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.

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.

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.

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.

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.

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.

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.

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.

Edwardsiella piscicida Interferes with Classical Endocytic Trafficking and Replicates in a Specialized Replication-Permissive Niche in Nonphagocytic Cells.

Edwardsiella piscicida is an intracellular pathogen within a broad spectrum of hosts. Essential to E. piscicida's virulence is its ability to invade and replicate inside host cells, yet the survival mechanisms and the nature of the replicative compartment remain unknown. Here, we characterized its intracellular lifestyle in nonphagocytic cells and showed that the intracellular replication of E. piscicida in nonphagocytic cells is dependent on its type III secretion system (T3SS) but not its type VI secretion system. Following internalization, E. piscicida is contained in vacuoles that transiently mature into early endosomes but subsequently bypasses the classical endosome pathway and fusion with lysosomes, which depend on its T3SS. Following rapid escape from the degradative pathway, E. piscicida was found to create a specialized replication-permissive niche characterized by endoplasmic reticulum (ER) markers. Furthermore, we found that a T3SS effector, EseJ, is responsible for the intracellular replication of E. piscicida by preventing endosome/lysosome fusion. In vivo experiments also confirmed that EseJ is necessary for bacterial colonization by E. piscicida in the epithelial layer, followed by systemic dissemination in both zebrafish and mice. Thus, this work elucidates the tactics used by E. piscicida to survive and proliferate within host nonphagocytic cells. IMPORTANCEE. piscicida is a facultative intracellular bacterium associated with septicemia and fatal infections in many animals, including fish and humans. However, little is known about its intracellular life, which is important for successful invasion of the host. The present study is the first comprehensive characterization of E. piscicida's intracellular lifestyle in host cells. Upon internalization, E. piscicida is transiently contained in Rab5-positive vacuoles, but the pathogen prevents further endosome maturation and fusion with lysosomes by utilizing a T3SS effector, EseJ. In addition, the bacterium creates a specialized replication niche for rapid growth via an interaction with the ER. Our study provides new insights into the strategies used by E. piscicida to successfully establish an intracellular lifestyle that contributes to its survival and dissemination during infection.

The role of UhpA in regulating the virulence gene expression in Edwardsiella piscicida.

Edwardsiella piscicida (E. piscicida) is an important fish pathogen. However, the mechanism of Glu6P transport regulatory protein UhpA how to affect the virulence gene expression in E. piscicida is still unclear. The results in this study showed that the metabolism-related gene expression of cysteine synthase (orf 1134) and sulphate transporter (ychM) in the uhpA mutant strain ΔuhpA was 0.76-fold and 0.68-fold lower than the ones in the wild strains (p < .05). The gene expression of ethA and ethB in the ΔuhpA strain was 0.80-fold and 0.72-fold lower than the ones in the wild strains (p < .05). However, the gene expression of fliC and flgN in the ΔuhpA was 1.51-fold and 1.21-fold higher than the ones in the wild strains (p < .05). The gene expression of T3SS (esrB and esrC) and T6SS (evpB and evpC) in the ΔuhpA was 1.27-fold, 1.13-fold, 1.28-fold and 1.23-fold higher than the ones in the wild strains (p < .05). This suggested that the uhpA gene could regulate the key virulence gene expression, and the uhpA gene was associated with the pathogenicity of E. piscicida in fish.

Genetic variability of Edwardsiella piscicida isolates from Mississippi catfish aquaculture with an assessment of virulence in channel and channel × blue hybrid catfish.

The bacterium Edwardsiella piscicida causes significant losses in global aquaculture, particularly channel (Ictalurus punctatus) × blue (I. furcatus) hybrid catfish cultured in the south-eastern United States. Emergence of E. piscicida in hybrid catfish is worrisome given current industry trends towards increased hybrid production. The project objectives were to assess intraspecific genetic variability of E. piscicida isolates recovered from diseased channel and hybrid catfish in Mississippi; and determine virulence associations among genetic variants. Repetitive extragenic palindromic sequence-based PCR (rep-PCR) using ERIC I and II primers was used to screen 158 E. piscicida diagnostic case isolates. A subsample of 39 E. piscicida isolates, representing predominant rep-PCR profiles, was further characterized using BOX and (GTG)5 rep-PCR primers, virulence gene assessment and multilocus sequence analysis (MLSA) targeting housekeeping genes gyrb, pgi and phoU. The MLSA provided greater resolution than rep-PCR, revealing 5 discrete phylogroups that correlated similarly with virulence gene profiles. Virulence assessments using E. piscicida representatives from each MLSA group resulted in 14-day cumulative mortality ranging from 22% to 54% and 63 to 72% in channel and hybrid fingerlings, respectively. Across all phylogroups, mortality was higher in hybrid catfish (p < .05), supporting previous work indicating E. piscicida is an emerging threat to hybrid catfish aquaculture in the south-eastern United States.

Genetic characterization of heterologous Edwardsiella piscicida isolates from diverse fish hosts and virulence assessment in a Chinook salmon Oncorhynchus tshawytscha model.

Edwardsiella piscicida is an emergent global fish pathogen with a wide host range, although host associations driving genetic diversity remain unclear. This study investigated the genetic and virulence diversity of 37 E. piscicida isolates recovered from 10 fish species in North America. Multilocus sequence analysis (MLSA) was conducted using concatenated alignments of the gyrB, pgi and phoU sequences. MLSA clustered the tested isolates into six discrete clades. In light of recent disease outbreaks in cultured salmonids, the virulence of each clade was evaluated in Chinook salmon Oncorhynchus tshawytscha fingerlings following intracoelomic challenge of ~106  CFU/fish. Challenged and control fish were monitored for 21d, and microbiological and histological examination was performed on dead and surviving fish. Peak mortality occurred 3-5 days post-challenge (dpc) regardless of isolate or genetic group. Edwardsiella piscicida was recovered from all moribund and dead animals. At 21 dpc, fish challenged with isolates from clades II, III and IV presented cumulative mortality ≥83.3%, whereas isolates from clade I, V and VI resulted in cumulative mortality ≤71.4%. This study suggests an underlying genetic basis for strain virulence and potential host associations. Further investigations using other fish models and variable challenge conditions are warranted.

Virulence and live vaccine potential of Edwardsiella piscicida phoP and phoQ mutants in catfish against edwardsiellosis.

Edwardsiella piscicida is a Gram-negative facultative intracellular bacterium causing edwardsiellosis in catfish, the largest aquaculture industry in the United States. A safe and effective vaccine is an urgent need to avoid economic losses associated with E. piscicida outbreaks. PhoP/PhoQ is a two-component signal transduction system (TCS) that plays an important role in bacterial pathogenesis through sense and response to environmental and host stress signals. This study aimed to explore the contribution of PhoQ/PhoP in E. piscicida virulence and develop live attenuated vaccines against E. piscicida infection in channel catfish (Ictalurus punctatus) and hybrid catfish (channel catfish ♀ × blue catfish (I. furcatus) ♂). In the current study, two in-frame deletion mutants were constructed by deleting phoP (ETAC_09785) and phoQ (ETAC_09790) genes in E. piscicida strain C07-087, and the virulence and protection efficacy of the constructed strains were evaluated in catfish following intraperitoneal injection. Both EpΔphoP and EpΔphoQ strains had a delayed adaptation to oxidative stress (0.2% H2 O2 ) compared to E. piscicida wild type. The EpΔphoP and EpΔphoQ mutants produced significantly less biofilm compared to wild-type E. piscicida. Notably, EpΔphoP and EpΔphoQ mutants were significantly attenuated in channel catfish compared with wild-type E. piscicida (6.63% and 4.17% versus 49.16% mortalities), and channel catfish vaccinated with EpΔphoP and EpΔphoQ were significantly protected (95.65% and 97.92% survival) against E. piscicida infection at 21 days post-vaccination. In hybrid catfish, EpΔphoP was significantly more attenuated than EpΔphoQ, but EpΔphoQ provided significantly better protection than EpΔphoP. EpΔphoP and EpΔphoQ strains both induced specific antibodies in channel catfish against E. piscicida at 14 and 21 days post-vaccination. This result indicated that EpΔphoP and EpΔphoQ mutants were safe and protective in channel catfish fingerlings, while EpΔphoP was safe in hybrid catfish. Our findings show that PhoP and PhoQ are required for adaptation to oxidative stress and biofilm formation and may help E. piscicida face tough environmental challenges; thus, functional PhoP and PhoQ are critical for a successful infection.

LuxS/AI-2 Quorum Sensing System in Edwardsiella piscicida Promotes Biofilm Formation and Pathogenicity.

LuxS/AI-2 is an important quorum sensing system which affects the growth, biofilm formation, virulence, and metabolism of bacteria. LuxS is encoded by the luxS gene, but how this gene is associated with a diverse array of physiological activities in Edwardsiella piscicida (E. piscicida) is not known. Here, we constructed an luxS gene mutant strain, the △luxS strain, to identify how LuxS/AI-2 affects pathogenicity. The results showed that LuxS was not found in the luxS gene mutant strain, and this gene deletion decreased E. piscicida growth compared to that of the wild-type strain. Meanwhile, the wild-type strain significantly increased penetration and motility in mucin compared to levels with the △luxS strain. The 50% lethal dose (LD50) of the E. piscicida △luxS strain for zebrafish was significantly higher than that of the wild-type strain, which suggested that the luxS gene deletion could attenuate the strain's virulence. The AI-2 activities of EIB202 were 56-fold higher than those in the △luxS strain, suggesting that the luxS gene promotes AI-2 production. Transcriptome results demonstrated that between cells infected with the △luxS strain and those infected with the wild-type strain 46 genes were significantly differentially regulated, which included 34 upregulated genes and 12 downregulated genes. Among these genes, the largest number were closely related to cell immunity and signaling systems. In addition, the biofilm formation ability of EIB202 was significantly higher than that of the △luxS strain. The supernatant of EIB202 increased the biofilm formation ability of the △luxS strain, which suggested that the luxS gene and its product LuxS enhanced biofilm formation in E. piscicida All results indicate that the LuxS/AI-2 quorum sensing system in E. piscicida promotes its pathogenicity through increasing a diverse array of physiological activities.

Critical role for a promoter discriminator in RpoS control of virulence in Edwardsiella piscicida.

Edwardsiella piscicida is a leading fish pathogen that causes significant economic loses in the aquaculture industry. The pathogen depends on type III and type VI secretion systems (T3/T6SS) for growth and virulence in fish and the expression of both systems is controlled by the EsrB transcription activator. Here, we performed a Tn-seq-based screen to uncover factors that govern esrB expression. Unexpectedly, we discovered that RpoS antagonizes esrB expression and thereby inhibits production of E. piscicida's T3/T6SS. Using in vitro transcription assays, we showed that RpoS can block RpoD-mediated transcription of esrB. ChIP-seq- and RNA-seq-based profiling, as well as mutational and biochemical analyses revealed that RpoS-repressed promoters contain a -6G in their respective discriminator sequences; moreover, this -6G proved critical for RpoS to inhibit esrB expression. Mutation of the RpoS R99 residue, an amino acid that molecular modeling predicts interacts with -6G in the esrB discriminator, abolished RpoS' capacity for repression. In a turbot model, an rpoS deletion mutant was attenuated early but not late in infection, whereas a mutant expressing RpoSR99A exhibited elevated fitness throughout the infection period. Collectively, these findings deepen our understanding of how RpoS can inhibit gene expression and demonstrate the temporal variation in the requirement for this sigma factor during infection.

An Edwardsiella piscicida esaS mutant reveals contribution to virulence and vaccine potential.

Edwardsiella piscicida is a Gram-negative pathogen that causes disease in diverse aquatic organisms. The disease leads to extensive losses in commercial aquaculture species, including farmed U.S. catfish. The type III secretion system (T3SS) often contributes to virulence of Gram-negative bacteria. The E. piscicida esaS gene encodes a predicted T3SS export apparatus protein. In the current study, an E. piscicida esaS mutant was constructed and characterized to increase our understanding of the role of T3SS in E. piscicida virulence. Deletion of esaS did not significantly affect biofilm formation and hemolytic activity of E. piscicida, but it had significant effects on expression of hemolysis and T3SS effector genes during biofilm growth. EpΔesaS showed significantly (P < 0.05) reduced virulence in catfish compared to the parent strain. No mortalities occurred in fish infected with EpΔesaS at 6.3 × 105 and 1.26 × 106 CFU/fish compared to 26% mortality in fish infected with wild-type E. piscicida at 7.5 × 105 CFU/fish. Bioluminescence imaging indicated that EpΔesaS invades catfish and colonizes for a short period in the organs. Furthermore, catfish immunized with EpΔesaS at 6.3 × 105 and 1.26 × 106 CFU provided 47% and 87% relative percent survival, respectively. These findings demonstrated that esaS plays a role in E. piscicida virulence, and the deletion mutant has vaccine potential for protection against wild-type E. piscicida infection.

Identification and study of InV as an inverse autotransporter family representative in Edwardsiella piscicida.

Invasins and intimins, members of virulence-related adhesin family which is involved in attachment and adherence to epithelial cells during infection, are found in various pathogens. These pathogens can attach to enterocytes and lead to the formation of a pedestal-like structure. Invasins and intimins belong to type Ve secretion systems, and the N-terminal ß-barrel domain acts as a translocation pore to secrete the C-terminal passenger domain. However, the relationship between invasins/intimins and type III secretion system (T3SS) has been poorly studied. Based on the transposon insertion mutant library of Edwardsiella piscicida, we got a transposon insertion mutant with significant T3SS defect and identified the mutated gene ETAE_0323 (named inV later). This gene encoded a protein with 2359 amino acid residues and was predicted to be an invasin. To study the relationship between InV and T3SS, strains with N-terminus or C-terminus deleted InV fragments were made. However, none of them was able to copy the phenotype of the transposon insertion mutant previously identified. The localization of InV in ΔT3SS strain was not significantly different from WT, suggesting that the T3SS defect in the transposon insertion mutant was likely to be caused by polar effect. Nevertheless, depletion of inV still showed dramatic internalization and virulence defect in HeLa cell and zebrafish model, respectively, suggesting InV as a virulence related protein.

The UhpA mutant of Edwardsiella piscicida enhanced its motility and the colonization in the intestine of tilapia.

Edwardsiella piscicida (E. piscicida) is a significant bacterial pathogen of cultured fish, which infected fish meanly through the intestine. Glucose 6-phosphate (Glu6P) in the intestine is nutritious to the pathogen, Meanwhile, Glu6P was found using as a virulent regulating signal for bacteria. The UhpA, one of the Glu6P transport system regulatory proteins could down-regulate the uhpC/uhpB/uhpA system and decrease its pathogenicity. However, the motility and the colonization of E. piscicida affected by UhpA were still unclear. In this study, the motility and the colonization of E. piscicida were monitored. The result demonstrated that the motility of EIB202 was significantly stronger than that of in ΔuhpA according to fractions 4, 8 and 9. However, the motility of ΔuhpA was significantly stronger than that of EIB202 according to the total number at the whole experiment. Although, there was no difference in the number of bacteria in the posterior intestine of tilapia after infected with E. piscicida EIB202 and ΔuhpA. The number of bacteria in the anterior and the middle intestine of fish infected with ΔuhpA were significantly higher than that of in fish infected with EIB202 at the whole experiment (P < 0.05). Interestingly, both E. piscicida strains colonized in the anterior intestine than that of in the middle and posterior intestines of tilapia. Besides, the gene expression of IL-1ß and TNF-α in the head-kidney of fish infected with ΔuhpA showed significantly higher (p < 0.05) than fish infected with EIB202 during the whole experimental period. Most importantly, the survival rate of E. piscicida EIB202 and ΔuhpA were 57% and 37% respectively. All results indicate that the uhpA gene mutant in E. piscicida could enhance its motility and the colonization in the intestine of tilapia, this illustrates the mechanism of UhpA decreases the pathogenesis of E. piscicida in fish.