Early Intracellular Trafficking and Subsequent Activity of Programmed Cell Death in Channel Catfish Macrophages Infected with Edwardsiella ictaluri.

The development of Edwardsiella-containing-vacuoles (ECV) and the ability of Edwardsiella ictaluri to survive and replicate within macrophages suggests a unique process relative to normal phagosomal/lysosomal maturation and programed cell death. Developing ECV showed that endosomal membrane markers Rab5, EEA1, and Rab7 were all detected in both the wild type (WT) and an E. ictaluri type-3 secretion system (T3SS) mutant, 65ST. Co-localization with Lamp1, however, was significantly lower in the WT. The host cell endoplasmic reticulum marker, calnexin, co-localized to 65ST ECV significantly more than WT ECV, while Golgi vesicle marker, giantin, was recruited to WT ECV significantly more than 65ST. The autophagosomal marker LC3 was significantly lower in WT than in 65ST and Western blotting demonstrated significantly greater induction of the membrane localized, lipidated form, LC3-II, in 65ST ECV than in WT ECV. Activity of the apoptosis initiator caspase-8 increased post-infection in 65ST and was significantly lower in WT-infected cells. Executioner caspase-3/7 activity also increased significantly in 65ST-infected cells compared to WT-infected cells. Repression of apoptosis was further demonstrated with flow cytometry using Alexa Fluor 647-labeled Annexin V and propidium iodide. Results indicate that WT ECV fused with early and late endosomes but that phagosomal/lysosomal fusion did not occur. Additionally, WT-infected cells recruited Golgi vesicles for vacuolar size increase and bacterial growth material, and both autophagy and apoptosis were repressed in the WT. This activity was all based on the function of the E. ictaluri T3SS.

Edwardsiella ictaluri type III secretion system (T3SS) effector EseN is a phosphothreonine lyase that inactivates ERK1/2.

EseN is a type III secretion system (T3SS) effector that is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase (PTL) activity, including OspF from Shigella and SpvC from Salmonella. A yeast-2-hybrid system was used to identify the major vault protein (MVP) as a specific host-cell binding partner for EseN, and the proximity ligation assay (PLA) confirmed the interaction. Similar to other pathogens, E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Structurally, EseN contains a highly conserved docking motif that is required for specific binding to mitogen-activated protein kinases, such as ERK1/2, and a motif that is essential for PTL activity. Immunoblotting and immunofluorescence analyses indicate that EseN inactivates ERK1/2 by dephosphorylation in vivo in the head kidney of infected fish and ex vivo in head kidney derived macrophages. Interaction of EseN with phosphorylated ERK1/2 (pERK1/2) was also confirmed using PLA, suggesting that MVP serves as a signaling scaffold for ERK1/2 and EseN. Channel catfish Ictalurus punctatus infected with E. ictaluri strains lacking the eseN gene had reduced numbers of E. ictaluri in the tissues following infection and reduced mortality compared to fish infected with the wild-type. Our results indicate that eseN encodes a PTL domain that interacts with MVP as a possible scaffold protein and inactivates pERK1/2 to ERK1/2, resulting in increased proliferation of E. ictaluri and, ultimately, death of the host.

Edwardsiella piscicida-like pathogen in cultured grouper.

An Edwardsiella sp. was isolated from the kidney of diseased groupers (Epinephelus aeneus and E. marginatus) cultured in Eilat (Israel, Red Sea). Affected fish presented a severe suppurative nephritis with large abscesses occasionally spreading into the surrounding musculature. Biochemical profiles and phenotypic comparisons failed to provide a clear identification to the species level, and genetic analysis of the 16S subunit failed to discriminate between Edwardsiella piscicida, E. tarda and E. ictaluri. Analysis of the gyrB gene, however, placed the grouper isolates into the E. piscicida-like group, a newly recognized taxon which also encompasses the non-motile strains previously classified as atypical E. tarda. Initial genomic analysis revealed the presence of the Edwardsiella type 3 secretion system (T3SS) but also revealed a pathogenicity island encoding a second T3SS with homology to the locus of enterocyte effacement of Escherichia coli. Further analysis revealed 3 different type 6 secretion systems that were also present in all sequenced isolates of Edwardsiella piscicida-like strains. Based on estimated DNA-DNA hybridization values and the average nucleotide index, the grouper strain fits into the E. piscicida-like phylogroup described as E. anguillarum sp. nov. The peculiarities associated with this isolate and the association of other conspecific piscine isolates from multiple marine and brackish water species suggest a link of the entire E. piscicida-like phylogroup to the marine environment.

Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System.

Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000 tons produced annually, and ESC is the leading cause of disease loss in the industry. We have demonstrated the survival and replication of E. ictaluri within channel catfish cells and identified a secretion system that is essential for E. ictaluri intracellular replication and virulence. We have also identified nine proteins encoded in the E. ictaluri genome that we believe are actively transferred from the bacterium to the cytoplasm of the host cell and act to manipulate host cell physiology to the advantage of the bacterium. The data presented here confirm that the proteins are actually transferred during an infection, which will lead to further work on approaches to preventing or controlling ESC.

Complete Genome Sequence of an Edwardsiella piscicida-Like Species Isolated from Diseased Grouper in Israel.

The Edwardsiella piscicida-like sp. is a Gram-negative facultative anaerobe that causes disease in some fish species. We report here the complete genome sequence of a virulent isolate from a diseased white grouper (Epinephelus aeneus) raised on the Red Sea in Israel, which contains a chromosome of 3,934,167 bp and no plasmids.

Modulation of vacuolar pH is required for replication of Edwardsiella ictaluri in channel catfish macrophages.

Previous in vitro work demonstrated that Edwardsiella ictaluri produces an acid-activated urease that can modulate environmental pH through the production of ammonia from urea. Additional work revealed that expression of the E. ictaluri type III secretion system (T3SS) is upregulated by acidic pH. Both the urease and the T3SS were previously shown to be essential to intracellular replication. In this work, fluorescence microscopy with LysoTracker Red DND-99 (LTR) indicated that E. ictaluri-containing vacuoles (ECV) became acidified following ingestion by head kidney-derived macrophages (HKDM). In vivo ratiometric imaging demonstrated a lowered ECV pH, which fell to as low as pH 4 but subsequently increased to pH 6 or greater. Inhibition of vacuolar H(+)-ATPases by use of the specific inhibitor bafilomycin A1 abrogated both ECV acidification and intracellular replication in HKDM. Failure of an E. ictaluri urease knockout mutant to increase the ECV pH in the in vivo ratiometric assay suggests that ammonia produced by the urease reaction mediates the pH increase. Additionally, when the specific arginase inhibitor l-norvaline was used to treat E. ictaluri-infected HKDM, the ECV failed to neutralize and E. ictaluri was unable to replicate. This indicates that the HKDM-encoded arginase enzyme produces the urea used by the E. ictaluri urease enzyme. Failure of the ECV to acidify would prevent both upregulation of the T3SS and activation of the urease enzyme, either of which would prevent E. ictaluri from replicating in HKDM. Failure of the ECV to neutralize would result in a vacuolar pH too low to support E. ictaluri replication.

Comparison of Vietnamese and US isolates of Edwardsiella ictaluri.

We compared Edwardsiella ictaluri from striped catfish in Vietnam with US channel catfish isolates. Biochemical analyses and sequencing of the 16S rRNA gene confirmed that the Vietnamese isolates were E. ictaluri. Comparison using rep-PCR fingerprinting demonstrated no significant differences between the isolates, but plasmid analysis indicated that the Vietnamese isolates grouped into 4 plasmid profiles, each different from the typical pEI1 and pEI2 plasmid profile found in the US isolates. Sequencing plasmids representative of the 4 profiles indicated that all contained derivatives of the E. ictaluri plasmid pEI1, whereas only 1 contained a plasmid derivative of the E. ictaluri plasmid pEI2. The pEI2 encoded type III secretion effector, EseI, and its chaperone, EscD, were found to be present on the chromosome in isolates lacking a pEI2 derivative. In addition, 1 isolate carried a 5023 bp plasmid that does not have homology to either pEI1 or pEI2. Furthermore, Vietnamese isolates were PCR positive for the type III and type VI secretion system genes esrC and evpC, respectively, and the urease enzyme, but were PCR-negative for the putative type IV secretion system gene virD4. A monoclonal antibody against the lipopolysaccharide of E. ictaluri ATCC 33202 did not react with the Asian isolates or with the more recent US isolates. Antibiotic resistance patterns were variable and did not correlate to the presence of any particular plasmid profile. Finally, the Vietnamese isolates were avirulent and had a significantly reduced capacity for intracellular replication within head-kidney-derived channel catfish macrophages.

Regulation of the Edwardsiella ictaluri type III secretion system by pH and phosphate concentration through EsrA, EsrB, and EsrC.

A recently described Edwardsiella ictaluri type III secretion system (T3SS) with functional similarity to the Salmonella pathogenicity island 2 T3SS is required for replication in channel catfish head-kidney-derived macrophages (HKDM) and virulence in channel catfish. Quantitative PCR and Western blotting identified low pH and phosphate limitation as conducive to expression of the E. ictaluri T3SS, growth conditions that mimic the phagosomal environment. Mutagenesis studies demonstrated that expression is under the control of the EsrAB two-component regulatory system. EsrB also induces upregulation of the AraC-type regulatory protein EsrC, which enhances expression of the EscB/EseG chaperone/effector operon in concert with EsrB and induces expression of the pEI1-encoded effector, EseH. EsrC also induces expression of a putative type VI secretion system translocon protein, EvpC, which is secreted under the same low-pH conditions as the T3SS translocon proteins. The pEI2-encoded effector, EseI, was upregulated under low-pH and low-phosphate conditions but not in an EsrB- or EsrC-dependent manner. Mutations of EsrA and EsrB both resulted in loss of the ability to replicate in HKDM and full attenuation in the channel catfish host. Mutation of EsrC did not affect intracellular replication but did result in attenuation in catfish. Although EsrB is the primary transcriptional regulator for E. ictaluri genes within the T3SS pathogenicity island, EsrC regulates expression of the plasmid-carried effector eseH and appears to mediate coordinated expression of the T6SS with the T3SS.

Edwardsiella ictaluri encodes an acid-activated urease that is required for intracellular replication in channel catfish (Ictalurus punctatus) macrophages.

Genomic analysis indicated that Edwardsiella ictaluri encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type E. ictaluri in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of wild-type E. ictaluri was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome.

Genomic organisation of the channel catfish Mx1 gene and characterisation of multiple channel catfish Mx gene promoters.

In order to further characterise channel catfish (Ictalurus punctatus) Mx1, studies were initiated to amplify and clone the Mx1 promoter into a reporter vector, pGL3basic. Initially the Mx1 gene was amplified from genomic DNA and was found to have 12 exons and 11 introns, spanning a region over 6 kilobases (kb) in length. The Mx1 promoter was amplified using genome walking and during this process four additional Mx promoters were identified, suggesting the presence of five Mx genes in the channel catfish. All five promoters possess an interferon stimulated response element (ISRE) and the Mx1 promoter possessed two potential NF-kappabeta transcription sites. Following cloning each construct was transiently transfected into COS-7 and EPC cells for 24h and treated with 5 microg/ml poly I:C for 24h. An increase in expression of the reporter gene in response to poly I:C was noted in both cell lines in the pGL3Mx1 construct only. However, the reporter gene was also constitutively expressed in these cells. Constitutive expression was also observed in channel catfish ovary cells transiently transfected with pGL3Mx1 only. Treatment with 5 microg/ml poly I:C did not increase this expression, which may be due to high levels of cell death in this difficult to transfect cell line. The constitutive expression observed implies that a repressor element is missing in the 390 base pair sequence of the Mx1 promoter used in this study. These results suggest that only channel catfish Mx1 is involved in the type I interferon pathway and that the presence of an ISRE in a regulatory region is not necessarily indicative of a role in the type I interferon response.

Signature-tagged mutagenesis of Edwardsiella ictaluri identifies virulence-related genes, including a salmonella pathogenicity island 2 class of type III secretion systems.

Edwardsiella ictaluri is the leading cause of mortality in channel catfish culture, but little is known about its pathogenesis. The use of signature-tagged mutagenesis in a waterborne infection model resulted in the identification of 50 mutants that were unable to infect/survive in catfish. Nineteen had minitransposon insertions in miscellaneous genes in the chromosome, 10 were in genes that matched to hypothetical proteins, and 13 were in genes that had no significant matches in the NCBI databases. Eight insertions were in genes encoding proteins associated with virulence in other pathogens, including three in genes involved in lipopolysaccharide biosynthesis, three in genes involved in type III secretion systems (TTSS), and two in genes involved in urease activity. With the use of a sequence from a lambda clone carrying several TTSS genes, Blastn analysis of the partially completed E. ictaluri genome identified a 26,135-bp pathogenicity island containing 33 genes of a TTSS with similarity to the Salmonella pathogenicity island 2 class of TTSS. The characterization of a TTSS apparatus mutant indicated that it retained its ability to invade catfish cell lines and macrophages but was defective in intracellular replication. The mutant also invaded catfish tissues in numbers equal to those of invading wild-type E. ictaluri bacteria but replicated poorly and was slowly cleared from the tissues, while the wild type increased in number.

Poly I:C induces an antiviral state against Ictalurid Herpesvirus 1 and Mx1 transcription in the channel catfish (Ictalurus punctatus).

In vivo studies were carried out to investigate the protective effect of the interferon inducer poly I:C against channel catfish virus (CCV). Channel catfish were stimulated by intraperitoneal injection of 50 microg of poly I:C or PBS at various days prior to immersion challenge with CCV. Mortality in the poly I:C group was significantly reduced from 70% to 3% at day 1 compared to the PBS controls. Mortality increased at day 3 but was still significantly different from the PBS controls. Mx1 transcription was significantly higher only at day 1. In an additional study Mx1 transcription was monitored in the liver, kidney, gills, spleen, and intestine at various time points post-stimulation with either poly I:C or CCV. Mx1 mRNA was significantly elevated in all organs only at day 1 post-injection with poly I:C. In response to CCV, Mx1 transcription was not significantly elevated until day 3 post-challenge, but remained elevated in certain organs until day 7.

Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene.

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C.

Construction of a safe, stable, efficacious vaccine against Photobacterium damselae ssp. piscicida.

Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.

Identification of a streptolysin S-associated gene cluster and its role in the pathogenesis of Streptococcus iniae disease.

Streptococcus iniae causes meningoencephalitis and death in cultured fish species and soft-tissue infection in humans. We recently reported that S. iniae is responsible for local tissue necrosis and bacteremia in a murine subcutaneous infection model. The ability to cause bacteremia in this model is associated with a genetic profile unique to strains responsible for disease in fish and humans (J. D. Fuller, D. J. Bast, V. Nizet, D. E. Low, and J. C. S. de Azavedo, Infect. Immun. 69:1994-2000, 2001). S. iniae produces a cytolysin that confers a hemolytic phenotype on blood agar media. In this study, we characterized the genomic region responsible for S. iniae cytolysin production and assessed its contribution to virulence. Transposon (Tn917) mutant libraries of commensal and disease-associated S. iniae strains were generated and screened for loss of hemolytic activity. Analysis of two nonhemolytic mutants identified a chromosomal locus comprising 9 genes with 73% homology to the group A streptococcus (GAS) sag operon for streptolysin S (SLS) biosynthesis. Confirmation that the S. iniae cytolysin is a functional homologue of SLS was achieved by PCR ligation mutagenesis, complementation of an SLS-negative GAS mutant, and use of the SLS inhibitor trypan blue. SLS-negative sagB mutants were compared to their wild-type S. iniae parent strains in the murine model and in human whole-blood killing assays. These studies demonstrated that S. iniae SLS expression is required for local tissue necrosis but does not contribute to the establishment of bacteremia or to resistance to phagocytic clearance.

Cloning and characterization of Edwardsiella ictaluri proteins expressed and recognized by the channel catfish Ictalurus punctatus immune response during infection.

An Edwardsiella ictaluri expression library was screened for clones expressing antigenic E. ictaluri proteins using anti-E. ictaluri serum, which resulted in the isolation of 32 clones. The clones were partially characterized and 4 were selected for complete analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-dimensional PAGE, Western blotting, and DNA sequencing were used to analyze expressed antigenic proteins and encoded genes. Sequence analysis identified 4 putative open reading frames (ORFs) in the insert of Clone 4d6, which corresponded to antigenic acidic proteins of 55, 20 and 18 kDa expressed by both the clone and E. ictaluri cells. The predicted gene products of these ORFs were similar to several products of the imp locus of Rhizobium leguminosarum bv. trifolii. The imp locus of R. leguminosarum contains 14 genes that encode proteins involved in a putative temperature-dependent protein secretion system. In addition there was significant amino acid identity for a variety of hypothetical proteins from R. solanacearum, Ps. aeruginosa, A. tumefaciens, Y. pestis, and Salmonella typhimurium. Overlapping inserts of Clones 1.4, 5d2, and 5d3 encoded ORFs similar to Escherichia coli partial genes serA and pgk, and complete genes rpiA, iciA, yggE, yggB and fda. These genes encode D-3-phosphoglycerate dehydrogenase (serA), ribose 5-phosphate isomerase (rpiA), a specific inhibitor of chromosomal initiation of replication (iciA), a hypothetical protein (yggE), a protein involved in responses to osmotic stress (yggB), fructose 1,6-bisphosphate aldolase (fda), and phosphoglycerate kinase (pgk). Cloned antigenic E. ictaluri proteins of 33, 27, 35 and 45 kDa appeared to be products of the ORFs similar to yggE, rpiA, iciA, and fda respectively. All the cloned antigenic proteins were recognized by antiserum from catfish that had recovered from enteric septicemia of catfish (ESC), indicating that these antigens are expressed during the infectious process. The cloned antigenic proteins were subsequently evaluated as subunit vaccines for protection against wild-type E. ictaluri. All vaccine treatments were protective against E. ictaluri in catfish, but results were inconclusive due to high levels of cross-reactive protection afforded by the E. coli host strain of the cloning vector.