Development of a real-time recombinase-aided amplification assay for rapid and sensitive detection of Edwardsiella piscicida.

Edwardsiella piscicida, a significant intracellular pathogen, is widely distributed in aquatic environments and causes systemic infection in various species. Therefore, it's essential to develop a rapid, uncomplicated and sensitive method for detection of E. piscicida in order to control the transmission of this pathogen effectively. The recombinase-aided amplification (RAA) assay is a newly developed, rapid detection method that has been utilized for various pathogens. In the present study, a real-time RAA (RT-RAA) assay, targeting the conserved positions of the EvpP gene, was successfully established for the detection of E. piscicida. This assay can be performed in a one-step single tube reaction at a temperature of 39°C within 20 min. The RT-RAA assay exhibited a sensitivity of 42 copies per reaction at a 95% probability, which was comparable to the sensitivity of real-time quantitative PCR (qPCR) assay. The specificity assay confirmed that the RT-RAA assay specifically targeted E. piscicida without any cross-reactivity with other important marine bacterial pathogens. Moreover, when clinical specimens were utilized, a perfect agreement of 100% was achieved between the RT-RAA and qPCR assays, resulting a kappa value of 1. These findings indicated that the established RT-RAA assay provided a viable alternative for the rapid, sensitive, and specific detection of E. piscicida.

Bacteriophage EPP-1, a potential antibiotic alternative for controlling edwardsiellosis caused by Edwardsiella piscicida while mitigating drug-resistant gene dissemination.

Edwardsiella piscicida causes significant economic losses to the aquaculture industry worldwide. Phage-based biocontrol methods are experiencing a renaissance because of the spread of drug-resistant genes and bacteria resulting from the heavy use of antibiotics. Here, we showed that the novel Edwardsiella phage EPP-1 could achieve comparable efficacy to florfenicol using a zebrafish model of Edwardsiella piscicida infection and could reduce the content of the floR resistance gene in zebrafish excreta. Specifically, phage EPP-1 inhibited bacterial growth in vitro and significantly improved the zebrafish survival rate in vivo (P = 0.0035), achieving an efficacy comparable to that of florfenicol (P = 0.2304). Notably, integrating the results of 16S rRNA sequencing, metagenomic sequencing, and qPCR, although the effects of phage EPP-1 converged with those of florfenicol in terms of the community composition and potential function of the zebrafish gut microbiota, it reduced the floR gene content in zebrafish excreta and aquaculture water. Overall, our study highlights the feasibility and safety of phage therapy for edwardsiellosis control, which has profound implications for the development of antibiotic alternatives to address the antibiotic crisis.

Edwardsiella piscicida causes iron storage disorders by an autophagy pathway in fish monocytes/macrophages.

Edwardsiella piscicida (E. piscicida) is a gram-negative pathogen that survives in intracellular environment. Currently, the interplay between E. piscicida and host cells has not been completely explored. In this study, we found that E. piscicida disturbed iron homeostasis in grass carp monocytes/macrophages to maintain its own growth. Further investigation revealed the bacteria induced an increase of intracellular iron, which was subjected to the degradation of ferritin. Moreover, the autophagy inhibitor impeded the degradation of ferritin and increase of intracellular iron in E. piscicida-infected monocytes/macrophages, implying possible involvement of autophagy response in the process of E. piscicida-broken iron homeostasis. Along this line, confocal microscopy observed that E. piscicida elicited the colocalization of ferritin with LC3-positive autophagosome in the monocytes/macrophages, indicating that E. piscicida mediated the degradation of ferritin possibly through the autophagic pathway. These results deepened our understanding of the interaction between E. piscicida and fish cells, hinting that the disruption of iron homeostasis was an important factor for pathogenicity of E. piscicida. They also indicated that autophagy was a possible mechanism governing intracellular iron metabolism in response to E. piscicida infection and might offer a new avenue for anti-E. piscicida strategies in the future.

Type III secretion system effector YfiD inhibits the activation of host poly(ADP-ribose) polymerase-1 to promote bacterial infection.

Modulation of cell death is a powerful strategy employed by pathogenic bacteria to evade host immune clearance and occupy profitable replication niches during infection. Intracellular pathogens employ the type III secretion system (T3SS) to deliver effectors, which interfere with regulated cell death pathways to evade immune defenses. Here, we reveal that poly(ADP-ribose) polymerase-1 (PARP1)-dependent cell death restrains Edwardsiella piscicida's proliferation in mouse monocyte macrophages J774A.1, of which PARP1 activation results in the accumulation of poly(ADP-ribose) (PAR) and enhanced inflammatory response. Moreover, E. piscicida, an important intracellular pathogen, leverages a T3SS effector YfiD to impair PARP1's activity and inhibit PAR accumulation. Once translocated into the host nucleus, YfiD binds to the ADP-ribosyl transferase (ART) domain of PARP1 to suppress its PARylation ability as the pharmacological inhibitor of PARP1 behaves. Furthermore, the interaction between YfiD and ART mainly relies on the complete unfolding of the helical domain, which releases the inhibitory effect on ART. In addition, YfiD impairs the inflammatory response and cell death in macrophages and promotes in vivo colonization and virulence of E. piscicida. Collectively, our results establish the functional mechanism of YfiD as a potential PARP1 inhibitor and provide more insights into host defense against bacterial infection.

Effect of polystyrene nanoplastics exposure on gene expression and pathogenesis of zoonotic pathogen, Edwardsiella piscicida.

A surge in the number of anthropogenic pollutants has been caused by increasing industrial activities. Nanoplastics are spotlighted as a new aquatic pollutant that are a threat to microbes and larger organisms. Our previous study showed that the subinhibitory concentrations of aquatic pollutants such as phenol and formalin act as signaling molecules and modulate global gene expression and metabolism. In this study, we aimed to investigate the impact of a new type of anthropogenic contaminant, polystyrene (PS) nanoplastics, on the expression of key virulence factors in zoonotic pathogen Edwardsiella piscicida and the assessment of potential changes in the susceptibility of zebrafish as a model host. The TEM data indicated a noticeable change in the cell membrane indicating that PS particles were possibly entering the bacterial cells. Transcriptome analyses performed to identify the differentially expressed genes upon PS exposure revealed that the genes involved in major virulence factor type VI secretion system (T6SS) were down-regulated. However, the expression of T6SS-related genes was recovered from the PS adapted E. piscicida when nanoplastics are free. This demonstrated the hypervirulence of pathogen in infection assays with both cell lines and in vivo zebrafish model. Therefore, this study provides experimental evidence elucidating the direct regulatory impact of nanoplastics influx into aquatic ecosystems on fish pathogenic bacteria, notably influencing the expression of virulence factors.

Transcriptome analysis of Edwardsiella piscicida during intracellular infection reveals excludons are involved with the activation of a mitochondrion-like energy generation program.

Phylogenetic evidence suggests a shared ancestry between mitochondria and modern Proteobacteria, a phylum including several genera of intracellular pathogens. Studying these diverse pathogens, particularly during intracellular infection of their hosts, can reveal characteristics potentially representative of the mitochondrial-Proteobacterial ancestor by identifying traits shared with mitochondria. While transcriptomic approaches can provide global insights into intracellular acclimatization by pathogens, they are often limited by excess host RNAs in extracts. Here, we developed a method employing magnetic nanoparticles to enrich RNA from an intracellular Gammaproteobacterium, Edwardsiella piscicida, within zebrafish, Danio rerio, fin fibroblasts, enabling comprehensive exploration of the bacterial transcriptome. Our findings revealed that the intracellular E. piscicida transcriptome reflects a mitochondrion-like energy generation program characterized by the suppression of glycolysis and sugar transport, coupled with upregulation of the tricarboxylic acid (TCA) cycle and alternative import of simple organic acids that directly flux into TCA cycle intermediates or electron transport chain donors. Additionally, genes predicted to be members of excludons, loci of gene pairs antagonistically co-regulated by overlapping antisense transcription, are significantly enriched in the set of all genes with perturbed sense and antisense transcription, suggesting a general but important involvement of excludons with intracellular acclimatization. Notably, genes involved with the activation of the mitochondrion-like energy generation program, specifically with metabolite import and glycolysis, are also members of predicted excludons. Other intracellular Proteobacterial pathogens appear to employ a similar mitochondrion-like energy generation program, suggesting a potentially conserved mechanism for optimized energy acquisition from hosts centered around the TCA cycle.IMPORTANCEPhylogenetic evidence suggests that mitochondria and Proteobacteria, a phylum encompassing various intracellular pathogens, share a common ancestral lineage. In this study, we developed a novel method employing magnetic nanoparticles to explore the transcriptome of an aquatic Gammaproteobacterium, Edwardsiella piscicida, during intracellular infection of host cells. We show that the strategy E. piscicida uses to generate energy strikingly mirrors the function of mitochondria-energy generators devoid of glycolytic processes. Notably, several implicated genes are members of excludons-gene pairs antagonistically co-regulated by overlapping antisense transcription. Other intracellular Proteobacterial pathogens appear to adopt a similar mitochondrion-like energy generation program, indicating a possibly conserved strategy for optimized energy acquisition from hosts centered around the tricarboxylic acid cycle.

The cyclic AMP (cAMP) phosphodiesterase CpdA required for growth, biofilm formation, motility and pathogenicity of Edwardsiella piscicida.

Edwardsiella piscicida is a severe fish pathogen with wide host range, causing the huge economic losses in the aquaculture industry. Cyclic adenosine monophosphate (cAMP) as an important second messenger regulates the physiological and behavioral responses to environmental cues in eukaryotic and prokaryotic. The intracellular level of cAMP for effective activity is tightly controlled by the synthesis of adenylate cyclase, excretion and degradation of phosphodiesterase. In this study, we identified and characterized a class III cAMP phosphodiesterase, named as CpdA, in the E. piscicida. To investigate the role of CpdA in the physiology and pathogenicity, we constructed the in-frame deletion mutant of cpdA of E. piscicida, TX01ΔcpdA. The results showed that TX01ΔcpdA accumulated the higher intracellular cAMP concentration than TX01, indicating that CpdA exerted the hydrolysis of cAMP. In addition, compared to the TX01, the TX01ΔcpdA slowed growth rate, diminished biofilm formation and lost motility. More importantly, pathogenicity analysis confirmed that TX01ΔcpdA significantly impaired the ability of invading the epithelial cells, reproduction in macrophages, tissues dissemination and lethality for healthy tilapias. The most of lost properties of TX01ΔcpdA were restored partially or fully by the introduction of cpdA gene. These results suggest that cpdA is required for regulation of the physiology and virulence of E. piscicida.

CRISPR/Cas9-mediated knockout of tnf-α1 in zebrafish reduces disease resistance after Edwardsiella piscicida bacterial infection.

Tumor necrosis factor (TNF) is an important cytokine involved in immune responses to bacterial infections in vertebrates, including fish. Although Tnf-α is a well-studied cytokine, there are contradictory findings about Tnf-α function following bacterial infection. In this study, we analyzed the expression and function of the Tnf-α-type I isoform (Tnf-α1) in zebrafish by knockout experiments using the CRISPR/Cas9 gene-editing tool. The open reading frame of tnf-α1 encodes a 25.82 kDa protein with 234 amino acids (aa). The expression of tnf-α1 in the early stages of zebrafish was observed from the 2-cell stage. Adult zebrafish spleens showed the highest expression of tnf-α1. To evaluate the function of Tnf-α1, an 8 bp deletion in the target region, resulting in a short truncated protein of 55 aa, was used to create the tnf-α1 knockout mutant. The pattern of downstream gene expression in 7-day larvae in wild-type (WT) and tnf-α1 knockout fish was examined. We also verified the fish mortality rate after Edwardsiella piscicida challenge and found that it was much higher in tnf-α1 knockout fish than in WT fish. Additionally, downstream gene expression analyses after E. piscicida exposure revealed a distinct expression pattern in tnf-α1 knockout fish compared to that in WT fish. Overall, our study using tnf-α1 deletion in zebrafish confirmed that Tnf-α1 is critical for immune regulation during bacterial infection.

The QseE-QseF two-component system: A key mediator of epinephrine-regulated virulence in the marine pathogen Edwardsiella piscicida.

Edwardsiella piscicida is a widespread pathogen that infects various fish species and causes massive hemorrhagic septicemia, resulting in significant property damage to the global aquaculture industry. Type III and VI secretion systems (T3/T6SS), controlled by the master regulator EsrB, are important virulence factors of E. piscicida that enable bacterial colonization and evasion from host immune clearance. In this study, we demonstrate that the QseE-QseF two-component system negatively regulated esrB expression by reanalysis of Tn-seq data. Moreover, the response regulator QseF directly bound to esrB promoter and inhibited the expression of T3/T6SS genes, especially in the presence of epinephrine. Furthermore, in response to the prompt increasing of epinephrine level, the host immune genes were delayed repressed and QseE-QseF timely inhibited the expression of T3/T6SS genes to evade immune clearance. In summary, this study enhances our understanding and knowledge of the conditional pathogenesis mechanism and virulence regulation network of E. piscicida.

The pathway of Edwardsiella piscicida infecting Lateolabrax maculatus via the immersion bath.

Edwardsiella piscicida, an infectious bacterium, causes great economic losses to the aquaculture industry. Immersion bath which is the closest way to how the fish infect bacterial pathogens in the natural environment is an effective route of artificial infection. In this study, the dynamic process of E. piscicida infection, in the spotted sea bass (Lateolabrax maculatus) was evaluated via the immersion bath. The results showed that soaking the spotted sea bass with 3 × 106 CFU mL-1 E. piscicida for 30 min could artificially induce edwardsiellosis. The higher culture temperature (28.5 ± 0.5°C) or the longer bath time (30 min) would lead to higher mortality of fish. E.piscicida first invaded the gill, then entered the blood circulation to infect the spleen and kidney, where it is colonized, and gradually multiplied in the liver and brain. Meanwhile, the fluorescence in situ hybridization showed that the localization of E. piscicida in the gill and foregut after the immersion challenge proceeded from the exterior to the interior. The invasion of pathogens triggers the immune response of fish and causes tissue damage to the host. The quantitative real-time PCR results displayed an increase in the relative expression level of immune genes (NK-lysin, LZM, IgM and IgD). Otherwise, the most notable histopathological changes of the infected spotted sea bass were multifocal necrosis. Findings in this study broaden our understanding of the infection conditions of E. piscicida and its pathogenicity to the spotted sea bass.

Zebrafish nos2a benefits bacterial proliferation via suppressing ROS and inducing NO production to impair the expressions of inflammatory cytokines and antibacterial genes.

The enzyme nitric oxide synthase 2 or inducible NOS (NOS2), reactive oxygen species (ROS) and nitric oxide (NO) are important participants in various inflammatory and immune responses. However, the functional significances of the correlations among piscine NOS2, ROS and NO during pathogen infection remain unclear. In teleost, there are two nos2 genes (nos2a and nos2b). It has been previously reported that zebrafish nos2a behaves as a classical inducible NOS, and nos2b exerts some functions similar to mammalian NOS3. In the present study, we reported the functional characterization of zebrafish nos2a during bacterial infection. We found that zebrafish nos2a promoted bacterial proliferation, accompanied by an increased susceptibility to Edwardsiella piscicida infection. The nagative regulation of zebrafish nos2a during E. piscicida infection was characterized by the impaired ROS levels, the induced NO production and the decreased expressions of proinflammatory cytokines, antibacterial genes and oxidant factors. Furthermore, although both inducing ROS and inhibiting NO production significantly inhibited bacterial proliferation, only inhibiting NO production but not inducing ROS significantly increased resistance to E. piscicida infection. More importantly, ROS supplementation and inhibition of NO completely abolished this detrimental consequence mediated by zebrafish nos2a during E. piscicida infection. All together, these results firstly demonstrate that the innate response mediated by zebrafish nos2a in promoting bacterial proliferation is dependent on the lower ROS level and higher NO production. The present study also reveals that inhibition of NO can be effective in the protection against E. piscicida infection.

Unraveling and characterization of novel T3SS effectors in Edwardsiella piscicida.

Type III secretion system (T3SS) facilitates survival and replication of Edwardsiella piscicida in vivo. Identifying novel T3SS effectors and elucidating their functions are critical in understanding the pathogenesis of E. piscicida. E. piscicida T3SS effector EseG and EseJ was highly secreted when T3SS gatekeeper-containing protein complex EsaB-EsaL-EsaM was disrupted by EsaB deficiency. Based on this observation, concentrated secretomes of ΔesaB strain and ΔesaBΔesaN strain were purified by loading them into SDS-PAGE gel for a short electrophoresis to remove impurities prior to the in-the gel digestion and mass spectrometry. Four reported T3SS effectors and two novel T3SS effector candidates EseQ (ETAE_2009) and Trx2 (ETAE_0559) were unraveled by quantitative comparison of the identified peptides. EseQ and Trx2 were revealed to be secreted and translocated in a T3SS-dependent manner through CyaA-based translocation assay and immunofluorescent staining, demonstrating that EseQ and Trx2 are the novel T3SS effectors of E. piscicida. Trx2 was found to suppress macrophage apoptosis as revealed by TUNEL staining and cleaved caspase-3 of infected J774A.1 monolayers. Moreover, Trx2 has been shown to inhibit the p65 phosphorylation and p65 translocation into the nucleus, thus blocking the NF-κB pathway. Furthermore, depletion of Trx2 slightly but significantly attenuates E. piscicida virulence in a fish infection model. Taken together, an efficient method was established in unraveling T3SS effectors in E. piscicida, and Trx2, one of the novel T3SS effectors identified in this study, was demonstrated to suppress apoptosis and block NF- κB pathway during E. piscicida infection. IMPORTANCE Edwardsiella piscicida is an intracellular bacterial pathogen that causes intestinal inflammation and hemorrhagic sepsis in fish and human. Virulence depends on the Edwardsiella type III secretion system (T3SS). Identifying the bacterial effector proteins secreted by T3SS and defining their role is key to understanding Edwardsiella pathogenesis. EsaB depletion disrupts the T3SS gatekeeper-containing protein complex, resulting in increased secretion of T3SS effectors EseG and EseJ. EseQ and Trx2 were shown to be the novel T3SS effectors of E. piscicida by a secretome comparison between ∆esaB strain and ∆esaB∆esaN strain (T3SS mutant), together with CyaA-based translocation assay. In addition, Trx2 has been shown to suppress macrophage apoptosis and block the NF-κB pathway. Together, this work expands the known repertoire of T3SS effectors and sheds light on the pathogenic mechanism of E. piscicida.

Inoculation of Freund's adjuvant in European eel (Anguilla anguilla) revealed key KEGG pathways and DEGs of host anti-Edwardsiella anguillarum infection.

Freund's complete (FCA) and incomplete adjuvants (FIA), generally applied in subunit fishery vaccine, have not been explored on the molecular mechanism of the nonspecific immune enhancement. In this study, we examined the RNA-seq in the spleen of European eel (Anguilla anguilla) inoculated with FCA and FIA (FCIA group) to elucidate the key KEGG pathways and differential expressed genes (DEGs) in the process of Edwardsiella anguillarum infection and A. anguilla anti-E. anguillarum infection using genome-wide transcriptome. After eels were challenged by E. anguillarum at 28 d post the first inoculation (dpi), compared to the control uninfected eels (Con group), the control infected eels (Con_inf group) showed severe pathological changes in the liver, kidney and spleen, although infected eels post the inoculation of FCIA (FCIA_inf group) also formed slight bleeding. Compared to the FCIA_inf group, there was more than 10 times colony forming unit (cfu) in the Con_inf group per 100 µg spleen, kidney or blood, and the relative percent survival (RPS) of eels was 44.4% in FCIA_inf vs Con_inf. Compared to the Con group, the SOD activity in the FCIA group increased significantly in the liver and spleen. Using high-throughput transcriptomics, DEGs were identified and 29 genes were verified using fluorescence real-time polymerase chain reaction (qRT-PCR). The result of DEGs clustering showed 9 samples in 3 groups of Con, FCIA and FCIA_inf were similar, contrast to distinct differences of 3 samples in the Con_inf group. We found 3795 up and 3548 down regulated DEGs in the compare of FCIA_inf vs Con_inf, of which 5 enriched KEGG pathways of "Lysosome", "Autophagy", "Apoptosis", "C-type lectin receptor signaling" and "Insulin signaling" were ascertained, and 26 of 30 top GO terms in the compare were significantly enriched. Finally, protein-protein interactions between the DEGs of the 5 KEGG pathways and other DEGs were explored using Cytoscape 3.9.1. The compare of FCIA_inf vs Con_inf showed 110 DEGs from the 5 pathways and 718 DEGs from other pathways formed total of 9747° in a network, of which 9 hub DEGs play vital roles in anti-infection or apoptosis. Together, the interaction networks revealed that 9 DEGs involved in the 5 pathways underlies the key process of A. anguilla anti-E. anguillarum infection or host cell apoptosis.

Comparative genomics of Edwardsiella anguillarum and Edwardsiella piscicida isolated in Taiwan enables the identification of distinctive features and potential virulence factors using Oxford-Nanopore MinION® sequencing.

Edwardsiella tarda (ET) and Edwardsiella anguillarum (EA) are the most harmful bacterial fish pathogens in Taiwan. However, there is confusion regarding the genotypic identification of E. tarda and E. piscicida (EP). Therefore, we used a novel Nanopore MinION MK1C platform to sequence and compare the complete genomes of E. piscicida and E. anguillarum. The number of coding genes, rRNA, and tRNA recorded for E. anguillarum and E. piscicida were 8322, 25, and 98, and 5458, 25, and 98, respectively. Ribosomal multilocus sequence typing (rMLST) for E. piscicida indicated 35 rps. The shared clusters between E. anguillarum and E. piscicida indicated several unique clusters for the individual genomes. The phylogenetic tree analysis for all complete genomes indicated that E. anguillarum and E. piscicida were placed into two species-specific genotypes. Distribution of subsystems for annotated genomes found that genes related to virulence, defence, and disease for E. anguillarum were 103 and those for E. piscicida were 60 and pathogenic islands (PI) were 498 and 225, respectively. Vaccine candidates were identified in silico from the core genes using high antigenic, solubility, and secretion probabilities. Altogether, the genome data revealed distinctive features between E. anguillarum and E. piscicida, which suggest different pathogenicity and thus the need for separate preventive strategies.

Evaluation of Edwardsiella piscicida basS and basR mutants as vaccine candidates in catfish against edwardsiellosis.

Catfish farming is the largest aquaculture industry in the United States and an important economic driver in several southeastern states. Edwardsiella piscicida is a Gram-negative pathogen associated with significant losses in catfish aquaculture. Several Gram-negative bacteria use the BasS/BasR two-component system (TCS) to adapt to environmental changes and the host immune system. Currently, the role of BasS/BasR system in E. piscicida virulence has not been characterized. In the present study, two mutants were constructed by deleting the basS and basR genes in E. piscicida strain C07-087. Both mutant strains were characterized for virulence and immune protection in catfish hosts. The EpΔbasS and EpΔbasR mutants were more sensitive to acidic environments and produced significantly less biofilm than the wild-type. In vivo studies in channel catfish (Ictalurus punctatus) revealed that both EpΔbasS and EpΔbasR were significantly attenuated compared with the parental wild-type (3.57% and 4.17% vs. 49.16% mortalities). Moreover, there was significant protection, 95.2% and 92.3% relative percent survival (RPS), in channel catfish vaccinated with EpΔbasS and EpΔbasR against E. piscicida infection. Protection in channel catfish was associated with a significantly higher level of antibodies and upregulation of immune-related genes (IgM, IL-8 and CD8-α) in channel catfish vaccinated with EpΔbasS and EpΔbasR strains compared with non-vaccinated fish. Hybrid catfish (channel catfish ♀ × blue catfish ♂) challenges demonstrated long-term protection against subsequent challenges with E. piscicida and E. ictaluri. Our findings demonstrate BasS and BasR contribute to acid tolerance and biofilm formation, which may facilitate E. piscicida survival in harsh environments. Further, our results show that EpΔbasS and EpΔbasR mutants were safe and protective in channel catfish fingerlings, although their virulence and efficacy in hybrid catfish warrant further investigation. These data provide information regarding an important mechanism of E. piscicida virulence, and it suggests EpΔbasS and EpΔbasR strains have potential as vaccines against this emergent catfish pathogen.

Phenotypic and genotypic analysis of Edwardsiella isolates from Taiwan indicates wide variation with a particular reference to Edwardsiella tarda and Edwardsiella anguillarum.

Edwardsiella spp. is a gram-negative, facultatively anaerobic, intracellular bacteria threatening the aquaculture industry worldwide. Noticeably, E. tarda is now genotypically classified into three distinct groups (E. tarda, E. piscicida and E. anguillarum), but morphologically, it is unclear due to varying degrees of virulence in different fish hosts. Hence, to reclassify E. tarda, we investigated differences in genotypes, phenotypes and pathogenicity. We collected Edwardsiella isolates from five different counties of Taiwan between 2017 and 2021. At first, gyrB gene was amplified for a phylogenetic tree from 40 isolates from different fish and one reference isolate, BCRC10670, from the human. Thirty-nine strains clustered into E. anguillarum, 1 strain into E. piscicida and 1 strain into E. tarda from human strain. Second, all isolates were characterized using various phenotypic (API 20E biochemical profiles) and genotypic (pulsed-field gel electrophoresis [PFGE], and virulence-related gene detection). SpeI digestion revealed 10 pulsotypes and I-CeuI into 7 pulsotypes. Virulent genes (citC, gadB, katB, mukF and fimA) confirmed in 35, 31, 28, 37 and 38 isolates, respectively. Finally, in vivo challenge test in milkfish (Chanos chanos) indicated the highest mortality from E. anguillarum. Overall, results revealed unique features with Edwardsiella spp. genotypes and pathogenicity, which are relevant to the host and provide useful insights for future vaccine development.

Comprehensive Relationship Analysis of the Long Noncoding RNAs (lncRNAs) and the Target mRNAs in Response to the Infection of Edwardsiella anguillarum in European eel (Anguilla anguilla) Inoculated with Freund's Adjuvant.

Freund's complete adjuvant (FCA) and incomplete adjuvant (FIA), generally applied in subunit fishery vaccine, have not been explored on the molecular mechanism of the non-specific immune enhancement. As long noncoding RNAs (lncRNAs) play vital regulating roles in various biological activities, in this study, we examined the genome-wide expression of transcripts in the liver of European eel (Anguilla anguilla, Aa) inoculated with FCA and FIA (FCIA) to elucidate the regulators of lncRNAs in the process of Edwardsiella anguillarum (Ea) infection and Aa anti-Ea infection using strand-specific RNA-seq. After eels were challenged by Ea at 28 days post the first inoculation (dpi), compared to the control uninfected eels (Li group), the control infected eels (Con_Li group) showed severe bleeding, hepatocyte atrophy, and thrombi formed in the hepatic vessels of the liver, although eels inoculated with FCIA (FCIA_Li group) also formed slight thrombi in the hepatic vessels. Compared to the FCIA_Li group, there was about 10 times colony-forming unit (cfu) in the Con_Li group per 100 µg liver tissue, and the relative percent survival (RPS) of eels was 50% in FCIA_Li vs Con_Li. Using high-throughput transcriptomics, differential expressed genes (DEGs) and transcripts were identified and the results were verified using fluorescence real-time polymerase chain reaction (qRT-PCR). Interactions between the differential expressed lncRNAs (DE-lncRNAs) and the target DEGs were explored using Cytoscape according to their co-expression and co-location relationship. We found 13,499 lncRNAs (10,176 annotated and 3423 novel lncRNAs) between 3 comparisons of Con_Li vs Li, FCIA_Li vs Li, and FCIA_Li vs Con_Li, of which 111, 110, and 129 DE-lncRNAs were ascertained. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs targeted by DE-lncRNAs revealed these DEGs mainly involved in single-organism cellular process in BP, membrane in CC and binding in MF, and KEGG pathways showed that the target DEGs in co-expression and co-location enriched in cell adhesion molecules. Finally, 118 DE-lncRNAs target 1161 DEGs were involved in an interaction network of 8474 co-expression and 333 co-location-related links, of which 16 DE-lncRNAs play vital roles in anti-Ea infection. Taken together, the interaction networks revealed that DE-lncRNAs underlies the process of Ea infection and Aa anti-Ea infection.

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.

Pathology and virulence of Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella anguillarum in channel (Ictalurus punctatus), blue (Ictalurus furcatus), and channel × blue hybrid catfish.

In the mid-2010s, Edwardsiella tarda was reaffiliated into three discrete taxa (E. anguillarum, E. piscicida, and E. tarda), obscuring previous descriptions of E. tarda-induced pathology in fish. To clarify ambiguity regarding the pathology of E. tarda, E. piscicida, and E. anguillarum infections in US farm-raised catfish, channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and channel × blue catfish hybrids were challenged with comparable doses of each bacterium. The most severe pathology and mortality occurred in fish challenged with E. piscicida, supporting previous reports of increased pathogenicity in commercially important ictalurids, while E. anguillarum and E. tarda warrant only minimal concern. Acute pathologic lesions among bacterial species were predominantly necrotizing and characteristic of gram-negative sepsis but became progressively granulomatous over time. After 100 days, survivors were exposed to the approximate median lethal doses of E. piscicida and E. ictaluri, revealing some cross-protective effects among E. piscicida, E. anguillarum, and E. ictaluri. In contrast, no fish that survived E. tarda challenge demonstrated any protection against E. piscicida or E. ictaluri. This work supports reports of increased susceptibility of channel, blue, and hybrid catfish to E. piscicida, while highlighting potential cross-protective affects among fish associated Edwardsiella spp.

Therapeutic efficacies of two newly isolated Edwardsiella phages against Edwardsiella piscicida infection.

The spread of multi-drug resistant (MDR) bacteria has posed a threat to the development of aquaculture. Due to its effective bactericidal ability, phage therapy has been considered as an alternative to antibiotics to reduce infection caused by MDR bacteria. In this study, two Edwardsiella piscicida phages were newly-isolated and characterized to prevent or treat infection in aquaculture. The phages were designated as vB_EpM_ZHS and vB_EpP_ZHX belonging to Myoviridae and Podoviridae families, respectively, in terms of genome sequence and morphology analyses. The combination of vB_EpM_ZHS and vB_EpP_ZHX improved the therapeutic efficacy both in vitro and in vivo. The phage cocktail significantly inhibited bacterial growth in vitro and decreased approximately 40% of mortality rate and an order of magnitude of bacterial burden in zebrafish and turbot infected by E. piscicida. Moreover, the phage cocktail increased transcription levels of tumor necrosis factor-alpha (TNF-α), interleukin-12 (IL-12), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) and alleviated inflammatory levels in the hindgut and spleen of turbots. The results indicate that the phage has a promising potential for therapeutic use against E. piscicida as the antimicrobial alternative to antibiotics in aquaculture.