EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC-eseE Operon.

Edwardsiella tarda is an important Gram-negative pathogen that employs a type III secretion system (T3SS) to deliver effectors into host cells to facilitate bacterial survival and replication. These effectors are translocated into host cells through a translocon complex composed of three secreted proteins, namely, EseB, EseC, and EseD. The secretion of EseB and EseD requires a chaperone protein called EscC, whereas the secretion of EseC requires the chaperone EscA. In this study, we identified a novel protein (EseE) that also regulates the secretion of EseC. An eseE deletion mutant secreted much less EseC into supernatants, accompanied by increased EseC levels within bacterial cells. We also demonstrated that EseE interacted directly with EseC in a pulldown assay. Interestingly, EseC, EseE, and EscA were able to form a ternary complex, as revealed by pulldown and gel filtration assays. Of particular importance, the deletion of eseE resulted in decreased levels of EseB and EseD proteins in both the bacterial pellet and supernatant fraction. Furthermore, real-time PCR assays showed that EseE positively regulated the transcription of the translocon operon escC-eseE, comprising escC, eseB, escA, eseC, eseD, and eseE These effects of EseE on the translocon components/operon appeared to have a functional consequence, since the ΔeseE strain was outcompeted by wild-type E. tarda in a mixed infection in blue gourami fish. Collectively, our results demonstrate that EseE not only functions as a chaperone for EseC but also acts as a positive regulator controlling the expression of the translocon operon escC-eseE, thus contributing to the pathogenesis of E. tarda in fish.

Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish.

The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens.

Identification and functional characterization of the novel Edwardsiella tarda effector EseJ.

Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro- and extraintestinal infections in humans. The type III secretion system (T3SS) of E. tarda has been identified as a key virulence factor that contributes to pathogenesis in fish. However, little is known about the associated effectors translocated by this T3SS. In this study, by comparing the profile of secreted proteins of the wild-type PPD130/91 and its T3SS ATPase ΔesaN mutant, we identified a new effector by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. This effector consists of 1,359 amino acids, sharing high sequence similarity with Orf29/30 of E. tarda strain EIB202, and is renamed EseJ. The secretion and translocation of EseJ depend on the T3SS. A ΔeseJ mutant strain adheres to epithelioma papillosum of carp (EPC) cells 3 to 5 times more extensively than the wild-type strain does. EseJ inhibits bacterial adhesion to EPC cells from within bacterial cells. Importantly, the ΔeseJ mutant strain does not replicate efficiently in EPC cells and fails to replicate in J774A.1 macrophages. In infected J774A.1 macrophages, the ΔeseJ mutant elicits higher production of reactive oxygen species than wild-type E. tarda. The replication defect is consistent with the attenuation of the ΔeseJ mutant in the blue gourami fish model: the 50% lethal dose (LD50) of the ΔeseJ mutant is 2.34 times greater than that of the wild type, and the ΔeseJ mutant is less competitive than the wild type in mixed infection. Thus, EseJ represents a novel effector that contributes to virulence by reducing bacterial adhesion to EPC cells and facilitating intracellular bacterial replication.

Type III Secretion System Translocon Component EseB Forms Filaments on and Mediates Autoaggregation of and Biofilm Formation by Edwardsiella tarda.

The type III secretion system (T3SS) of Edwardsiella tarda plays an important role in infection by translocating effector proteins into host cells. EseB, a component required for effector translocation, is reported to mediate autoaggregation of E. tarda. In this study, we demonstrate that EseB forms filamentous appendages on the surface of E. tarda and is required for biofilm formation by E. tarda in Dulbecco's modified Eagle's medium (DMEM). Biofilm formation by E. tarda in DMEM does not require FlhB, an essential component for assembling flagella. Dynamic analysis of EseB filament formation, autoaggregation, and biofilm formation shows that the formation of EseB filaments occurs prior to autoaggregation and biofilm formation. The addition of an EseB antibody to E. tarda cultures before bacterial autoaggregation prevents autoaggregation and biofilm formation in a dose-dependent manner, whereas the addition of the EseB antibody to E. tarda cultures in which biofilm is already formed does not destroy the biofilm. Therefore, EseB filament-mediated bacterial cell-cell interaction is a prerequisite for autoaggregation and biofilm formation.

Edwardsiella tarda-Induced cytotoxicity depends on its type III secretion system and flagellin.

Many Gram-negative bacteria utilize a type III secretion system (T3SS) to translocate virulence proteins into host cells to cause diseases. In responding to infection, macrophages detect some of the translocated proteins to activate caspase-1-mediated cell death, called pyroptosis, and secretion of proinflammatory cytokines to control the infection. Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. In this study, we report that the T3SS of E. tarda facilitates its survival and replication in murine bone marrow-derived macrophages, and E. tarda infection triggers pyroptosis of infected macrophages from mice and fish and increased secretion of the cytokine interleukin 1ß in a T3SS-dependent manner. Deletion of the flagellin gene fliC of E. tarda results in decreased cytotoxicity for infected macrophages and does not attenuate its virulence in a fish model of infection, whereas upregulated expression of FliC in the fliC mutant strain reduces its virulence. We propose that the host controls E. tarda infection partially by detecting FliC translocated by the T3SS, whereas the bacteria downregulate the expression of FliC to evade innate immunity.

Secreted in a Type III Secretion System-Dependent Manner, EsaH and EscE Are the Cochaperones of the T3SS Needle Protein EsaG of Edwardsiella piscicida.

The intracellular EscE protein tightly controls the secretion of the type III secretion system (T3SS) middle and late substrates in Edwardsiella piscicida. However, the regulation of secretion by EscE is incompletely understood. In this work, we reveal that EscE interacts with EsaH and EsaG. The crystal structures of the EscE-EsaH complex and EscE-EsaG-EsaH complex were resolved at resolutions of 1.4 Å and 1.8 Å, respectively. EscE and EsaH form a hydrophobic groove to engulf the C-terminal region of EsaG (56 to 73 amino acids [aa]), serving as the cochaperones of T3SS needle protein EsaG in E. piscicida. V61, K62, M64, and M65 of EsaG play a pivotal role in maintaining the conformation of the ternary complex of EscE-EsaG-EsaH, thereby maintaining the stability of EsaG. An in vivo experiment revealed that EscE and EsaH stabilize each other, and both of them stabilize EsaG. Meanwhile, either EscE or EsaH can be secreted through the T3SS. The secondary structure of EsaH lacks the fourth and fifth α helices presented in its homologs PscG, YscG, and AscG. Insertion of the α4 and α5 helices of PscG or swapping the N-terminal 25 aa of PscG with those of EsaH starkly decreases the protein level of the chimeric EsaH, resulting in instability of EsaG and deactivation of the T3SS. To the best of our knowledge, these data represent the first reported structure of the T3SS needle complex of pathogens from Enterobacteriaceae and the first evidence for the secretion of T3SS needle chaperones. IMPORTANCE Edwardsiella piscicida causes severe hemorrhagic septicemia in fish. Inactivation of the type III secretion system (T3SS) increases its 50% lethal dose (LD50) by ~10 times. The secretion of T3SS middle and late substrates in E. piscicida is tightly controlled by the intracellular steady-state protein level of EscE, but the mechanism is incompletely understood. In this study, EscE was found to interact with and stabilize EsaH in E. piscicida. The EscE-EsaH complex is structurally analogous to T3SS needle chaperones. Further study revealed that EscE and EsaH form a hydrophobic groove to engulf the C-terminal region of EsaG, serving as the cochaperones stabilizing the T3SS needle protein EsaG. Interestingly, both EscE and EsaH are secreted. Our study reveals that the EscE-EsaH complex controls T3SS protein secretion by stabilizing EsaG, whose secretion in turn leads to the secretion of the middle and late T3SS substrates.

Tourette syndrome and excitatory substances: is there a connection?

BACKGROUND AND PURPOSE: The objective of this study is to investigate the relationship between excitatory substances by testing the urine in children with Tourette syndrome (TS). METHODS: We performed a control study involving 44 patients with TS and 44 normal children by investigating the children's daily eating habits. We used the gas chromatograph-mass spectrometer and liquid chromatograph-mass spectrometer from Agilent. Substances for detection included 197 excitatory substances prohibited by the International Olympic Committee and other substances with similar chemical structures or biological functions for urine samples. RESULTS: Forty-four patients who did not take any drugs in the past 2 weeks enrolled in the study. The positive rate in the experiment group was three cases, while it was negative in the control group. The level of 1-testosterone increased in one extremely severe TS patient who ate large amounts of puffed food and drank an average of 350 ml of cola per day. Cathine and other substances with similar chemical constitution or similar biological effects increased in one severe TS patient who ate bags of instant noodles daily, according to the high score of the Yale Global Tic Severity Scale. CONCLUSION: An increase in ephedrine type, testosterone, and stimulants may be related to the pathogenesis of TS. Unhealthy food possibly causes TS. The relationship between excitatory substances and TS needs to be explored with the goal of providing more information on diagnosing and treating TS.

[Effects of Huangqi on phagocytic activity of peritoneal macrophage of mice].

OBJECTIVE: To study the effects of Huangqi decoction (HQD) on phagocytic activity of peritoneal macrophage of mice. METHOD: One hundred Kunming mice, whose weight varied from 18 g to 22 g, were selected and divided into 10 groups randomly in eluding contrast group, groups conducted at different doses of HQD by ig, groups conducted in various ways of taking medicine, and groups conducted with comparative treat combining Huangqi and Dexamethasone. Mice in every group were taken medicine one time daily for 6 days. RESULT: Among the groups treated at different doses of HQD, phagocytic rate and phagocytic index of mice, which were taken HQD by ig at high, middle, and low doses, were significantly higher (P < 0.01) than that of mice in contrast group, at the same time the effect in group with high dose was the best. Among the groups treated in various ways of taking medicine, phagocytic rate of the ip group was significantly better (P < 0.01) than that of the sc group and that of the ig group respectively, but there was not significant difference (P > 0.05) of phagocytic index among them. Among the groups combining Huangqi and Dexamethasone, Huangqi could antagonize the immunosuppressive effect of Dexamethasone obviously (P < 0.01). CONCLUSION: HQD at different doses and with various ways of taking medicine could improve phagocytic activity of peritoneal macrophage of mice at different degree, and could antagonize the immunosuppressive effect of Dexamethasone.

[In vitro drug release profiles and mucoadhesive property of bioadhesive microspheres of metronidazole].

AIM: To prepare bioadhesive microspheres of metronidazole (Metro) with prolonging resident time in the stomach and sustaining drug release. METHODS: The microspheres were prepared by a drying-in-liquid method. The appearance, particle size and drug release in vitro were examined. The factors influencing bioadhesive property and drug release, such as ethyl cellulose (EC)/carbopol 934P (CP) ratio, particle size and Metro content were investigated. RESULTS: The average diameter of the Metro-EC-CP microspheres was 559.9 microns. The release profiles of metronidazole were shown to fit to first-order equations well. With the increase of CP content in the Metro-EC-CP microspheres, the microspheres showed better mucoadhesion and faster drug release. The drug release rate decreased with the increase of particle size and the decrease of Metro content. CONCLUSION: The Metro-EC-CP microspheres have a sound mucoadhsive property and sustained drug release when the ratio of EC and CP was 17:3 and Metro content was 25%. The drug release was shown to last for 8 h in 0.1 mol.L-1 hydrochloric acid.

Roles of plasmid-encoded proteins, EseH, EseI and EscD in invasion, replication and virulence of Edwardsiella ictaluri.

Native plasmids pEI1 and pEI2 were detected in Edwardsiella ictaluri HSN-1 isolated from diseased yellow catfish (Pelteobagrus fulvidraco). EseH encoded by pEI1 and other two proteins, EseI and EscD, encoded by pEI2, were found with homology to type III secretion system (T3SS) proteins. To investigate their roles in pathogenesis, the native plasmids were cured based on plasmid incompatibility by introducing a Kan positive and SacB negative selection marker into gene spacer of the native plasmids. Mutants with the deletion of the target genes were obtained by reverse PCR and self-ligation, and all mutants were examined for their virulence effect in yellow catfish. Compared with the HSN-1 strain, the two mutants ΔeseH and ΔeseI were attenuated, while mutant ΔescD had increased virulence with higher Competitive Index (CI) value. The adherence and invasion assays on fish EPC cells indicated that ΔeseH and ΔeseI had decreased ability in adherence. Using E. tarda as surrogate, EseH and EseI were detected in culture supernatants, but EscD was not, with the secretion of EseH depending on T3SS. In addition, EseH and EseI were found translocated into host cells, and by means of subcellular fractionation, EseH was localized in membrane fraction of ZF4 cells, and EseI in the cytosol fraction. Hence, the role of these three genes in adherence, invasion and cellular replication was revealed from the pathogenic bacterium E. ictaluri.