sequence that can be cleaved after amino acid residue 19 by type II signal peptidase. After sec-dependent translocation, the EscJ preprotein may undergo lipid modification and signal peptide cleavage to form a mature 19-kDa lipoprotein, which may be anchored to the inner membrane via its N-terminal lipid moiety (34). Although several Esc proteins, namely, EscR, EscS, EscT, and EscU, are predicted to be components of the basal body, their precise functions and localizations in the EPEC TTS apparatus remain unclear. In this study, we analyzed the structures of NCs isolated from EPEC by using centrifugation techniques, including CsCl density gradient centrifugation. Furthermore, protein components of NCs were examined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), Western blotting, and immunoelectron microscopy. In addition, the protein-protein interactions required for assembly of the EPEC TTS apparatus were analyzed using a glutathione S-transferase (GST) pulldown assay, and confirmation of the putative components of the TTS apparatus needed for NC assembly, as well as those needed for the secretion of Esp proteins and the Tir effector, was carried out by the construction of deletion mutants.

In Bordetella bronchiseptica, the functional type III secretion system (TTSS) is required for the induction of necrotic cell death in infected mammalian cells. To identify the factor(s) involved in necrotic cell death, type III-secreted proteins from B. bronchiseptica were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry. We identified a 69-kDa secreted protein designated BopC. The gene encoding BopC is located outside of the TTSS locus and is also highly conserved in both Bordetella parapertussis and Bordetella pertussis. The results of a lactate dehydrogenase release assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay demonstrated that BopC is required for necrotic cell death. It has been reported that tyrosine-phosphorylated proteins (PY) of host cells are dephosphorylated during B. bronchiseptica infection in a TTSS-dependent manner. We found that BopC is also involved in PY dephosphorylation in infected host cells. It appears that the necrotic cell death triggered by BopC occurs prior to the PY reduction in host cells, because Bordetella-induced cell death was not affected even in the presence of a dephosphorylation inhibitor. Furthermore, a translocation assay showed that the signal sequence for both secretion into culture supernatant and translocation into the host cell is located in 48 amino acid residues of the BopC N terminus. This report reveals for the first time that a novel type III effector, BopC, is required for the induction of necrotic cell death during Bordetella infection.

Enteropathogenic Escherichia coli (EPEC) infects intestinal epithelial cells and perturbs the intestinal barrier that limits the paracellular movement of molecules. The disruption of the barrier is mediated by the effectors translocated into the host cells through the bacterial type III secretion system (TTSS). A previous report has described the importance of a bacterial outer membrane protein, intimin, in EPEC-mediated disruption of the barrier, and proposed that intimin, in concert with a host intimin receptor, controls the activity of the translocated barrier-disrupting effectors [P. Dean, B. Kenny, Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein, Mol. Microbiol. 54 (2004) 665-675]. In this study, we found that the importance of intimin is in its ability to bind a bacterial intimin receptor, Tir. Additionally, the impaired ability of an intimin-negative mutant was not restored by co-infection with intimin-expressing TTSS mutants. Collectively, the results in this study favor an alternative scenario explaining the importance of intimin, that the binding of intimin with Tir on the bacterial surface triggers or promotes the translocation of factors required for the efficient disruption of the barrier. Thus, the interaction of intimin with Tir may serve as a molecular switch that controls the delivery of virulence factors into the host cells.

This is the case study of concurrent cytomegalovirus and respiratory syncytial virus infection in an infant who showed thrombocytosis, liver dysfunction and bronchiolitis. The combination of thrombocytosis with this co-infection is causally related to elevated levels of thrombopoietin and interleukin-6. This study represents the first such case ever recorded.

Bacterial pathogens cause a wide spectrum of diseases in human and other animals. Some virulence factors, which are referred to as effectors, are directly translocated into the host cell via an injection apparatus, i.e., the type-III secretion system. Most effectors mimic host molecules, and translocated effectors are thereby able to perturb or modulate host cell signaling, cytoskeletal rearrangement, vesicular traffic, and autophagy, thus eliciting disease. Effectors are roughly classified among exotoxins, but in most cases, their functions are exerted focally when they are translocated into the host cell.

The attachment of enteropathogenic Escherichia coli (EPEC) to host cells and the induction of attaching and effacing (A/E) lesions are prominent pathogenic features. EPEC infection also leads to host cell death and damage to the intestinal mucosa, which is partly dependent upon EspF, one of the effectors. In this study, we demonstrate that EspF is a mitochondrial import protein with a functional mitochondrial targeting signal (MTS), because EspF activity for importing into the mitochondria was abrogated by MTS deletion mutants. Substitution of the 16th leucine with glutamic acid (EspF(L16E)) completely abolished EspF activity. Infection of HeLa cells with wild type but not the espF mutant (ΔespF) decreased mitochondrial membrane potential (ΔΨm), leading to cell death. The ΔΨm decrease and cell death were restored in cells infected with ΔespF/pEspF but not ΔespF/pEspF(L16E), suggesting that the 16th leucine in the MTS is a critical amino acid for EspF function. To demonstrate the impact of EspF in vivo, we exploited Citrobacter rodentium by infecting C3H/HeJ mice with ΔespFCR, ΔespFCR/pEspFCR, or ΔespFCR/pEspF(L16E)CR. These results indicate that EspF activity contributes to bacterial pathogenesis, as judged by murine lethality and intestinal histopathology, and promotion of bacterial colonization of the intestinal mucosa.

A new guidance and control system for a spaceplane is presented. The dynamics of the spaceplane has strong nonlinearity, due to which it is difficult to determine the optimal trajectory analytically and to design a stable trajectory tracking system. Therefore, in this study, we attempt to design a guidance and control system using a state-space exact linearization method without any approximation. Then, a minimum acceleration guidance law is derived analytically by solving a two-point boundary-value problem. Lastly, a trajectory control system is designed to track the vehicle with respect to the reference trajectory generated by the guidance system. The numerical simulation results confirm the validity of the linearized model, the optimality of the guidance system and the good tracking property of the developed system.

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B . bronchiseptica type III secretion system. Although BopB, BopN, BopD, and Bsp22 have been identified as type III secreted proteins, these proteins remain to be characterized. In this study, in order to clarify the function of BopD during Bordetella infection, a BopD mutant was generated. Although secretion of BopD into the culture supernatant was completely abolished by the bopD mutation, the secretion of other type III secreted proteins was not affected by this mutation. It has been reported that severe cytotoxicity, including cell detachment from the substrata, and release of lactate dehydrogenase (LDH) into the supernatant are induced in L2 cells by wild-type B . bronchiseptica infection, and these phenotypes are dependent on the type III secretion system. In contrast, neither cell detachment nor LDH release was induced in L2 cells infected with the BopD mutant. Furthermore, the hemolytic activity of the BopD mutant was greatly impaired compared with that of the wild-type strain. On the basis of the results of coimmunoprecipitation assays with anti-BopB antibodies, we conclude that BopD has the ability to associate with BopB. Finally, we show that the BopD-BopB complex is responsible for the pore formation in the host plasma membrane that functions as the conduit for the transition of effector proteins into host cells.

Enteropathogenic Escherichia coli (EPEC) secretes several Esps (E. coli-secreted proteins) that are required for full virulence. Insertion of the bacterial protein Tir into the host epithelial cell membrane is facilitated by a type III secretion apparatus, and at least EspA and EspB are required for Tir translocation. An EPEC outer membrane protein, intimin, interacts with Tir on the host membrane to establish intimate attachment and formation of a pedestal-like structure. In this study, we identified a Tir chaperone, CesT, whose gene is located between tir and eae (which encodes intimin). A mutation in cesT abolished Tir secretion into culture supernatants and significantly decreased the amount of Tir in the bacterial cytoplasm. In contrast, this mutation did not affect the secretion of the Esp proteins. The level of tir mRNA was not affected by the cesT mutation, indicating that CesT acts at the post-transcriptional level. The cesT mutant could not induce host cytoskeletal rearrangements, and displayed the same phenotype as the tir mutant. Gel overlay and GST pulldown assays demonstrated that CesT specifically interacts with Tir, but not with other Esp proteins. Furthermore, by using a series of Tir deletion derivatives, we determined that the CesT binding domain is located within the first 100 amino-terminal residues of Tir, and that the pool of Tir in the bacterial cytoplasm was greatly reduced when this domain was disrupted. Interestingly, this domain was not sufficient for Tir secretion, and at least the first 200 residues of Tir were required for efficient secretion. Gel filtration studies showed that Tir-CesT forms a large multimeric complex. Collectively, these results indicate that CesT is a Tir chaperone that may act as an anti-degradation factor by specifically binding to its amino-terminus, forming a multimeric stabilized complex.

Bordetella bronchiseptica colonizes the respiratory tracts of a wide variety of mammals and causes a range of diseases, from lethal pneumonia to asymptomatic chronic infection. We report the complete genome sequence of Bordetella bronchiseptica strain S798, isolated from a pig with atrophic rhinitis in Japan.

Enteropathogenic Escherichia coli (EPEC) secretes several Esp proteins via the type III secretion system (secreton). EspA, EspB, and EspD are required for translocation of the effector proteins into host cells, in which EspB and EspD are thought to form a pore in the host membrane. Recent study has shown that EspA forms a filamentous structure that assembles as a physical bridge between bacteria and host cell surfaces, which then functions as a conduit for the translocation of bacterial effectors into host cells. To investigate the supermolecular structure of the type III secreton in EPEC, we partially purified it from the bacteria membrane and observed it via transmission electron microscopy. The EPEC type III secreton was composed of a basal body and a needle part and was similar to those of Salmonella and Shigella , except for a sheath-like structure at the tip of the needle. The length of sheath-like structures varied; it extended more than 600 nm and was 10 times longer than the Shigella needle part. The putative major needle component, EscF, was required for both secretion of Esp proteins and needle complex formation. Interestingly, elongation of the sheath-like structure was observed under constitutive expression of EspA but not of EscF. Furthermore, the transmission electron microscopy view with immunogold labeled anti-EspA antibodies clearly showed that EspA is a component of the sheath-like structure. This study revealed, to our knowledge for the first time, the supermolecular structure of the EPEC type III secreton and its direct association with the EspA-sheath-like structure.