Role of CD4+ and CD8α+ T cells in protective immunity against Edwardsiella tarda infection of ginbuna crucian carp,Carassius auratus langsdorfii

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Our previous study suggests that cell-mediated immunity (CMI) plays an essential role in protection against E. tarda infection. In the present study, we adoptively transferred T-cell subsets sensitized with E. tarda to isogenic naïve ginbuna crucian carp to determination the T-cell subsets involved in protecting fish from E. tarda infection. Recipients of CD4⁺ and CD8α⁺ cells acquired significant resistance to infection with E. tarda 8 days after sensitization, indicating that helper T cells and cytotoxic T lymphocytes plays crucial roles in protective immunity to E. tarda. Moreover, transfer of sensitized CD8α⁺ cells up-regulated the expression of genes encoding interferon-γ (IFN-γ) and perforin, suggesting that protective immunity to E. tarda involves cell-mediated cytotoxicity and interferon-γ-mediated induction of CMI. The results establish that CMI plays a crucial role in immunity against E. tarda. These findings provide novel insights into understanding the role of CMI to intracellular pathogens of fish.     

Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda,a potential vaccine candidate for fish,common carp

Outer membrane protein A (OmpA) is a component of the outer membrane of Edwardsiella tarda and is wildly distributed in Enterobacteriaceae family. The gene encoding the OmpA protein was cloned from E. tarda and expressed in Escherichia coli M15 cells. The recombinant OmpA protein containing His₆ residues was estimated to have a molecular weight of ∼38 kDa. In Western blot the native protein showed expression at ∼36 kDa molecular weight which was within the range of major outer membrane proteins (36–44 kDa) observed in this study. All E. tarda isolates tested harbored the ompA gene and the antibody raised to this protein was seen to cross react with other Gram negative bacteria. The OmpA protein characterized in this study was observed to be highly immunogenic in both rabbit and fish. In Enzyme linked immunosorbent assay, rabbit antisera showed an antibody titer of 1: 128,000. Common carp vaccinated with recombinant OmpA protein elicited high antibody production and immunized fish showed a relative percentage survival of 54.3 on challenge.     

FliC,a Flagellin Protein,Is Essential for the Growth and Virulence of Fish Pathogen Edwardsiella tarda

Edwardsiella tarda is a flagellated Gram-negative bacterium which causes edwardsiellosis in fish. FliC, as a flagellar filament structural protein, is hypothesized to be involved in the pathogenesis of infection. In this study, a fliC in-frame deletion mutant of a virulent isolate of E. tarda was constructed through double crossover allelic exchange by means of the suicide vector pRE112, and its virulence-associated phenotypes and pathogenicity were tested. It was found that the deletion of fliC significantly decreased the diameter of flagella filaments. In addition, the mutant showed reduced pathogenicity to fish by increasing the LD₅₀ value for 100-fold compared to the wild-type strain, as well as showed impaired bacterial growth, reduced motility, decreased biofilm formation and reduced levels of virulence-associated protein secretion involved in the type III secretion system (TTSS). The phenotypic characteristics of the fliC deletion mutant uncovered in this investigation suggest that fliC plays an essential role in normal flagellum function, bacterial growth, protein secretion by TTSS and bacterial virulence.     

Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp

Outer membrane protein A (OmpA) is a component of the outer membrane of Edwardsiella tarda and is wildly distributed in Enterobacteriaceae family. The gene encoding the OmpA protein was cloned from E. tarda and expressed in Escherichia coli M15 cells. The recombinant OmpA protein containing His₆ residues was estimated to have a molecular weight of ∼38 kDa. In Western blot the native protein showed expression at ∼36 kDa molecular weight which was within the range of major outer membrane proteins (36–44 kDa) observed in this study. All E. tarda isolates tested harbored the ompA gene and the antibody raised to this protein was seen to cross react with other Gram negative bacteria. The OmpA protein characterized in this study was observed to be highly immunogenic in both rabbit and fish. In Enzyme linked immunosorbent assay, rabbit antisera showed an antibody titer of 1: 128,000. Common carp vaccinated with recombinant OmpA protein elicited high antibody production and immunized fish showed a relative percentage survival of 54.3 on challenge.     

Genome Sequence of the Versatile Fish Pathogen Edwardsiella tarda Provides Insights into its Adaptation to Broad Host Ranges and Intracellular Niches

Background

Edwardsiella tarda is the etiologic agent of edwardsiellosis, a devastating fish disease prevailing in worldwide aquaculture industries. Here we describe the complete genome of E. tarda, EIB202, a highly virulent and multi-drug resistant isolate in China.

Methodology/Principal Findings

E. tarda EIB202 possesses a single chromosome of 3,760,463 base pairs containing 3,486 predicted protein coding sequences, 8 ribosomal rRNA operons, and 95 tRNA genes, and a 43,703 bp conjugative plasmid harboring multi-drug resistant determinants and encoding type IV A secretion system components. We identified a full spectrum of genetic properties related to its genome plasticity such as repeated sequences, insertion sequences, phage-like proteins, integrases, recombinases and genomic islands. In addition, analysis also indicated that a substantial proportion of the E. tarda genome might be devoted to the growth and survival under diverse conditions including intracellular niches, with a large number of aerobic or anaerobic respiration-associated proteins, signal transduction proteins as well as proteins involved in various stress adaptations. A pool of genes for secretion systems, pili formation, nonfimbrial adhesions, invasions and hemagglutinins, chondroitinases, hemolysins, iron scavenging systems as well as the incomplete flagellar biogenesis might feature its surface structures and pathogenesis in a fish body.

Conclusion/Significance

Genomic analysis of the bacterium offered insights into the phylogeny, metabolism, drug-resistance, stress adaptation, and virulence characteristics of this versatile pathogen, which constitutes an important first step in understanding the pathogenesis of E. tarda to facilitate construction of a practical effective vaccine used for combating fish edwardsiellosis.     

Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method

Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65°C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.     

Immune responses and expression profiles of some immune-related genes in Indian major carp,Labeo rohita to Edwardsiella tarda infection

Edwardsiella tarda is an important Gram-negative bacterium that causes systemic infections in a wide range of hosts including fish. The pathogenic mechanisms in this disease are still poorly understood in fish. Indian major carp, Labeo rohita were intraperitoneally challenged with a pathogenic isolate of E. tarda to measure sequential changes in immunity level. A significant decrease in the superoxide production, myeloperoxidase, alternative complement activity, total protein levels and antiprotease activity of serum was marked in the infected fish. However, the serum lysozyme activity and haemagglutination titre were raised in the infected fish. Similarly, a significant rise in specific antibody titre was noticed on and after 10 days post-challenge. This study also elucidates the changes in the relative expression of some immune-related genes viz., interleukin 1-beta (IL-1β), inducible nitric oxide synthase (iNOS), complement component C3, β₂-microglobulin, CXCa, tumor necrosis factor-alpha (TNFα), and C-type and G-type lysozymes during the infection. Significant up-regulation of IL-1β, iNOS, C3, CXCa and expression of both types of lysozyme genes was noticed at 6–12 h post-challenge (h.p.c.) whereas down-regulation of β₂-microglobulin and TNFα genes was observed after 48 h p.c. The results obtained here strengthen the understanding on molecular pathogenesis of edwardsiellosis in L. rohita.     

Precise feeding of probiotics in the treatment of edwardsiellosis by accurate estimation of <Emphasis Type="Italic">Edwardsiella tarda</Emphasis>

Edwardsiella tarda is one of the leading fish pathogens for the aquaculture industry. To realize efficient disease control of edwardsiellosis, a predictive model for E. tarda in seawater was developed. The modified logistic model was used to regress the growth curves of E. tarda JN at five different temperatures (range from 10 to 30 °C) and four organic nutrient concentrations (range from 5 to 40 mg l^?1 measured by chemical oxygen demand (COD)). The modeling effects of temperature and COD on the specific growth rate (μ) were developed by square-root model and saturation-growth rate model, respectively. The growth model was validated in turbot aquaculture tanks by estimating the dynamics of inoculated E. tarda. The accurate feeding of probiotic Bacillus pumilus strain H2 was calculated based on the estimation of E. tarda. Results showed that the logistic model produced a good fit to the growth curves of E. tarda JN (average R²?=?0.962). The overall predictions based on above models agreed well with the growth curve of E. tarda JN observed by plate counting in the validation tests (average Af?=?1.16; average Bf?=?1.32). The use of predicted amount of B. pumilus (5.66 log CFU ml^?1) successfully prevent the deterioration of disease for turbot with 13.3% mortality rate in a recirculating aquaculture system (RAS), while the feeding of 0 and 3.0 log CFU ml^?1 of B. pumilus resulted in 53.7 and 75.3% of turbot mortality rate, respectively. In conclusion, accurate estimation of E. tarda realized the precise feeding of probiotics, which successfully prevent the rapid progression of the edwardsiellosis.     

Characterization of Edwardsiella tarda waaL: roles in lipopolysaccharide biosynthesis, stress adaptation, and virulence toward fish

Edwardsiella tarda is the causative agent of edwardsiellosis in fish. The genome sequence of a virulent strain EIB202 has been determined. According to the genome sequence, the lipopolysaccharide (LPS) synthesis cluster containing a putative O-antigen ligase gene waaL was identified. Here, the in-frame deletion mutant ΔwaaL was constructed to analyze the function of WaaL in E. tarda EIB202. The ΔwaaL mutant displayed absence in O-antigen side chains in the LPS production. The ΔwaaL mutant exhibited an increased sensitivity to hydrogen peroxide indicating that the LPS was involved in the endurance to the oxidative stress in hosts during infection. In addition, the resistance of ΔwaaL to serum and polymyxin B decreased remarkably. The ΔwaaL mutant was also attenuated in virulence, showed an impaired ability in internalization of epithelioma papulosum cyprinid (EPC) cells and a comparatively poor ability of proliferation in vivo, which was in line with the increased LD₅₀ value. These results indicated that waaL gene was a functional member of the gene cluster involved in LPS synthesis and highlighted the importance of the O-antigen side chains to stress adaption and virulence in E. tarda, signifying the gene as a potential target for live attenuated vaccine against this bacterium.     

Phenotypic characterization, virulence, and immunogenicity of Edwardsiella tarda LSE40 aroA mutant

Bacterial aro mutants are frequently used as live attenuated vaccines for domestic animals. In this study, we characterized Edwardsiella tarda strain LSE40 with a deletion in the aroA gene. In addition to autotrophy, the aroA mutant appeared to have delayed cell division and reductions in its swarming motility, biofilm formation, and production of translocator proteins in the type III secretory system. The mutant exhibited high virulence attenuation in turbot fish, Scophthalmus maximus (L.), where the 50 % lethal dose increased by more than 3 log₁₀ via intraperitoneal (i.p.) injection and by >2 log₁₀ via immersion exposure compared with the wild-type parent strain. A tissue persistence study showed that the mutant retained the ability to invade and spread in turbot and viable cells could be detected up to 28 days after i.p. infection and 21 days after immersion exposure. These results suggested a pleiotropic role for aroA in the physiological behavior of E. tarda. Turbot exhibited a good humoral response and the enhanced expression of innate immune factors, interleukin 1β and lysozyme, when vaccinated with aroA mutant at 10⁵?CFU via i.p. injection and at 10⁸?CFU via immersion exposure. However, the aroA mutant did not provide effective protection for turbot against edwardsiellosis following i.p. vaccination at doses of 10⁴–10⁶?CFU or immersion vaccination at doses of 10⁶–10⁸?CFU?ml^?1. We hypothesized that the aroA mutant did not trigger an appropriate T cell-immune response in turbot against infection of E. tarda.     

Functional characterization of Edwardsiella tarda twin-arginine translocation system and its potential use as biological containment in live attenuated vaccine of marine fish

Bacterial twin-arginine translocation (Tat) system contributes to translocate folded proteins to the periplasm and plays pleiotropic roles in physiological fitness. Here, we showed that the fish pathogen Edwardsiella tarda Tat pathway was functional and was essential for H₂S production and hemolytic activity. E. tarda Tat mutant was more susceptible to diverse stresses such as high temperature, SDS, ethanol, and high-salt conditions. However, E. tarda Tat mutant displayed marginal in vivo virulence attenuation in fish models. Comparative proteomics analysis using two-dimensional gel electrophoresis (2-DGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry were performed to identify proteins undergoing changes in expression levels under high-salt conditons when the Tat pathway was mutilated. Of the 96 differently expressed proteins on the 2-DGE map, 15 proteins were successfully identified with a MASCOT score >45 (p?<?0.05) and fold change higher than 2. These significantly differentially expressed proteins were functionally related to basal metabolism and the biosynthesis of proteins and macromolecules. The results of plate counting further confirmed that the Tat mutant was high-salt-sensitive, indicating that Tat mutant merits as a novel salt-sensitive biological containment system for live attenuated vaccine (LAV) in marine fish vaccinology. To test this, we deleted the type III secretion system genes and cured endogenous plasmid pEIB202 to construct a LAV candidate in the context of Tat abrogation in E. tarda. The results indicated that the LAV candidate was highly attenuated when injected intraperitoneally and elicited significant protection against challenge of wild-type E. tarda in turbot while being rapidly eliminated in seawater.     

Analysis of Edwardsiella tarda DegP,a serine protease and a protective immunogen

Edwardsiella tarda is a severe aquaculture pathogen with a broad host range that includes humans, animal, and fish. A gene (degP_Et) encoding a DegP homologue was cloned from TX01, a pathogenic E. tarda strain isolated from diseased fish. DegP_Et shares high sequence identities with the DegP proteins of several bacterial species. Functional analyses showed that degP_Et could complement the temperature-sensitive phenotype of an Escherichia coli degP null mutant. Expression of degP_Et in TX01 was modulated by growth phase and temperature, the latter possibly through the action of the σ^E-like factor. Overexpression of degP_Et (i) enhanced the ability of TX01 to disseminate in fish blood at the advanced stage of infection, (ii) heightened the activity of type 2 autoinducer, and (iii) increased the expression of luxS and the genes encoding components of the virulence-associated type III secretion system. Recombinant DegP_Et purified from E. coli was a serine protease that exhibited maximum activity at 40 °C and pH8.0. The proteolytic activity of recombinant DegP_Et depended on the catalytic triad and the PDZ domains. Immunoprotective analyses showed that purified recombinant DegP_Et was a protective immunogen that could induce the production of specific serum antibodies and elicit strong protective immunity in fish vaccinated with DegP_Et.     

Asd-based balanced-lethal system in attenuated Edwardsiella tarda to express a heterologous antigen for a multivalent bacterial vaccine

Edwardsiella tarda is an enteric Gram-negative invasive intracellular pathogen, which causes enteric septicemia in fish. It could be potentially used to develop a recombinant attenuated E. tarda vaccine for the aquaculture industry. Because live vaccine strains can potentially be released into the environment upon vaccination, medical and environmental safety issues must be considered. Deletion of the asdB gene in E. tarda resulted in a diaminopimelic acid (DAP)-dependent mutant. The wild type asdB gene was inserted in place of the antibiotic-resistance gene in the plasmid, and the resultant non-antibiotic resistant vector was transformed into the attenuated and DAP-dependent E. tarda vaccine strain (WEDΔasdB) to obtain a balanced-lethal system for heterologous antigen expression. The balanced-lethal expression system was further optimized by comparing plasmid replicons with different Shine–Dalgarno sequences and start codons for the asdB gene. Utilizing the optimized balanced-lethal expression system, the protective antigen gene gapA34 from the fish pathogen Aeromonas hydrophila LSA34 was expressed in the attenuated E. tarda to generate the multivalent vaccine candidate WEDΔasdB/pUTta4DGap. This vaccine was shown to evoke an effective immune response against both E. tarda and A. hydrophila LSA34 by vaccinating turbot via a simple immersion route. This multivalent E. tarda vector vaccine has great potential for broad applications in aquaculture.     

OmpA: Gating and dynamics via molecular dynamics simulations

Outer membrane proteins (OMPs) of Gram-negative bacteria have a variety of functions including passive transport, active transport, catalysis, pathogenesis and signal transduction. Whilst the structures of ∼ 25 OMPs are currently known, there is relatively little known about their dynamics in different environments. The outer membrane protein, OmpA from Escherichia coli has been studied extensively in different environments both experimentally and computationally, and thus provides an ideal test case for the study of the dynamics and environmental interactions of outer membrane proteins. We review molecular dynamics simulations of OmpA and its homologues in a variety of different environments and discuss possible mechanisms of pore gating. The transmembrane domain of E. coli OmpA shows subtle differences in dynamics and interactions between a detergent micelle and a lipid bilayer environment. Simulations of the crystallographic unit cell reveal a micelle-like network of detergent molecules interacting with the protein monomers. Simulation and modelling studies emphasise the role of an electrostatic-switch mechanism in the pore-gating mechanism. Simulation studies have been extended to comparative models of OmpA homologues from Pseudomonas aeruginosa (OprF) and Pasteurella multocida (PmOmpA), the latter model including the periplasmic C-terminal domain.     

Identification of the interactome between fish plasma proteins and Edwardsiella tarda reveals tissue-specific strategies against bacterial infection

Elucidating the complex pathogen-host interaction is essential for a comprehensive understanding of how these remarkable agents invade their hosts and how the hosts defend against these invaders. During the infection, pathogens interact intensively with host to enable their survival, which can be revealed through their interactome. Edwardsiella tarda is a Gram-negative bacterial pathogen causing huge economic loss in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. E. tarda is an ideal model for host-pathogen investigation as it infects fish in three distinct steps: entering the host, circulating through the blood and establishing infection. We adopted a previous established proteomic approach that inactivated E. tarda cells and covalent crosslink fish plasma proteins were used to capture plasma proteins and bacterial outer membrane proteins, respectively. By the combinatorial use of proteomic and biochemical approaches, six plasma proteins and seven outer membrane proteins (OMPs) were identified. Interactions among these proteins were validated with protein-array, far-Western blotting and co-immunoprecipitation. At last, seventeen plasma protein-bacteria protein⿿protein interaction were confirmed to be involved in the interaction network, forming a complex interactome. Compared to our previous results, different host proteins were detected, whereas some of the bacterial proteins were similar, which indicates that hosts adopt tissue-specific strategies to cope with the same pathogen during infection. Thus, our results provide a robust demonstration of both bacterial initiators and host receptors or interacting proteins to further explore infection and anti-infective mechanisms between hosts and microbes.     

Effects of dietary supplementation of Lactobacillus pentosus PL11 on the growth performance,immune and antioxidant systems of Japanese eel Anguilla japonica challenged with Edwardsiella tarda

The aim of this study was to determine the efficacy of dietary administration of Lactobacillus pentosus PL11 on growth performance and the immune and antioxidant systems in Japanese eel Anguilla japonica challenged with Edwardsiella tarda. A total of 75 Japanese eels (24.63 ± 0.83 g) were grouped into 5 treatment diets which were a control diet (C) without E. tarda and 4 treatment diets with E. tarda challenge, including C for E. tarda challenge (NC), C plus L. pentosus PL11 supplemented diet (10⁸ cfu g^?1) (T-PL11), C plus L. pentosus KCCM 40997 supplemented diet (10⁸ cfu g^?1) (T-Lp) and C plus Weissella hellenica DS-12 supplemented diet (10⁸ cfu g^?1) (T-Wh) for 5 weeks (4 week before and 1 week after challenge). The results showed enhanced growth performance in fish fed the diet containing L. pentosus PL11 compared to others. The growth performance parameters including specific growth rate (SGR) and weight gain (WG), feed intake (FI), feed conversion ratio (FCR) and survival were significantly (P < 0.05) higher in fish maintained on L. pentosus PL11 supplemented diet compared to C and NC. T-PL11 group also shows a significant increase in the levels of plasma immunoglobulin M, CAT and SOD activities compared to NC. Hematological parameters and mieloperoxidase were significantly better in fish fed the L. pentosus PL11 supplemented diet than in the control. L. pentosus PL11 supplementation recover the reduced expression of SOD, CAT and heat shock protein 70 genes in liver and intestine in pathogen challenged fishes. In conclusion the result of the current study demonstrated L. pentosus PL11 potential as an alternative to antibiotic supplementation to improve the growth and health performance of Japanese eel (A. japonica).     

Direct antibacterial activity of CD8+/CD4+ T-cells in ginbuna crucian carp,Carassius auratus langsdorfii

Cytotoxic T cells (CTLs) constitute an important component of the specific effector mechanism in killing against microbial-infected or transformed cells. In addition to these activities, recent studies in mammals have suggested that CTLs can exhibit direct antimicrobial activity. Therefore, the present investigation was conducted to find out the microbicidal activity of CD8α⁺ T cells of ginbuna crucian carp, Carassius auratus langsdorfii. The CD8α⁺ T cells from immunised ginbuna exhibited the antibacterial activity against both facultative intracellular bacteria and extracellular bacteria. The maximum reduction of viable count of pathogens was recorded with effector (sensitized) cells and target (bacteria) ratio of 10:1 co-incubated for a period of 1–2 h at 26 °C when effector cells were derived from ginbuna 7 days after one booster dose at 15th day of primary sensitization/immunisation. Sensitized CD8α⁺ T cells are found to kill 92.1 and 98.9% of Lactococcus garvieae and Edwardsiella tarda, respectively. No significant difference in the bacterial killing activity could be recorded against facultative intracellular bacteria and extracellular bacteria. The specificity study indicated the non-specific killing of bacteria. CD8α⁺ T cells from E. tarda immunised ginbuna exhibited 40% of non-specific killing activity against L. garvieae and those from L. garvieae immunised ginbuna showed 42.7% of non-specific killing activity against E. tarda. Furthermore, CD4⁺ T cells also killed 88% and 95.7% of L. garvieae and E. tarda, respectively. In addition to T cell subsets, surface IgM⁺ cells also killed both types of pathogens. Therefore, the present study demonstrated the direct antibacterial activity of CD8α⁺, CD4⁺ T-cells and surface IgM⁺ cells in fish.     

Inv1: an Edwardsiella tarda invasin and a protective immunogen that is required for host infection

Invasin is an outer membrane protein that is known to mediate entry of enteric bacteria into mammalian cells. In this study, we analyzed the function and immunoprotective potential of the invasin Inv1 from Edwardsiella tarda, a serious fish pathogen that can also infect humans. In silico analysis indicated that Inv1 possesses a conserved N-terminal DUF3442 domain and a C-terminal group 1 bacterial Ig-like domain. Subcellular localization analysis showed that Inv1 is exposed on cell surface and could be recognized by specific antibodies. Mutation of inv1 had no effect on bacterial growth but attenuates overall bacterial virulence and impaired the ability of E. tarda to attach and invade into host cells. Consistent with these observations, antibody blocking of Inv1 inhibited E. tarda infection of host cells. To examine the immunoprotective potential of Inv1, recombinant Inv1 (rInv1) corresponding to the DUF3442 domain was purified and used to vaccinate Japanese flounder (Paralichthys olivaceus). The results showed that rInv1 induced strong protection against lethal-dose challenge of E. tarda. ELISA analysis showed that rInv1-vaccinated fish produced specific serum antibodies that could enhance the serum bactericidal activity against E. tarda. Taken together, these results indicate that Inv1 is a surface-localized virulence factor that is involved in host infection and can induce effective immunoprotection when used as a subunit vaccine.     

The trimeric periplasmic chaperone Skp of Escherichia coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions

The interactions of outer membrane proteins (OMPs) with the periplasmic chaperone Skp from Escherichia coli are not well understood. We have examined the binding of Skp to various OMPs of different origin, size, and function. These were OmpA, OmpG, and YaeT (Omp85) from Escherichia coli, the translocator domain of the autotransporter NalP from Neisseria meningitides, FomA from Fusobacterium nucleatum, and the voltage-dependent anion-selective channel, human isoform 1 (hVDAC1) from mitochondria. Binding of Skp was observed for bacterial OMPs, but neither for hVDAC1 nor for soluble bovine serum albumin. The Skp trimer formed 1:1 complexes, OMP·Skp₃, with bacterial OMPs, independent of their size or origin. The dissociation constants of these OMP·Skp₃ complexes were all in the nanomolar range, indicating that they are stable. Complexes of Skp₃ with YaeT displayed the smallest dissociation constants, complexes with NalP the largest. OMP binding to Skp₃ was pH-dependent and not observed when either Skp or OMPs were neutralized at very basic or very acidic pH. When the ionic strength was increased, the free energies of binding of Skp to OmpA or OmpG were reduced. Electrostatic interactions were therefore necessary for formation and stability of OMP·Skp₃ complexes. Light-scattering and circular dichroism experiments demonstrated that Skp₃ remained a stable trimer from pH 3 to pH 11. In the OmpA·Skp₃ complex, Skp efficiently shielded tryptophan residues of the transmembrane strands of OmpA against fluorescence quenching by aqueous acrylamide. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, bound to OmpA·Skp₃ complexes at low stoichiometries. Acrylamide quenching of fluorescence indicated that in this ternary complex, the tryptophan residues of the transmembrane domain of OmpA were located closer to the surface than in binary OmpA·Skp₃ complexes. This may explain previous observations that folding of Skp-bound OmpA into lipid bilayers is facilitated in presence of LPS.