Elevated proton motive force is a tetracycline resistance mechanism that leads to the sensitivity to gentamicin in Edwardsiella tarda.

Tetracycline is a commonly used human and veterinary antibiotic that is mostly discharged into environment and thereby tetracycline-resistant bacteria are widely isolated. To combat these resistant bacteria, further understanding for tetracycline resistance mechanisms is needed. Here, GC-MS based untargeted metabolomics with biochemistry and molecular biology techniques was used to explore tetracycline resistance mechanisms of Edwardsiella tarda. Tetracycline-resistant E. tarda (LTB4-RTET ) exhibited a globally repressed metabolism against elevated proton motive force (PMF) as the most characteristic feature. The elevated PMF contributed to the resistance, which was supported by the three results: (i) viability was decreased with increasing PMF inhibitor carbonylcyanide-3-chlorophenylhydrazone; (ii) survival is related to PMF regulated by pH; (iii) LTB4-RTET were sensitive to gentamicin, an antibiotic that is dependent upon PMF to kill bacteria. Meanwhile, gentamicin-resistant E. tarda with low PMF are sensitive to tetracycline is also demonstrated. These results together indicate that the combination of tetracycline with gentamycin will effectively kill both gentamycin and tetracycline resistant bacteria. Therefore, the present study reveals a PMF-enhanced tetracycline resistance mechanism in LTB4-RTET and provides an effective approach to combat resistant bacteria.

Stimulatory effect of dietary alpha-lipoic acid on growth performance, antioxidant capacity, liver enzymes, immunity and protection of African catfish, Clarias gariepinus (B.), Edwardsiella tarda infection.

Edwardsiella tarda is one of the most common causes of fish diseases that hinder aquaculture. Oxidative stress in farm animals can induce a number of pathological disorders, production and general animal welfare. The use of exogenous dietary nonenzymatic antioxidants such as alpha-lipoic acid (ALA) can stop a pro-oxidant state and thus appears to have the potential to modulate the immune system and protect fish from bacterial infection. Thus, this study investigates the stimulatory effect of dietary ALA on growth performance, antioxidant capacity, liver enzymes, immunity and protection of African catfish, Clarias gariepinus (B.), against an infection with E. tarda. Five isonitrogenous and isocaloric diets (400 g/kg of crude protein) containing ALA at doses of 0.0 (control), 500, 1000, 1500 or 2000 mg/kg diet were served to 300 juveniles of African catfish (mean weight = 8.2 ± 0.2 g) adequately thrice per day for 12 weeks. Thereafter, 0.1 mL of E. tarda (ATCC 15947; 1.0 × 108 CFU/mL) was intraperitoneally injected into 10 fish from each tank and was monitored for 14 days. The results showed that ALA-fortified diets significantly boosted the fish growth, feed consumption and utilization and feed conversion ratio but no did not affect fish survival rate. The highest final fish weight (g), weight growth (g) and weight gain (%) were all considerably higher in fish fed with ALA-fortified diets (p < 0.05), especially from 1000 to 200 mg/kg ALA than the control group. Also, an enhanced hemato-biochemical, antioxidant and immune indices were noticed in African catfish-fed ALA-enriched diets. In a dose-dependent order, the levels of haematological indices such Ht, Hb, RBCs, WBCs and platelets were markedly increased (p < 0.05). Additionally, fish fed with ALA-based diets showed substantial (p < 0.05) declines in aspartate and alanine aminotransferase values, with the lowest values being found in the 2000 mg/kg diet while control group had highest values. Further, African catfish fed the feed fortified with 2000 mg ALA/kg diet showed the highest levels of lysozyme, respiratory burst, proteases and esterase activities (p < 0.05). Following exposure of fish to E. tarda infection, a significant reduction in the mortality was obtained in African catfish fed with ALA-based diets, especially from 1500 to 2000 mg ALA/kg diet (3.3%); while fish fed with the control diet had highest mortality (86.7%). Therefore, diets supplemented with ALA evoked fish growth performance, antioxidants and nonspecific immunity of African catfish. Also, resistance of African catfish to E. Tarda infection were raised when fed ALA-fortified diets at optimum inclusion rate of 1300 mg ALA/kg diet.

Functional Proteomics Analysis of Norfloxacin-Resistant Edwardsiella tarda.

Multidrug-resistant Edwardsiella tarda threatens both sustainable aquaculture and human health, but the control measure is still lacking. In this study, we adopted functional proteomics to investigate the molecular mechanism underlying norfloxacin (NOR) resistance in E. tarda. We found that E. tarda had a global proteomic shift upon acquisition of NOR resistance, featured with increased expression of siderophore biosynthesis and Fe3+-hydroxamate transport. Thus, either inhibition of siderophore biosynthesis with salicyl-AMS or treatment with another antibiotic, kitasamycin (Kit), which was uptake through Fe3+-hydroxamate transport, enhanced NOR killing of NOR-resistant E. tarda both in vivo and in vitro. Moreover, the combination of NOR, salicyl-AMS, and Kit had the highest efficacy in promoting the killing effects of NOR than any drug alone. Such synergistic effect not only confirmed in vitro and in vivo bacterial killing assays but also applicable to other clinic E. tarda isolates. Thus, our data suggest a proteomic-based approach to identify potential targets to enhance antibiotic killing and propose an alternative way to control infection of multidrug-resistant E. tarda.

Edwardsiella tarda TraT is an anti-complement factor and a cellular infection promoter.

Edwardsiella tarda is a well-known bacterial pathogen with a broad range of host, including fish, amphibians, and mammals. One eminent virulence feature of E. tarda is its strong ability to resist the killing of host serum complement, but the involving mechanism is unclear. In this report, we identified E. tarda TraT as a key player in both complement resistance and cellular invasion. TraT, a surface-localized protein, bound and recruited complement factor H onto E. tarda, whereby inhibiting complement activation via the alternative pathway. TraT also interacted with host CD46 in a specific complement control protein domain-dependent manner, whereby facilitating the cellular infection and tissue dissemination of E. tarda. Thus, by acting as an anti-complement factor and a cellular infection promoter, TraT makes an important contribution to the complement evasion and systemic infection of E. tarda. These results add insights into the pathogen-host interaction mechanism during E. tarda infection.

AroC, a chorismate synthase, is required for the formation of Edwardsiella tarda biofilms.

Biofilms contribute to the resistance of Edwardsiella tarda to antibiotics and host immunity. AroC in the shikimate pathway produces chorismate to synthesize crucial intermediates such as indole. In this study, the differences between biofilms produced by aroC mutants (△aroC), wild-type (WT) strains, and △aroC complementary strains (C△aroC) were detected both in vitro with 96-well plates, tubes, or coverslips and in vivo using a mouse model of subcutaneous implants. When examining potential mechanisms, we found that the diameters of the movement rings in soft agar plates and the flagellar sizes and numbers determined by silver staining were all lower for △aroC than for WT and C△aroC. Moreover, qRT-PCR showed that the transcription levels of flagellar synthesis genes, fliA and fliC, were reduced in △aroC. AroC, FliC, or FliA may accompany the motility of △aroC strains. In addition, compared with the WT and C△aroC, the amounts of indole in △aroC were significantly decreased. Notably, the formation of biofilms by these strains could be promoted by exogenous indole. Therefore, the aroC gene could affect the biofilm formation of E. tarda concerning its impact on flagella and indole.

Selection of References for microRNA Quantification in Japanese Flounder (Paralichthys olivaceus) Normal Tissues and Edwardsiella tarda-Infected Livers.

MicroRNA (miRNA) plays essential roles in post-transcriptional regulation of protein coding genes, and the quantitative real-time polymerase chain reaction (qRT-PCR) is the powerful and broadly employed tool to conduct studies of miRNA expression. Identifying appropriate references to normalize quantitative data is a prerequisite to ensure the qRT-PCR accuracy. Until now, there has been no report about miRNA reference for qRT-PCR in Japanese flounder (Paralichthys olivaceus), one important marine cultured fish along the coast of Northern Asia. In this study, combined with miRNA-Seq analysis and literature search, 10 candidates (miR-34a-5p, miR-205-5p, miR-101a-3p, miR-22-3p, miR-23a-3p, miR-210-5p, miR-30c-5p, U6, 5S rRNA, and 18S rRNA) were chosen as potential references to test their expression stability among P. olivaceus tissues, and in livers of P. olivaceus infected with Edwardsiella tarda at different time points. The expression stability of these candidates was analyzed by qRT-PCR and evaluated with Delta CT, BestKeeper, geNorm, as well as NormFinder methods, and RefFinder was employed to estimate the comprehensive ranking according to the four methods. As the result, miR-22-3p and miR-23a-3p were proved to be the suitable combination as reference miRNAs for both P. olivaceus normal tissues and livers infected with E. tarda, and they were successfully applied to normalize miR-7a and miR-221-5p expression in P. olivaceus livers in response to E. tarda infection. All these results provide valuable information for P. olivaceus miRNA quantitative expression analysis in the future.

Cytokines Induced by Edwardsiella tarda: Profile and Role in Antibacterial Immunity.

Edwardsiella tarda is a Gram-negative bacterial pathogen with a broad range of hosts, including fish and mammals. In the present study, we used an advanced antibody array technology to identify the expression pattern of cytokines induced by E. tarda in a mouse infection model. In total, 31 and 24 differentially expressed cytokines (DECs) were identified in the plasma at 6 h and 24 h post-infection (hpi), respectively. The DECs were markedly enriched in the Gene Ontology (GO) terms associated with cell migration and response to chemokine and in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with immunity, diseases, and infection. Ten key DECs, including IL6 and TNF-α, were found to form extensive protein-protein interaction networks. IL6 was demonstrated to inhibit E. tarda infection and be required for E. tarda-induced inflammatory response. TNF-α also exerted an inhibitory effect on E. tarda infection, and knockdown of fish (Japanese flounder) TNF-α promoted E. tarda invasion in host cells. Together, the results of this study revealed a comprehensive profile of cytokines induced by E. tarda, thus adding new insights into the role of cytokine-associated immunity against bacterial infection and also providing the potential plasma biomarkers of E. tarda infection for future studies.

Lipopolysaccharide-induced TNFα factor (LITAF) promotes inflammatory responses and activates apoptosis in zebrafish Danio rerio.

Lipopolysaccharide-induced TNFα factor (LITAF) is an important transcription factor which activates the transcription of TNFα and regulates cell apoptosis and inflammatory response. In the present study, a LITAF gene homologue was identified in zebrafish (Danio rerio) and was shown to be well conserved in the protein sequence, genomic organization and synteny with human LITAF. DrLITAF was constitutively expressed in tissues, with the highest expression detected in the gills. Its expression could be modulated by LPS, poly(I:C), and infection with Edwardsiella tarda, Aeromonus hydrophila and septicemia viremia of carp virus (SVCV). DrLITAF, when overexpressed, was shown to be located on the cellular membrane and nuclear membrane of HEK293T and ZF4 cells and was associated with the endoplasmic reticulum. Stimulation with LPS resulted in rapid translocation of DrLITAF into the nucleus. In addition, DrLITAF was able to induce cell apoptosis and the expression of caspase 3. The results demonstrate that DrLITAF is involved in the immune defence against bacterial and viral infection and plays a role in regulating inflammation and apoptosis.

Enteroendocrine cells sense bacterial tryptophan catabolites to activate enteric and vagal neuronal pathways.

The intestinal epithelium senses nutritional and microbial stimuli using epithelial sensory enteroendocrine cells (EEC). EECs communicate nutritional information to the nervous system, but whether they also relay signals from intestinal microbes remains unknown. Using in vivo real-time measurements of EEC and nervous system activity in zebrafish, we discovered that the bacteria Edwardsiella tarda activate EECs through the receptor transient receptor potential ankyrin A1 (Trpa1) and increase intestinal motility. Microbial, pharmacological, or optogenetic activation of Trpa1+EECs directly stimulates vagal sensory ganglia and activates cholinergic enteric neurons by secreting the neurotransmitter 5-hydroxytryptamine (5-HT). A subset of indole derivatives of tryptophan catabolism produced by E. tarda and other gut microbes activates zebrafish EEC Trpa1 signaling. These catabolites also directly stimulate human and mouse Trpa1 and intestinal 5-HT secretion. These results establish a molecular pathway by which EECs regulate enteric and vagal neuronal pathways in response to microbial signals.

Triclosan induces immunosuppression and reduces survivability of striped catfish Pangasianodon hypophthalmus during the challenge to a fish pathogenic bacterium Edwardsiella tarda.

Toxicological studies on the emergent pollutant, triclosan (TCS) have established the wide-ranging effects of the compound on fish and other aquatic organisms. Although the available literature describes the standalone effects of TCS on growth and metabolism of fish yet, reports about the combined effects of TCS with microbial pathogens are scarce. In a real environment, a combined exposure to TCS and pathogens is of common occurrence, therefore, such investigation facilitates in developing a better understanding about the gross effects of pollutants and microbial pathogens on aquatic organisms including fish. In this context, the experimental fish (striped catfish, Pangasianodon hypophthalmus) were exposed to three different concentrations of TCS viz. 10, 20 and 30% of 96 h LC50 (1177 µg L-1) for 45 days including two control group firstly solvent control (without TCS) group and another one (without solvent and TCS) group in triplicate. Sampling was performed fortnightly and blood, serum and tissues (liver, and gills) samples were collected for evaluating immunological and biochemical parameters. Following 45 days of the experiments, the experimental fish in each treatment group including controls were challenged with a fish pathogenic bacterium Edwardsiella tarda (LD50 dose) and fish mortality was daily monitored for calculating cumulative mortality till 7 days and further, relative per cent survivable was estimated. A significant reduction in cellular immune responses i.e. respiratory burst activity (RBA), myeloperoxidase activity (MPO), phagocytic activity (PA) and humoral immune components viz. serum lysozyme activity, total immunoglobulin in serum, ceruloplasmin level, serum total protein, albumin and globulin level was evident in TCS exposed groups in comparison to control during the experimental periods. Further, oxidative stress parameters viz. superoxide dismutase (SOD), catalase (CAT), glutathione-s-transferase (GST) activity in liver and gill tissue exhibited a dose-dependent increase in activity with related to TCS concentration during the experimental periods. A significant reduction in relative percentage survival was observed with increasing TCS concentration. The present study reveals that TCS can inhibit the cellular and humoral components of the innate immune system of the fish and can elevate the mortality due to TCS mediated immunosuppression in fish during the bacterial infection.

Fish borne Edwardsiella tarda eha involved in the bacterial biofilm formation, hemolytic activity, adhesion capability and pathogenicity.

Edwardsiella tarda (E. tarda) is distributed widely in a variety of hosts including humans, other mammals and fish, and it is worthwhile to notice that E. tarda -caused fish infections lead to the most important bacterial disease in fish. Considering Eha acting as a transcriptional regulator in E. tarda strain ET13 have been reported previously, to better understand its pathogenesis due to this, a type of cell of epithelial cell line (Caco-2) infection model for the pathogen was established in the laboratory. We focused on studying various parameters such as lactate dehydrogenase release (to measure cytotoxicity) and cell adhesions, both of which are related to the bacterial pathogenesis. Furthermore biofilm formation, hemolytic activity, and adhesion to Caco-2 cells were decreased in an E.tarda mutant strain with deletion in-frame isogenic gene eha (∆eha) compared to the wild-type and the complementary strain eha+ (an engineered construct of ∆eha expressing eha); Meanwhile, we found that hemolytic activity and biofilm formation were significantly enhanced in the strain eha+. Moreover, the ∆eha strain had attenuated pathogenicity in the zebrafish infection model. The data also demonstrated that the series of genes fimA, esrB, gadB, mukF, katB, and katG are regulated by eha based on a quantitative reverse transcription polymerase chain reaction tests and analysis. Thus our research data indicated that eha has an impact on hemolytic activity, biofilm formation, adhesion, and pathogenicity of pathogenic strain ET13 and plays an essential role in manifesting the virulence factors.

Desialylation by Edwardsiella tarda is the initial step in the regulation of its invasiveness.

Edwardsiella tarda is a gram-negative bacterium causing significant economic losses to aquaculture. E. tarda possesses NanA sialidase which removes sialic acids from α2-3 sialo-glycoprotein of host cells. However, the relationship between NanA sialidase activity and E. tarda invasiveness remains poorly understood. Furthermore, the pathway of sialic acid metabolism in E. tarda remains to be elucidated. We studied sialidase activity in several E. tarda strains and found that the pathogenic strains exhibited higher sialidase activity and greater up-regulation of the NanA mRNA level than non-pathogenic strain. Pathogenic strains also showed higher rates of infection in GAKS cells, and the infection was drastically suppressed by sialidase inhibitor. Additionally, NanA gene overexpression significantly increased infection and treatment of E. tarda with free sialic acid enhanced the rate of infection in GAKS cells. Sialic acid treatment enhanced mRNA levels of two N-acetylneuraminate lyases and one N-acetylneuraminate cytidylyltransferase. E. tarda uses sialic acid as a carbon source for growth via N-acetylneuraminate lyases. The strains with high N-acetylneuraminate cytidylyltransferase level showed greater sialylation of the lipopolysaccharides and glycoproteins. Our study establishes the significance of desialylation by E. tarda sialidase in the regulation of its invasiveness.

Characterization of balofloxacin-stressed proteomics and identification of balofloxacin-binding proteins pre-peptidase and integration host factor in Edwardsiella tarda.

The overuse of antibiotics to control bacterial pathogens leads to the generation of their antibiotic-resistant strains including Edwardsiella tarda. Understanding of mechanisms of the antibiotic resistance is crucial to develop novel methods to manage the infection. Here, two-dimensional electrophoresis-based proteomics was used to characterize balofloxacin-responsive proteins. The altered proteome consisted of 19 proteins with differential abundance, where six metabolic pathways were enriched. The metabolic modulation activated the central carbon metabolism with elevation of NADH, PMF, and ATP. Among the 19 proteins, ETAE_1987 (pre-peptidase) and ETAE_2174 (integration host factor beta subunit) were bound with balofloxacin directly. This was further confirmed by the binding of balofloxacin with recombinant ETAE_1987 and ETAE_2174 using Oxford cup method. Compared with bovine serum albumin, a known balofloxacin-binding protein, ETAE_1987 and ETAE_2174 increased the binding capability by 3.3- and 22-fold, respectively. The combination was validated by microscale thermophoresis. These data characterize the balofloxacin-stressed proteome as a result of the increased central carbon metabolism and energy metabolism and determine ETAE_1987 and ETAE_2174 as balofloxacin-binding proteins. These findings have significant implications in understanding bacterial antibiotic-resistant and drug action mechanisms based on balofloxacin-binding proteins. SIGNIFICANCE: Antibiotic-resistant Edwardsiella tarda strains are frequently isolated and cause a great loss in aquaculture since these bacterial strains are insensitivity to antibiotics. The present study showed that the increased central carbon metabolism forms a characteristic feature of the balofloxacin-stressed proteomics. Furthermore, two proteins, ETAE_1987 (pre-peptidase) and ETAE_2174, of the balofloxacin-stressed proteome were identified as balofloxacin-binding proteins. The binding capability is 0.39 ±â€¯0.017 and 2.67 ±â€¯0.066 ng/µg proteins for ETAE_1987 and ETAE_2174, respectively. These results reveal the elevated central carbon metabolism as a key feature of the balofloxacin-stressed proteomics and pre-peptidase and integration host factor as balofloxacin-binding proteins in E. tarda. These findings are useful in the understanding of bacterial balofloxacin-stressed mechanisms and providing new targets for controlling antibiotic-resistant bacteria.

De novo whole transcriptome profiling of Edwardsiella tarda isolated from infected fish (Labeo catla).

Edwardsiella tarda belongs to the genera of Gram negative bacterium mainly associated with edwardsiellosis, the most commonly found infectious fish disease throughout the globe. E. tarda is also a widespread pathogen which cause infections such as cellulitis or gas gangrene and generalized infections in humans. To control the escalating infection of E. trada on various species, it is essential to decoded the mysterious mechanism behind the bacterial infection at transcript level. In this present study, we carry out a de novo E. tarda Whole transcriptome sequencing, isolated from infected fish intestine using SOLiD sequencing platform. RNA-Seq data analysis was performed using various bioinformatics pipelines. Protein-protein interaction study for pathway enrichment and gene ontology study were executed for further investigation. Assembly statistics for E. tarda dataset showed that the number of transcript contigs was 9657 out of which 6749 were GO annotated whereas 1528 were not assigned any GO terms. GO analysis showed that the expressed genes were enhanced with molecular function, cellular component and biological process. A KEGG enrichment study showed that pathway's that are directly linked with immune diseases like Rheumatoid arthritis (0.2%), Tuberculosis (0.3%) Endocytosis (0.6%) was considerably enriched. Protein-protein interaction study showed that most of the expressed proteins were involved in metabolic pathways, flagellar assembly, Propanoate metabolism, Microbial metabolism in diverse environments, Butanoate metabolism and Carbon. The present study provides novel E. tarda transcriptome sequence data, allowing us to identify biologically significant genes and their functional relationship with fish diseases, and will be useful in recognize the reliable therapeutic targets in near feature.

Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria.

The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.

Proteomic analysis of macrophage in response to Edwardsiella tarda-infection.

Edwardsiella tarda is an important facultative intracellular pathogen infecting a wide range of host from fish to humans. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. E. tarda -macrophage interaction is vital in determining the outcome of edwardsiellasis. To fully elucidate the pathogenesis of E. tarda, the differential proteomes of RAW264.7 cells in response to E. tarda-infection, were analyzed at different time points with two-dimensional gel electrophoresis (2-DE) followed by liquid-chromatography-tandem mass spectrometry (LC-MS/MS) identification. 26 altered proteins (18 up-regulated and 8 down-regulated proteins) were successfully identified, which are mainly involved in formation of phagosomes, macrophage microbicidal activity and anti-apoptosis of macrophage. Moreover, 6 corresponding genes of the differentially expressed proteins were quantified by quantitative real-time PCR (qPCR) to examine the transcriptional profiles. Western blot analysis further confirmed the differential expression of 5 proteins in the proteomic profiles. Based on these findings, we hypothesize that these differentially expressed proteins likely play a pivotal role in determining the course of E. tarda-infection. The result suggested that E. tarda could develop some strategies to achieve a successful intracellular lifestyle, including modulation of phagosome biogenesis, resistance to macrophage microbicidal agent and anti-apoptosis of macrophages. Thus, this work effectively provides useful and novel protein-related information to further understand the underlying pathogenesis of E. tarda-infection.

Regulation of Type III Secretion of Translocon and Effector Proteins by the EsaB/EsaL/EsaM Complex in Edwardsiella tarda.

The type III secretion system (T3SS) plays a crucial role in the pathogenesis of many Gram-negative bacteria, including Edwardsiella tarda, an important fish pathogen. Within the E. tarda T3SS, there are three proteins (EsaB/EsaL/EsaM) that are homologous to proteins present in many other bacteria, including SpiC/SsaL/SsaM in Salmonella, SepD/SepL/CesL in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), and YscB/YopN/SycN in Yersinia EsaL was found to interact with both EsaB and EsaM within the bacterial cell, as revealed by a coimmunoprecipitation assay. Moreover, EsaM is required for EsaB stability, and the two proteins interact with each other. EsaB, EsaL, and EsaM are all indispensable for the secretion of the T3SS translocon protein EseC into supernatants under pH 5.5 and pH 7.2 conditions. Unlike EseC, EseG is a T3SS effector whose secretion is suppressed by EsaL at pH 7.2 while it is promoted at pH 5.5 condition. Despite this finding, mutant strains lacking EsaB, EsaL, or EsaM (i.e., the ΔesaB, ΔesaL, or ΔesaM strain, respectively) were all outcompeted by wild-type E. tarda during a coinfection model. These results demonstrate that EsaB/EsaL/EsaM form a ternary complex controlling the secretion of T3SS translocon and effector proteins and contributing to E. tarda pathogenesis.

The Bacterial T6SS Effector EvpP Prevents NLRP3 Inflammasome Activation by Inhibiting the Ca2+-Dependent MAPK-Jnk Pathway.

Inflammasome activation is an important innate immune defense mechanism against bacterial infection, and in return, bacteria express virulence determinants that counteract inflammasome activation. Many such effectors are secreted into host cells via specialized bacterial secretion systems. Here, the intracellular pathogenic bacterium Edwardsiella tarda was demonstrated to activate NLRC4 and NLRP3 inflammasomes via a type III secretion system (T3SS), and to inhibit NLRP3 inflammasome via a type VI secretion system (T6SS), indicating the antagonistic roles of these systems in inflammasome signaling. Furthermore, a non-VgrG T6SS effector, EvpP, was identified that significantly inhibited NLRP3 inflammasome activation. Subsequent studies revealed that EvpP significantly suppressed Jnk activation, thus impairing oligomerization of the inflammasome adaptor ASC. Moreover, EvpP counteracted cytoplasmic Ca2+ increase, which works upstream of Jnk activation to regulate the NLRP3 inflammasome. Finally, EvpP-mediated inflammasome inhibition promoted bacterial colonization in vivo. This work expands our understanding of bacterial T6SS in counteracting host immune responses.

Three-dimensional visualization of green fluorescence protein-labelled Edwardsiella tarda in whole Medaka larvae.

The invasive fish pathogen Edwardsiella tarda is common in aquatic environments and causes the environmentally and economically destructive emphysematous putrefactive disease called edwardsiellosis. In order to understand the organism's infection pathway, medaka larvae (Oryzias latipes) were immersion-infected with E. tarda labelled with green fluorescence protein (GFP) and then visualized in three dimensions under confocal laser microscopy and light-sheet fluorescence microscopy. Confocal microscopy revealed GFP-labelled E. tarda in the mouth, head, gill bridges, gill cover, skin, membrane fin, gastrointestinal tract and air bladder, and in the caudal vein, somite veins, caudal artery and caudal capillaries. Light-sheet microscopy additionally showed GFP-labelled E. tarda in the pharyngeal cavity, muscle of the pectoral fin and cardiac atrium and ventricle. These findings suggest that during its infection of fish, E. tarda initially adheres to, and invades, the epithelial cells of the skin, gills and gastrointestinal tract (through the pharyngeal cavity); E. tarda then enters the blood vessels to access organs, including the air bladder and heart.

Quantitative proteomic analysis of Edwardsiella tarda in response to oxytetracycline stress in biofilm.

Edwardsiella tarda is a virulent fish pathogen that causes extensive economic losses in the aquaculture industry worldwide. The antibiotic resistance status of E. tarda is high, especially in the biofilm status; however, the mechanisms underlying its resistance remain largely unknown. In this study, isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods were used to compare the differential expression of E. tarda in response to oxytetracycline (OXY) stress in biofilm. Additional bioinformatics analysis demonstrated an increasing abundance of translation-related proteins, especially ribosomal subunits, and a decreasing abundance of key metabolic pathways underlying the adaptation of E. tarda to OXY in biofilm. We performed Western blotting and quantitative PCR (qPCR) analyses to validate selected proteomics results, and measured enzyme activity to verify the antibiotic resistance functions of central metabolic pathways. In addition, we examined the antibiotic susceptibility of a mutant of an NADP-dependent malic enzyme (MaeB), which is involved in the bacterial tricarboxylic acid cycle, and found significantly increased resistance to OXY in biofilm. Our findings demonstrate the importance of central metabolic pathways in the antibiotic resistance of E. tarda to bacterial biofilms and provide insight into the prevention of this resistance, which would aid in disease control. BIOLOGICAL SIGNIFICANCE: The antibiotics resistance mechanisms in E. tarda have been well documented recently; however, its response to antibiotics in biofilms remains elusive. Our current study is the first exploratory report investigating this aspect via an iTARQ-based quantitative proteomics method. Several important proteins, related processes, and metabolic pathways were found to be involved in OXY fitness in biofilm status. Most importantly, the depletion of the maeB gene decreased the susceptibility of E. tarda to OXY indicating the important role of central metabolic pathways in antibiotics resistance in biofilm.