ABSTRACT
OBJECTIVES: The pathogenic microorganisms that cause intestinal diseases can significantly jeopardize people's health. Currently, there are no authorized treatments or vaccinations available to combat the germs responsible for intestinal disease. METHODS: Using immunoinformatics, we developed a potent multi-epitope Combination (combo) vaccine versus Salmonella and enterohemorrhagic E. coli. The B and T cell epitopes were identified by performing a conservancy assessment, population coverage analysis, physicochemical attributes assessment, and secondary and tertiary structure assessment of the chosen antigenic polypeptide. The selection process for vaccine development included using several bioinformatics tools and approaches to finally choose two linear B-cell epitopes, five CTL epitopes, and two HTL epitopes. RESULTS: The vaccine had strong immunogenicity, cytokine production, immunological properties, non-toxicity, non-allergenicity, stability, and potential efficacy against infections. Disulfide bonding, codon modification, and computational cloning were also used to enhance the stability and efficacy of expression in the host E. coli. The vaccine's structure has a strong affinity for the TLR4 ligand and is very durable, as shown by molecular docking and molecular modeling. The results of the immunological simulation demonstrated that both B and T cells had a heightened response to the vaccination component. CONCLUSIONS: The comprehensive in silico analysis reveals that the proposed vaccine will likely elicit a robust immune response against pathogenic bacteria that cause intestinal diseases. Therefore, it is a promising option for further experimental testing.
Subject(s)
Epitopes, B-Lymphocyte , Epitopes, T-Lymphocyte , Vaccinology , Humans , Epitopes, T-Lymphocyte/immunology , Vaccinology/methods , Epitopes, B-Lymphocyte/immunology , Vaccines, Combined/immunology , Genomics/methods , Enterohemorrhagic Escherichia coli/immunology , Salmonella/immunology , Animals , Computational Biology/methods , Molecular Docking Simulation , Escherichia coli Vaccines/immunology , Escherichia coli Infections/prevention & control , Escherichia coli Infections/immunology , Salmonella Infections/immunology , Salmonella Infections/prevention & control , Antigens, Bacterial/immunology , Vaccine Development/methods , Bacterial Vaccines/immunologyABSTRACT
Inflammatory caspase-dependent cytosolic lipopolysaccharide (LPS) sensing is a critical arm of host defense against bacteria. How pathogens overcome this pathway to establish infections is largely unknown. Enterohemorrhagic Escherichia coli (EHEC) is a clinically important human pathogen causing hemorrhagic colitis and hemolytic uremic syndrome. We found that a bacteriophage-encoded virulence factor of EHEC, Shiga toxin (Stx), suppresses caspase-11-mediated activation of the cytosolic LPS sensing pathway. Stx was essential and sufficient to inhibit pyroptosis and interleukin-1 (IL-1) responses elicited specifically by cytosolic LPS. The catalytic activity of Stx was necessary for suppression of inflammasome responses. Stx impairment of inflammasome responses to cytosolic LPS occurs at the level of gasdermin D activation. Stx also suppresses inflammasome responses in vivo after LPS challenge and bacterial infection. Overall, this study assigns a previously undescribed inflammasome-subversive function to a well-known bacterial toxin, Stx, and reveals a new phage protein-based pathogen blockade of cytosolic immune surveillance.
Subject(s)
Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli Infections/immunology , Inflammasomes/immunology , Shiga Toxin 1/metabolism , Shiga Toxin 2/metabolism , Animals , Bacteriophages/immunology , Bacteriophages/metabolism , Caspases, Initiator/genetics , Caspases, Initiator/metabolism , Chlorocebus aethiops , Citrobacter rodentium/immunology , Citrobacter rodentium/pathogenicity , Disease Models, Animal , Enterohemorrhagic Escherichia coli/immunology , Enterohemorrhagic Escherichia coli/virology , Escherichia coli Infections/microbiology , Female , Humans , Immunologic Surveillance , Inflammasomes/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Lipopolysaccharides/immunology , Male , Mice , Mice, Knockout , Phosphate-Binding Proteins/metabolism , Shiga Toxin 1/immunology , Shiga Toxin 2/immunology , Vero Cells , Viral Proteins/immunology , Viral Proteins/metabolism , Virulence Factors/immunology , Virulence Factors/metabolismABSTRACT
BACKGROUND: For successful colonization, enterohaemorrhagic Escherichia coli (EHEC) injects virulence factors, called effectors, into target cells through the type three secretion system (T3SS), which is composed of a needle and basal body. Under anaerobic conditions, the T3SS machinery remains immature and does not have a needle structure. However, activation of nitrate respiration enhances the completion of the T3SS machinery. Because nitric oxide released by the host inflammatory response increases nitrate concentration, we sought to determine the effect of the inflammatory response on initiation of EHEC microcolony-formation. RESULTS: The colony-forming capacity was increased in accordance with the increase of nitrate in the medium. The addition of the nitric oxide-producing agent NOR-4 also enhanced the adherence capacity, which was dependent on nitrate reductase encoded by the narGHJI genes. Culture supernatant of epithelial cells, which was stimulated by a cytokine mixture, enhanced the colony-forming capacity of wild-type EHEC but not of the narGHJI mutant. Finally, colony formation by wild-type EHEC on epithelial cells, which were preincubated with heat-killed bacteria, was higher than the narGHJI mutant, and this effect was abolished by aminoguanidine hydrochloride, which is an iNOS (inducible nitric oxide synthase) inhibitor. CONCLUSIONS: These results indicate that the inflammatory response enhances EHEC adherence by increasing nitrate concentration.
Subject(s)
Cytokines/metabolism , Enterohemorrhagic Escherichia coli/physiology , Mutation , Nitrates/metabolism , Type III Secretion Systems/immunology , Bacterial Adhesion , Bacteriological Techniques , Caco-2 Cells , Enterohemorrhagic Escherichia coli/immunology , Escherichia coli Proteins/genetics , Escherichia coli Proteins/immunology , Gene Expression Regulation, Bacterial , HeLa Cells , Humans , Nitric Oxide/metabolism , Type III Secretion Systems/genetics , Virulence Factors/genetics , Virulence Factors/immunologyABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) are known as the gastrointestinal pathogens and major causes of enterohemorrhagic colitis since decades ago. There is no efficient approved vaccine against EHEC O157 and non-O157. In the present study, a recombinant candidate vaccine against enterohemorrhagic E. coli (EHEC) O157:H7 entrapped in the sodium alginate and PLGA nanoparticles and the efficiency of the immunization of these formulations were investigated. nanoparticles due to their properties like controlled cargoes release, adjuvanticity, cargo protection, increased bioavailability, etc have been noticed for drug delivery. A chimeric protein composed of HcpA, EspA, Tir and Stx2B antigens was designed, recombinantly expressed, purified and entrapped in nanoparticles. BALB/c mice were administrated with nano-formulated and free proteins. IgG titer, EHEC fecal shedding and the ability of the immune sera to neutralize Stx toxin and inhibit the bacterial attachment to Caco-2 cells were analyzed. Fecal shedding analysis demonstrated that the colonization of the bacteria in the intestine of the mice was reduced significantly (Pâ¯>â¯0.01). Immune mice were able to tolerate up to 200 LD50 of the active Stx toxin. About 80% of the bacterial binding capacity to Caco-2 cells was declined, especially in groups immunized with nano-formulations. Considering the importance of EHEC, especially O157 serotype, on public health and the other hand, the lack of an efficient vaccine in this regard, delivery of HETS candidate vaccine with NPs can be applied to prevent the infection by the pathogen.
Subject(s)
Alginates/chemistry , Antibody Formation/immunology , Escherichia coli Infections/immunology , Escherichia coli O157/immunology , Nanoparticles/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Recombinant Fusion Proteins/immunology , Animals , Antibodies, Bacterial/immunology , Caco-2 Cells , Cell Line, Tumor , Enterohemorrhagic Escherichia coli/immunology , Escherichia coli Proteins/immunology , Female , Humans , Immunization/methods , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , Polylactic Acid-Polyglycolic Acid Copolymer/immunology , Recombinant Fusion Proteins/chemistry , Vaccines, Synthetic/immunologyABSTRACT
Here we exploit the natural properties of a synthetic nanoparticle (NP) scaffold as a subunit vaccine against enterohemorrhagic Escherichiacoli (EHEC). Two EHEC-specific immunogenic antigens, namely, LomW and EscC, either alone or in combination, were covalently linked on the surface of gold nanoparticles (AuNPs) and used to immunize mice prior to challenge with EHEC O157:H7 strain 86-24. LomW is a putative outer membrane protein encoded in bacteriophage BP-933W, while EscC is a structural type III secretion system protein which forms a ring in the outer membrane. The resulting AuNP preparations, AuNP-LomW and AuNP-EscC, showed that the nanoparticles were able to incorporate the antigens, forming stable formulations that retained robust immunogenicity in vivo after subcutaneous immunization. When administered subcutaneously, AuNP-LomW or AuNP-EscC or a combination containing equivalent amounts of both candidates resulted in higher IgG titers in serum and secretory IgA titers in feces. The serum IgG titers correlated with a significant reduction in EHEC intestinal colonization after 3 days postinoculation. In addition, we showed that serum from antigen-coated AuNP-immunized mice resulted in a reduction of adherence to human intestinal epithelial cells for EHEC, as well as for two other E. coli pathotypes (enteropathogenic E. coli [EPEC], encoding EscC, and enteroaggregative E. coli [EAEC], encoding LomW). Further, the serum had antigen-specific bactericidal properties, engaging the classical complement pathway. Overall, our results demonstrate the immunogenicity and stability of a novel nanovaccine against EHEC. These results also strengthen the prospect of development of a synthetic nanoparticle vaccine conjugated to E. coli antigens as a promising platform against other enteric pathogens.IMPORTANCE Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis, which can progress to hemolytic uremic syndrome. These complications represent a serious global public health problem that requires laborious public health interventions and safety control measures to combat recurrent outbreaks worldwide. Today, there are no effective interventions for the control of EHEC infections, and, in fact, the use of antibiotics is counterindicated for EHEC disease. Therefore, a viable alternative for the prevention of human infections is the development of vaccines; however, no such vaccines are approved for human use. In this study, we developed a novel gold nanoparticle platform which acts as a scaffold for the delivery of various antigens, representing a nanovaccine technology which can be applied to several disease models.
Subject(s)
Escherichia coli Infections/prevention & control , Escherichia coli O157/immunology , Escherichia coli Vaccines/immunology , Gold/chemistry , Metal Nanoparticles/chemistry , Animals , Antibodies, Bacterial/immunology , Antibodies, Bacterial/metabolism , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Bacterial Adhesion , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Caco-2 Cells , Cross Protection , Disease Models, Animal , Enterohemorrhagic Escherichia coli/immunology , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Escherichia coli O157/physiology , Escherichia coli Vaccines/administration & dosage , Female , Gastrointestinal Tract/microbiology , Humans , Immunization , Immunoglobulin A, Secretory/metabolism , Immunoglobulin G/blood , Mice , Mice, Inbred BALB C , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunologyABSTRACT
INTRODUCTION: Severe intestinal infections caused by V. cholerae, ETEC and EHEC have contributed to the mortality rate in developing countries. Vibrio Cholera, ETEC and EHEC bacterium with the production of CT, LT and Stx2 toxins respectively lead to severe watery and bloody diarrhea. This study aimed to investigate a trimeric vaccine candidate containing recombinant chimeric protein, encapsulate the protein in chitosan nanoparticles and assess its immunogenicity. METHODS: The LSC recombinant gene was used. It is composed of LTB (L), STXB (S) and CTXB (C) subunits respectively. The LSC recombinant protein was expressed and purified and confirmed by western blotting. The purified protein was encapsulated in chitosan nanoparticles, and its size was measured. BalB/c mice were immunized in four groups through oral and injection methods by LSC protein. The antibody titer was then evaluated by ELISA, and finally, the challenge test of the toxins from all three bacteria was done on the immunized mouse. RESULTS: After expression and purification LSC protein size of nanoparticles containing protein was measured at 104.6â¯nm. Nanoparticles were able to induce systemic and mucosal immune responses by generating a useful titer of IgG and IgA. The challenge results with LT, CT and Stx toxins showed that the LSC protein might partially neutralize the effect of toxins. CONCLUSION: LSC chimeric protein with the simultaneous three essential antigens have a protective effect against the toxins produced by ETEC, EHEC and Vibrio cholera bacteria and it can be used in vaccines to prevent Diarrhea caused by these three bacteria.
Subject(s)
Bacterial Toxins/immunology , Bacterial Vaccines/immunology , Chitosan/pharmacology , Immunization , Nanoparticles/chemistry , Recombinant Fusion Proteins/immunology , Vaccination , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Bacterial Toxins/genetics , Bacterial Toxins/isolation & purification , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/genetics , Cholera Toxin/genetics , Cholera Toxin/immunology , Diarrhea/microbiology , Diarrhea/prevention & control , Disease Models, Animal , Enterohemorrhagic Escherichia coli/genetics , Enterohemorrhagic Escherichia coli/immunology , Enterotoxigenic Escherichia coli/genetics , Enterotoxigenic Escherichia coli/immunology , Escherichia coli Infections/immunology , Escherichia coli Infections/prevention & control , Gene Expression Regulation, Bacterial , Immunity, Mucosal , Immunoglobulin A/blood , Immunoglobulin G/blood , Mice, Inbred BALB C , Particle Size , Recombinant Fusion Proteins/genetics , Shiga Toxins/genetics , Shiga Toxins/immunology , Survival Analysis , Vibrio cholerae/genetics , Vibrio cholerae/immunologyABSTRACT
Microlipid vesicles (MLV) have a broad spectrum of applications for the delivery of molecules, ranging from chemical compounds to proteins, in both in vitro and in vivo conditions. In the present study, we developed a new set of nanosize multilayer lipid vesicles (NMVs) containing a unique combination of lipids. The NMVs enable the adsorption of histidine-tagged proteins at the vesicle surface and were demonstrated to be suitable for the in vivo delivery of antigens. The NMVs contained a combination of neutral (DOPC) and anionic (DPPG) lipids in the inner membrane and an external layer composed of DOPC, cholesterol, and a nickel-containing lipid (DGS-NTA [Ni]). NMVs combined with a recombinant form of the B subunit of the Shiga toxin (rStx2B) produced by certain enterohemorragic Escherichia coli (EHEC) strains enhanced the immunogenicity of the antigen after parenteral administration to mice. Mice immunized with rStx2B-loaded NMVs elicited serum antibodies capable of neutralizing the toxic activities of the native toxin; this result was demonstrated both in vitro and in vivo. Taken together, these results demonstrated that the proposed NMVs represent an alternative for the delivery of antigens, including recombinant proteins, generated in different expression systems.
Subject(s)
Antibodies, Bacterial/immunology , Drug Delivery Systems/methods , Enterohemorrhagic Escherichia coli/immunology , Escherichia coli Infections/immunology , Lipids/chemistry , Shiga Toxin/immunology , Animals , Antibody Formation , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Drug Delivery Systems/instrumentation , Enterohemorrhagic Escherichia coli/genetics , Escherichia coli Infections/microbiology , Escherichia coli Infections/prevention & control , Female , Humans , Immunization , Mice , Mice, Inbred BALB C , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Shiga Toxin/administration & dosage , Shiga Toxin/chemistryABSTRACT
Nucleotide-binding oligomerization domain 2 (NOD2), a member of the NOD-like receptors (NLRs) family that is well-known to play a key role in innate immune responses and is involved in innate antibacterial responses. In this study, rabbit NOD2 (rNOD2) was cloned from rabbit kidney (RK) cells. It was distributed in various tissues, and the highest level of rNod2 was detected in spleen. Moreover, the expression of rNod2 was significantly upregulated in the heart, liver, and spleen induced by enterohemorrhagic Escherichia coli (EHEC). Overexpression of rNOD2 induced the expression of pro-inflammatory cytokine, including Il1ß, Il6, Ifn-γ, and Tnf, as well as defensins, including Defb124, Defb125, and Defb128 through the nuclear factor (NF)-κB signaling pathway. Furthermore, overexpression of rNOD2 inhibited the growth of EHEC, and knockdown of rNOD2 or inhibition of the NF-κB pathway promoted its replication. In addition, our results suggest that rNOD2 can significantly activate NF-κB signaling and trigger antibacterial defenses to increase the expression of pro-inflammatory cytokine and defensins after stimulation by EHEC. These findings are useful to further understanding the innate immune system of rabbits and providing a new perspective for the prevention of bacterial diseases in rabbits.
Subject(s)
Enterohemorrhagic Escherichia coli/immunology , Escherichia coli Infections/immunology , Immunity, Innate , Nod2 Signaling Adaptor Protein/metabolism , Signal Transduction , Animal Structures/pathology , Animals , Cytokines/analysis , Disease Models, Animal , Escherichia coli Infections/pathology , Gene Knockdown Techniques , Immunologic Factors/analysis , Nod2 Signaling Adaptor Protein/genetics , RabbitsABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) or other attaching/effacing pathogen infections often cause host intestinal inflammation and pathology, which is thought to result in part from a host aggressive innate immune response. However, few effectors that play an important role in this pathology change have been reported. In this study, we discovered a previously unknown EHEC effector, Stk (putative serine/threonine kinase), which induces host aggressive inflammatory response during EHEC infection. Interestingly, homologous proteins of Stk are widely distributed in many pathogens. After translocating into the infected host cells, Stk efficiently phosphorylates IκBα and activates the NF-κB pathway. In EHEC-infected mice, Stk increases serum keratinocyte-derived cytokine (KC) levels and hyperactivates the inflammatory response of the colon, intensifying pathological injury of the colon. The virulence of Stk is based on its eukaryotic-like kinase activity. In conclusion, our data suggest that Stk is a new effector that induces the host aggressive inflammatory response during EHEC infection.
Subject(s)
Enterohemorrhagic Escherichia coli/immunology , Enterohemorrhagic Escherichia coli/metabolism , Escherichia coli Infections/immunology , Escherichia coli Infections/metabolism , Host-Pathogen Interactions/immunology , Protein Serine-Threonine Kinases/immunology , Protein Serine-Threonine Kinases/metabolism , Eukaryota/immunology , Eukaryota/metabolism , Humans , Inflammation/immunology , Inflammation/metabolismABSTRACT
Innate immunity is an essential component in the protection of a host against pathogens. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are known to modulate the innate immune responses of infected cells. The interference is dependent on their type III secretion system (T3SS) and T3SS-dependent effector proteins. Furthermore, these cytosolically injected effectors have been demonstrated to engage multiple immune signaling pathways, including the IFN/STAT, MAPK, NF-κB, and inflammasome pathways. In this review, recent work describing the interaction between EPEC/EHEC and the inflammasome pathway will be discussed.
Subject(s)
Enterohemorrhagic Escherichia coli/pathogenicity , Enteropathogenic Escherichia coli/pathogenicity , Host-Pathogen Interactions , Immune Evasion , Inflammasomes/antagonists & inhibitors , Signal Transduction , Virulence Factors/metabolism , Animals , Enterohemorrhagic Escherichia coli/immunology , Enteropathogenic Escherichia coli/immunology , Escherichia coli Proteins/metabolism , Humans , Type III Secretion Systems/metabolismABSTRACT
BACKGROUND: In contrast to atypical haemolytic uraemic syndrome (aHUS), only single case reports and limited data have been published on systemic activation of the complement system and mutations in complement genes in paediatric enterohaemorrhagic Escherichia coli-induced HUS (EHEC-HUS). METHODS: Complement activation (CH50, APH50, C3d, sC5b-9) was analysed at four timepoints (Week 1, Week 2, Month 3 and Month 6 after primary diagnosis of HUS) in 25 children with EHEC-HUS. Seven patients received the complement C5 inhibitor eculizumab. Targeted next generation sequencing for a total of 89 genes involved in complement regulation and coagulation and haemostasis was performed in all patients. RESULTS: Activity of classical (CH50) and alternative (APH50) complement pathways was normal or even elevated throughout the observation time, except for patients under eculizumab treatment. In contrast, the mean concentration of the soluble terminal complement complex (sC5b-9) was significantly elevated at the first timepoint (mean 498 ng/mL), dropping to normal values after 2 weeks. Initially elevated (42 mU/L) median C3d concentration reached normal levels from Week 2. Levels of sC5b-9 >320 ng/mL at the time of HUS diagnosis were associated with arterial hypertension, oedema and lower platelet counts, but not with the duration of dialysis. Genetic analysis revealed various changes that may have had a modifying impact on the clinical course. CONCLUSIONS: Complement activation at the acute phase of EHEC-HUS, indicated by increased levels of sC5b-9, predicts a poor outcome. Complement alterations appear to be more frequent in patients with EHEC-HUS than previously thought and are suspected to have a role in the severity of the disease.
Subject(s)
Complement Activation , Complement System Proteins/genetics , Enterohemorrhagic Escherichia coli/immunology , Hemolytic-Uremic Syndrome/immunology , Adolescent , Antibodies, Monoclonal, Humanized/therapeutic use , Biomarkers/blood , Child , Child, Preschool , Complement Inactivating Agents/therapeutic use , Complement System Proteins/metabolism , Female , Hemolytic-Uremic Syndrome/blood , Hemolytic-Uremic Syndrome/drug therapy , Humans , Male , Transcriptome/immunologyABSTRACT
Diarrheal diseases represent a major health problem in developing countries. Several viruses and bacterial agents, such as Enterotoxigenic Escherichia coli (ETEC) and Enterohemorrhagic Escherichia coli (EHEC) are responsible for human enteric infections. In humans, EHEC infections result in bloody or non-bloody diarrhea, which may be complicated by haemorrhagic colitis and haemolytic uraemic syndrome (HUS). Infection by ETEC is accompanied by a non inflammatory watery diarrhea. E. coli follows a common strategy of infection: colonization on a mucosal site, evasion of host defenses, multiplication, and host damage. Intimin, Stx, Lt and Cfa proteins are the virulence factors expressed by these strains. Antibiotic treatment is generally not recommended for most cases of diarrhea, since antibiotic usage may lead to antibiotic resistance in ETEC and may also change the intestinal flora. We hypothesized that the chimeric forms of these effectors as vaccine candidates would reduce the colonization of bacteria. This study is based on an in silico analysis of chimeric protein structure and its stability and solubility. The secondary and tertiary structures of selected domains were also predicted. Moreover, T and B cell epitopes were mapped. Protein structure Prediction showed that each domain of antigen was separated completely also stable for recombinant expression. We believe that this chimeric vaccine candidate is effective for prevention of bacteria caused diarrheal diseases.
Subject(s)
Enterohemorrhagic Escherichia coli/immunology , Enterotoxigenic Escherichia coli/immunology , Epitopes, B-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/chemistry , Escherichia coli Vaccines/chemistry , Virulence Factors/chemistry , Computer Simulation , Diarrhea/microbiology , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Escherichia coli Infections/microbiology , Escherichia coli Infections/prevention & control , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/immunology , Escherichia coli Vaccines/immunology , Humans , Models, Molecular , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Virulence Factors/immunologyABSTRACT
Shiga toxin-producing Escherichia coli (STEC) serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS) to secrete effector proteins (T3SPs) that result in the formation of attaching and effacing (A/E) lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes.
Subject(s)
Antibodies, Bacterial/pharmacology , Bacterial Secretion Systems/immunology , Enterohemorrhagic Escherichia coli/immunology , Escherichia coli Proteins/immunology , Animals , Antibodies, Bacterial/immunology , Bacterial Adhesion/immunology , Cattle , Cell Line , Cross Reactions , Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli Vaccines/immunology , Escherichia coli Vaccines/pharmacology , Humans , Immunoglobulin G/immunology , Mice , RabbitsABSTRACT
Non-O157 enterohemorrhagic Escherichia coli in priority serogroups O26, O45, O103, O111, O121, and O145 are increasingly recognized as important human pathogens. In the present study, a panel of monoclonal antibodies (MAbs) to the lipopolysaccharide O antigens of E. coli in serogroups O26, O45, O103, O111, O121, and O145 was produced. The specificity was evaluated by examining the reactivity of the MAbs with 50 E. coli strains and 42 non-E. coli bacteria, and several MAbs highly specific for E. coli strains in each of the six non-O157 priority serogroups were identified. The use of these highly specific MAbs may be of considerable value for determining whether an E. coli isolate belongs to one of the six priority non-O157 serogroups, for developing specific detection assays for these organisms, and for characterizing the lipopolysaccharide O antigens of isolates in these serogroups.
Subject(s)
Antibodies, Monoclonal/immunology , Enterohemorrhagic Escherichia coli/classification , Enterohemorrhagic Escherichia coli/immunology , O Antigens/immunology , Antibody Specificity , Enterohemorrhagic Escherichia coli/isolation & purification , Escherichia coli Infections/microbiology , Escherichia coli Proteins , Humans , Serogroup , SerotypingABSTRACT
Four invariant chain (Ii) isoforms assist the folding and trafficking of human MHC class II (MHCIIs). The main isoforms, Iip33 and Iip35, assemble in the ER into homo- and/or hetero-trimers. The sequential binding of up to three MHCII αß heterodimers to Ii trimers results in the formation of pentamers, heptamers and nonamers. MHCIIs are required to overcome the p35-encoded di-arginine (RxR) ER retention motif and to allow anterograde trafficking of the complex. Here, we show that inactivation of the RxR motif requires a direct cis interaction between p35 and the MHCII, precluding ER egress of some unsaturated Ii trimers. Interestingly, as opposed to MHCII/p33 complexes, those including p35 remained in the ER when co-expressed with the NleA protein of enterohaemorrhagic Escherichia coli. Taken together, our results demonstrate that p35 influences distinctively MHCII/Ii assembly and trafficking.
Subject(s)
Antigens, Differentiation, B-Lymphocyte/metabolism , Endoplasmic Reticulum/metabolism , Enterohemorrhagic Escherichia coli/immunology , Escherichia coli Proteins/metabolism , Histocompatibility Antigens Class II/metabolism , Virulence Factors/metabolism , Antigens, Differentiation, B-Lymphocyte/genetics , Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli Proteins/genetics , HEK293 Cells , HLA Antigens/metabolism , Histocompatibility Antigens Class II/genetics , Humans , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Multimerization , Protein Sorting Signals/genetics , Protein Transport/drug effects , Protein Transport/genetics , Virulence Factors/geneticsABSTRACT
Enteric bacterial pathogens commonly use a type III secretion system (T3SS) to successfully infect intestinal epithelial cells and survive and proliferate in the host. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC; EHEC) colonize the human intestinal mucosa, form characteristic histological lesions on the infected epithelium and require the T3SS for full virulence. T3SS effectors injected into host cells subvert cellular pathways to execute a variety of functions within infected host cells. The EPEC and EHEC effectors that subvert innate immune pathways--specifically those involved in phagocytosis, host cell survival, apoptotic cell death and inflammatory signalling--are all required to cause disease. These processes are reviewed within, with a focus on recent work that has provided insights into the functions and host cell targets of these effectors.
Subject(s)
Enterohemorrhagic Escherichia coli/immunology , Enteropathogenic Escherichia coli/immunology , Host-Pathogen Interactions , Immune Evasion , Immune Tolerance , Bacterial Secretion Systems , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Escherichia coli Proteins/metabolism , Humans , Virulence Factors/metabolismABSTRACT
Effectors are virulence factors that are secreted by bacteria during an infection in order to subvert cellular processes or induce the surveillance system of the host. Pathogenic microorganisms encode effectors, toxins and components of secretion systems that inject the effectors to the host. Escherichia coli is part of the innocuous commensal microbial flora of the gastrointestinal tract. However, pathogenic E. coli can cause diarrheal and extraintestinal diseases. Pathogenic E. coli uses secretion systems to inject an array of effector proteins directly into the host cells. Herein, we discuss the effectors secreted by different pathotypes of E. coli and provide an overview of strategies employed by effectors to target the host cellular and subcellular processes as well as their role in triggering host immune response.
Subject(s)
Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Escherichia coli/pathogenicity , Virulence Factors/metabolism , Animals , Enterohemorrhagic Escherichia coli/immunology , Enterohemorrhagic Escherichia coli/metabolism , Enterohemorrhagic Escherichia coli/pathogenicity , Enteropathogenic Escherichia coli/immunology , Enteropathogenic Escherichia coli/metabolism , Enteropathogenic Escherichia coli/pathogenicity , Escherichia coli/immunology , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Host-Pathogen Interactions , Humans , Signal TransductionABSTRACT
Enteropathogenic Escherichia coli (EPEC) are a major cause of infant diarrhoea in developing countries and a significant public health issue in industrialized countries. Currently there are no simple tests available for the diagnosis of EPEC. Serology of O-antigens is widely used routinely in many laboratories throughout the world, even though it has been known for many years to be an unreliable indicator of EPEC virulence. We have developed a simple, low-cost immunodiagnostic test based on the EspA filament, an essential virulence factor of EPEC and the related enterohaemorrhagic E. coli (EHEC). Using recombinant proteins of the five major variants of EspA as immunogens, we raised a panel of three monoclonal antibodies in mice that detects all variants of the native target in bacterial cultures. The antibodies proved suitable for application in sandwich-type assays, including ELISA and lateral flow immunoassays (LFI). Prototypes for both assays were specific for EPEC and EHEC strains when tested against a panel of control micro-organisms. We have also developed a simple, affordable culture medium, A/E medium, which optimizes expression of EspA allowing improved sensitivity of detection compared with standard Dulbecco's modified Eagle's medium. Together these reagents form the basis of robust, informative tests for EPEC for use especially in developing countries but also for routine screening in any clinical laboratory.
Subject(s)
Antibodies, Monoclonal , Diagnostic Tests, Routine/methods , Enterohemorrhagic Escherichia coli/isolation & purification , Enteropathogenic Escherichia coli/isolation & purification , Escherichia coli Infections/diagnosis , Escherichia coli Proteins/analysis , Gastroenteritis/diagnosis , Animals , Antibodies, Monoclonal/isolation & purification , Enterohemorrhagic Escherichia coli/immunology , Enteropathogenic Escherichia coli/immunology , Escherichia coli Infections/microbiology , Escherichia coli Proteins/immunology , Gastroenteritis/microbiology , Humans , Immunologic Tests/methods , Mice, Inbred BALB C , Virulence Factors/analysis , Virulence Factors/immunologyABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) is an extracellular pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. The proinflammatory cytokine, interleukin-1ß, has been linked to hemolytic uremic syndrome. Here we identify the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome as an essential mediator of EHEC-induced IL-1ß. Whereas EHEC-specific virulence factors were dispensable for NLRP3 activation, bacterial nucleic acids such as RNA:DNA hybrids and RNA gained cytosolic access and mediated inflammasome-dependent responses. Consistent with a direct role for RNA:DNA hybrids in inflammasome activation, delivery of synthetic EHEC RNA:DNA hybrids into the cytosol triggered NLRP3-dependent responses, and introduction of RNase H, which degrades such hybrids, into infected cells specifically inhibited inflammasome activation. Notably, an E. coli rnhA mutant, which is incapable of producing RNase H and thus harbors increased levels of RNA:DNA hybrid, induced elevated levels of NLRP3-dependent caspase-1 activation and IL-1ß maturation. Collectively, these findings identify RNA:DNA hybrids of bacterial origin as a unique microbial trigger of the NLRP3 inflammasome.
Subject(s)
Carrier Proteins/metabolism , DNA, Single-Stranded/metabolism , Enterohemorrhagic Escherichia coli/immunology , Hemolytic-Uremic Syndrome/immunology , Inflammasomes/immunology , Interleukin-1beta/immunology , RNA/metabolism , Animals , Base Sequence , Carrier Proteins/immunology , Caspase 1/immunology , DNA, Single-Stranded/genetics , Enzyme-Linked Immunosorbent Assay , Escherichia coli Proteins/genetics , Immunoblotting , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Molecular Sequence Data , NLR Family, Pyrin Domain-Containing 3 Protein , RNA/genetics , Ribosomal Proteins/geneticsABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains are major human food-borne pathogens, responsible for bloody diarrhea and hemolytic-uremic syndrome worldwide. Thus far, there is no vaccine for humans against EHEC infections. In this study, a comparative genomics analysis was performed to identify EHEC-specific antigens useful as potential vaccines. The genes present in both EHEC EDL933 and Sakai strains but absent in nonpathogenic E. coli K-12 and HS strains were subjected to an in silico analysis to identify secreted or surface-expressed proteins. We obtained a total of 65 gene-encoding protein candidates, which were subjected to immunoinformatics analysis. Our criteria of selection aided in categorizing the candidates as high, medium, and low priority. Three members of each group were randomly selected and cloned into pVAX-1. Candidates were pooled accordingly to their priority group and tested for immunogenicity against EHEC O157:H7 using a murine model of gastrointestinal infection. The high-priority (HP) pool, containing genes encoding a Lom-like protein (pVAX-31), a putative pilin subunit (pVAX-12), and a fragment of the type III secretion structural protein EscC (pVAX-56.2), was able to induce the production of EHEC IgG and sIgA in sera and feces. HP candidate-immunized mice displayed elevated levels of Th2 cytokines and diminished cecum colonization after wild-type challenge. Individually tested HP vaccine candidates showed that pVAX-12 and pVAX-56.2 significantly induced Th2 cytokines and production of fecal EHEC sIgA, with pVAX-56.2 reducing EHEC cecum colonization. We describe here a bioinformatics approach able to identify novel vaccine candidates potentially useful for preventing EHEC O157:H7 infections.