T3SS protein EsrC binds to the lacI-like operator of type 1 fimbrial operon to suppress adhesion of Edwardsiella piscicida.

Type 1 fimbria, the short hair-like appendage assembled on the bacterial surface, plays a pivotal role in adhesion and invasion in Edwardsiella piscicida. The type III secretion system (T3SS), another bacterial surface appendage, facilitates E. piscicida's replication in vivo by delivering effectors into host cells. Our previous research demonstrated that E. piscicida T3SS protein EseJ inhibits adhesion and invasion of E. piscicida by suppressing type 1 fimbria. However, how EseJ suppresses type 1 fimbria remains elusive. In this study, a lacI-like operator (nt -245 to -1 of fimA) upstream of type 1 fimbrial operon in E. piscicida was identified, and EseJ inhibits type 1 fimbria through the lacI-like operator. Moreover, through DNA pull-down and electrophoretic mobility shift assay, an AraC-type T3SS regulator, EsrC, was screened and verified to bind to nt -145 to -126 and nt -50 to -1 of fimA, suppressing type 1 fimbria. EseJ is almost abolished upon the depletion of EsrC. EsrC and EseJ impede type 1 fimbria expression. Intriguingly, nutrition and microbiota-derived indole activate type 1 fimbria through downregulating T3SS, alleviating EsrC or EseJ's inhibitory effect on lacI-like operator of type 1 fimbrial operon. By this study, it is revealed that upon entering the gastrointestinal tract, rich nutrients and indole downregulate T3SS and thereof upregulate type 1 fimbria, stimulating efficient adhesion and invasion; upon being internalized into epithelium, the limit in indole and nutrition switches on T3SS and thereof switches off type 1 fimbria, facilitating effector delivery to guarantee E. piscicida's survival/replication in vivo.IMPORTANCEIn this work, we identified the lacI-like operator of type 1 fimbrial operon in E. piscicida, which was suppressed by the repressors-T3SS protein EseJ and EsrC. We unveiled that E. piscicida upregulates type 1 fimbria upon sensing rich nutrition and the microbiota-derived indole, thereof promoting the adhesion of E. piscicida. The increase of indole and nutrition promotes type 1 fimbria by downregulating T3SS. The decrease in EseJ and EsrC alleviates their suppression on type 1 fimbria, and vice versa.

Accumulation and Phagocytosis of Fluorescently Visualized Macrophages Against Edwardsiella piscicida Infection in Established mpeg1.1-Transgenic Japanese Medaka Oryzias latipes.

Intracellular bacteria such as those belonging to the genus Edwardsiella can survive and proliferate within macrophages. However, the detailed mechanisms underlying the host macrophage immune response and pathogen evasion strategies remain unknown. To advance the field of host macrophage research, we successfully established transgenic (Tg) Japanese medaka Oryzias latipes that possesses fluorescently visualized macrophages. As a macrophage marker, the macrophage-expressed gene 1.1 (mpeg1.1) was selected because of its predominant expression across various tissues in medaka. To validate the macrophage characteristics of the fluorescently labeled cells, May-Grünwald Giemsa staining and peroxidase staining were conducted. The labeled cells exhibited morphological features consistent with those of monocyte/macrophage-like cells and tested negative for peroxidase activity. Through co-localization studies, the fluorescently labeled cells co-localized with E. piscicida in the intestines and kidneys of infected medaka larvae, confirming the ingestion of bacteria through phagocytosis. In addition, the labeled cells expressed macrophage markers but lacked a neutrophil marker. These results suggested that the fluorescently labeled cells of Tg[mpeg1.1:mCherry/mAG] medaka were monocytes/macrophages, which will be useful for future studies aimed at understanding the mechanisms of macrophage-mediated bacterial infections.

Long-chain unsaturated fatty acids sensor controlling the type III/VI secretion system is essential for Edwardsiella piscicida infection.

Edwardsiella piscicida is an acute marine pathogen that causes severe damage to the aquaculture industry worldwide. The pathogenesis of E. piscicida is dependent mainly on the type III secretion system (T3SS) and type VI secretion system (T6SS), both of which are critically regulated by EsrB and EsrC. In this study, we revealed that fatty acids influence T3SS expression. Unsaturated fatty acids (UFAs), but not saturated fatty acids (SFAs), directly interact with EsrC, which abolishes the function of EsrC and results in the turn-off of T3/T6SS. Moreover, during the in vivo colonization of E. piscicida, host fatty acids were observed to be transported into E. piscicida through FadL and to modulate the expression of T3/T6SS. Furthermore, the esrCR38G mutant blocked the interaction between EsrC and UFAs, leading to dramatic growth defects in DMEM and impaired colonization in HeLa cells and zebrafish. In conclusion, this study revealed that the interaction between UFAs and EsrC to turn off T3/T6SS expression is essential for E. piscicida infection.

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.

Fur-mediated regulation of hydrogen sulfide synthesis, stress response, and virulence in Edwardsiella piscicida.

The production of endogenous hydrogen sulfide (H2S) is an important phenotype of bacteria. H2S plays an important role in bacterial resistance to ROS and antibiotics, which significantly contributes to bacterial pathogenicity. Edwardsiella piscicida, the Gram-negative pathogen causing fish edwardsiellosis, has been documented to produce hydrogen sulfide. In the study, we revealed that Ferric uptake regulator (Fur) controlled H2S synthesis by activating the expression of phsABC operon. Besides, Fur participated in the bacterial defense against ROS and cationic antimicrobial peptides and modulated T3SS expression. Furthermore, the disruption of fur exhibited a significant in vivo colonization defect. Collectively, our study demonstrated the regulation of Fur in H2S synthesis, stress response, and virulence, providing a new perspective for better understanding the pathogenesis of Edwardsiella.

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 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.

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.

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.

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.

A genome-wide association study of the occurrence of genetic variations in Edwardsiella piscicida, Vibrio harveyi, and Streptococcus parauberis under stressed environments.

Bacterial mutation and genetic diversity in aquaculture have led to increasing phenotypic variances, which can weaken or invalidate strategies for controlling diseases. However, few studies have monitored the degree of mutation in fish bacterial pathogens caused by environmental pressure within a short period. In this study, transcriptomic sequences from Edwardsiella piscicida, Vibrio harveyi and Streptococcus parauberis under stressed environments were used for investigating the emergence of variants. In detail, a sub-inhibitory concentration of formalin and phenol for E. piscicida, sea water at 30°C for V. harveyi and flounder serum for S. parauberis were used as stressed environments, and significant single-nucleotide polymorphisms (SNPs) and/or mutation sites were investigated after culture in the ordinary liquid media (control) and the stressed environment through a genome-wide association study. As results, several SNPs or mutations during incubation were observed under different environments in E. piscicida and/or V. harveyi in the genes relevant to flagella, fimbria type 3 secretion systems, and outer and inner membranes that have been directly exposed to external environments. In particular, given that flagella and fimbriae are considered important factors in differentiating the serotypes in some bacterial pathogens, it can be speculated that different environmental pressures are the source of phenotypic or serotypic differentiation from the same origin. On the other hands, S. parauberis did not exhibit notable changes for 4 h when inoculated in the serum from olive flounder. The results presented in this study provide examples of possible molecular evolution in pathogens relevant to the aquaculture industry as a response to different environmental pressure.

Involvement of N-acetylneuraminate cytidylyltransferase in Edwardsiella piscicida pathogenicity.

Edwardsiella piscicida is a gram-negative bacterium that causes Edwardsiellosis in cultured fish. Edwardsiellosis is accompanied by symptoms such as skin lesions, hemorrhage, and necrosis in fish organs, which leads to significant economic losses in the aquaculture industry. Recently, we found that bacterial sialoglycoconjugates may be involved in the infectivity of E. piscicida. The more infectious strains of E. piscicida contain more sialic acid in the bacterial body, and the mRNA level of putative CMP-Neu5Ac synthase (css) is upregulated compared to that in the non-pathogenic strain. However, this putative css gene is yet to be cloned, and the involvement of CSS in E. piscicida pathogenicity remains unclear. Here, we cloned and transferred the css gene from E. piscicida into the FPC498 strain. CSS promoted infection in cultured cells originating from different fish species, and enhanced the mortality of E. piscicida-infected zebrafish larvae. CSS enhanced cell attachment and motility in E. piscicida, which differs from the decreased bacterial growth observed with the sialic acid-supplemented M9 medium. Both fractions (chloroform-methanol)-soluble and -insoluble fraction) prepared from E. piscicida pellet exhibited the increment of sialo-conjugates induced by CSS. Further, lectin blotting revealed the increment of Sia α2-3- and α2-6-, but not α2-8-, -linked glycoprotein in CSS-overexpressing E. piscicida. Overall, these findings indicate the physiological significance of CSS and the role of sialylation in E. piscicida pathogenicity.

PurA facilitates Edwardsiella piscicida to escape NF-κB signaling activation.

The host NF-κB signaling pathway plays critical role in defensing against bacterial infection. However, bacteria also evolve strategies to escape from host clearance. Edwardsiella piscicida is a threatening pathogen in aquaculture, while the molecular mechanism of E. piscicida in inhibiting NF-κB signaling remains largely unknown. Herein, using E. piscicida transposon insertion mutant library combined with a NF-κB luciferase reporter system, we identified forty-six genes of E. piscicida, which were involved in inhibiting the NF-κB signaling activation in vitro. Moreover, we further explored the top 10 significantly changed mutants through zebrafish larvae infection model and validated that six genes were involved in inhibiting NF-κB activation in vivo. Specifically, we identified the adenylosuccinate synthase mutated strain (ΔpurA) infection exhibited a robust activation of NF-κB signaling, along with higher expression of cxcl8a and cxcl8b to mediate the recruitment of neutrophils in vivo. Taken together, these results identified the key factors of E. piscicida in inhibiting NF-κB activation, which will contribute to better understanding the pathogenesis of this important pathogen.

Multiplex PCR assay for correct identification of the fish pathogenic species of Edwardsiella genus reveals the presence of E. anguillarum in South America in strains previously characterized as E. tarda.

AIMS: Develop a species-specific multiplex PCR to correctly identify Edwardsiella species in routine diagnostic for fish bacterial diseases. METHODS AND RESULTS: The genomes of 62 Edwardsiella spp. isolates available from the National Center for Biotechnology Information (NCBI) database were subjected to taxonomic and pan-genomic analyses to identify unique regions that could be exploited by species-specific PCR. The designed primers were tested against isolated Edwardsiella spp. strains, revealing errors in commercial biochemical tests for bacterial classification regarding Edwardsiella species. CONCLUSION: Some of the genomes of Edwardsiella spp. in the NCBI platform were incorrectly classified, which can lead to errors in some research. A functional mPCR was developed to differentiate between phenotypically and genetically ambiguous Edwardsiella, with which, we detected the presence of Edwardsiella anguillarum affecting fish in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the misclassification of Edwardsiella spp in Brazil concealed the presence of E. anguillarum in South America. Also, this review of the taxonomic classification of the Edwardsiella genus is a contribution to the field to help researchers with their sequencing and identification of genomes, showing some misclassifications in online databases that must be corrected, as well as developing an easy assay to characterize Edwardsiella species in an end-point mPCR.

Xenogeneic nucleoid-associated EnrR thwarts H-NS silencing of bacterial virulence with unique DNA binding.

Type III and type VI secretion systems (T3/T6SS) are encoded in horizontally acquired genomic islands (GIs) that play crucial roles in evolution and virulence in bacterial pathogens. T3/T6SS expression is subjected to tight control by the host xenogeneic silencer H-NS, but how this mechanism is counteracted remains to be illuminated. Here, we report that xenogeneic nucleoid-associated protein EnrR encoded in a GI is essential for virulence in pathogenic bacteria Edwardsiella and Salmonella. We showed that EnrR plays critical roles in T3/T6SS expression in these bacteria. Various biochemical and genetic analyses demonstrated that EnrR binds and derepresses the promoter of esrB, the critical regulator of T3/T6SS, to promote their expression by competing with H-NS. Additionally, EnrR targets AT-rich regions, globally modulates the expression of ∼363 genes and is involved in various cellular processes. Crystal structures of EnrR in complex with a specific AT-rich palindromic DNA revealed a new DNA-binding mode that involves conserved HTH-mediated interactions with the major groove and contacts of its N-terminal extension to the minor groove in the symmetry-related duplex. Collectively, these data demonstrate that EnrR is a virulence activator that can antagonize H-NS, highlighting a unique mechanism by which bacterial xenogeneic regulators recognize and regulate foreign DNA.

CRISPR/Cas9-mediated generation of auxotrophic Edwardsiella piscicida mutants and immunization in olive flounder (Paralichthys olivaceus).

Edwardsiella piscicida has been a cause of mass mortality in cultured fish. In this study, to produce auxotrophic E. piscicida mutants, a CRISPR/Cas9 system was used instead of the traditional sacB-based allelic exchange method. Under the optimal CRISPR engineering condition, we could efficiently produce either alr or asd gene knockout E. piscicida auxotrophic mutants, and this genome editing process was much simpler and faster than the allelic exchange method. The simultaneous knockout of double auxotrophic genes (alr and asd) and the insertion of a foreign gene expression cassette in E. piscicida chromosome were also successfully performed using the established CRISPR/Cas9 system. Furthermore, to enhance the possibility to get permission as a commercial vaccine, we produced an auxotrophic E. piscicida mutant having only one nucleotide-deleted alr gene (E. piscicida △alr-1). Olive flounder (Paralichthys olivaceus) fingerlings immunized with 1 × 106 and 1 × 105 CFU/fish of E. piscicida △alr-1 showed the superior ability in the induction of serum agglutination activity and in the protection against E. piscicida compared to killed E. piscicida. However, olive flounder immunized with 1 × 107 CFU/fish of E. piscicida △alr-1 showed high mortality far before the challenge, and the isolated E. piscicida from moribund and dead fish had the wild type alr gene, suggesting the reversion of one base-deleted alr gene to original form by a second mutation in olive flounder. Therefore, investigation on the minimum number of edited nucleotide for stable maintenance of E. piscicida mutants should be further conducted.

Characterization of Edwardsiella piscicida CK108 flagellin genes and evaluation of their potential as vaccine targets in the zebrafish model.

The control of bacterial pathogens, including Edwardsiella piscicida, in the aquaculture industry has high economic importance. This study aimed to identify a potential live vaccine candidate against E. piscicida infection to minimize the side effects and elicit immunity in the host. This study evaluated the virulence factors of E. piscicida CK108, with a special focus on the flagella. E. piscicida has two important homologous flagellin genes, namely flagellin-associated protein (fap) and flagellin domain-containing protein (fdp). CK226 (Δfap), CK247 (Δfdp) and CK248 (Δfap, fdp) mutant strains were constructed. Both CK226 and CK247 displayed decreased length and thickness of flagellar filaments, resulting in reduced bacterial swimming motility, while CK248 was non-motile as it lacked flagella. The loss of flagella and decreased motility was expected to decrease the pathogenicity of CK248. However, the median lethal dose (LD50 ) of CK248 against zebrafish was lower than those of the wild-type, CK226 and CK247 strains. The protective immunity and cytokine gene expression levels in the CK248-infected zebrafish were lower than those in the wild type-infected zebrafish. In conclusion, Fap and Fdp are essential for flagella formation and motility, and for stimulating fish immune response, which can be utilized as a potential adjuvants for E. piscicida vaccination.

Versatile lifestyles of Edwardsiella: Free-living, pathogen, and core bacterium of the aquatic resistome.

Edwardsiella species in aquatic environments exist either as individual planktonic cells or in communal biofilms. These organisms encounter multiple stresses, include changes in salinity, pH, temperature, and nutrients. Pathogenic species such as E. piscicida, can multiply within the fish hosts. Additionally, Edwardsiella species (E. tarda), can carry antibiotic resistance genes (ARGs) on chromosomes and/or plasmids, that can be transmitted to the microbiome via horizontal gene transfer. E. tarda serves as a core in the aquatic resistome. Edwardsiela uses molecular switches (RpoS and EsrB) to control gene expression for survival in different environments. We speculate that free-living Edwardsiella can transition to host-living and vice versa, using similar molecular switches. Understanding such transitions can help us understand how other similar aquatic bacteria switch from free-living to become pathogens. This knowledge can be used to devise ways to slow down the spread of ARGs and prevent disease outbreaks in aquaculture and clinical settings.

Interplay between ferric uptake regulator Fur and horizontally acquired virulence regulator EsrB coordinates virulence gene expression in Edwardsiella piscicida.

Edwardsiella piscicida mediates hemorrhagic septicemia and is a leading pathogen of fish. E. piscicida invades and colonizes macrophages using type III and VI secretion systems (T3/T6SS) that are controlled by a two-component system (TCS) EsrA-EsrB. Iron acquisition is essential for E. piscicida pathogenesis and coordination between iron and TCS signaling in modulating bacterial virulence is not well understood. Here, we show that iron uptake systems are co-regulated by ferric uptake regulator (Fur) in E. piscicida. Fur bound to 98 genes that harbored conserved Fur-box to globally control the expression of ∼755 genes, including those encoding iron uptake systems, T3/T6SS, and Icc, cAMP phosphodiesterase that represses biofilm formation. Additionally, Fur, in complex with iron, bound to the esrB promoter to repress expression and ultimately attenuated virulence. Conversely, EsrB activated the expression of T3/T6SS and iron uptake systems to mitigate a shortage of intracellular iron during iron scarcity. Furthermore, EsrB directly bound to and activated the fur promoter, leading to Fur-ferrous ion-dependent esrB repression in the presence of iron. Finally, Fur-EsrB interplay was essential for bacterial fitness during in vivo infection and survival in seawater environments. Collectively, we highlight the mechanisms that underlie the reciprocal regulatory networks of iron homeostasis and virulence systems in E. piscicida.