ABSTRACT
SignificanceYersinia pestis, the etiologic agent of plague, has been responsible for high mortality in several epidemics throughout human history. This plague bacillus has been used as a biological weapon during human history and is currently one of the deadliest biological threats. Currently, no licensed plague vaccines are available in the Western world. Since an array of immunogens are enclosed in outer membrane vesicles (OMVs), immune responses elicited by OMVs against a diverse range of antigens may reduce the likelihood of antigen circumvention. Therefore, self-adjuvanting OMVs from a remodeled Yersinia pseudotuberculosis strain as a type of plague vaccine could diversify prophylactic choices and solve current vaccine limitations.
Subject(s)
Antigens, Bacterial , Lipid A , Plague Vaccine , Plague , Pore Forming Cytotoxic Proteins , Yersinia pseudotuberculosis , Animals , Antibodies, Bacterial/blood , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Lethal Dose 50 , Lipid A/genetics , Lipid A/immunology , Mice , Plague/prevention & control , Plague Vaccine/administration & dosage , Plague Vaccine/genetics , Plague Vaccine/immunology , Plasmids/genetics , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/immunology , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis/immunologyABSTRACT
Yersinia pseudotuberculosis is a foodborne pathogen that subverts immune function by translocation of Yersinia outer protein (Yop) effectors into host cells. As adaptive γδ T cells protect the intestinal mucosa from pathogen invasion, we assessed whether Y. pseudotuberculosis subverts these cells in mice and humans. Tracking Yop translocation revealed that the preferential delivery of Yop effectors directly into murine Vγ4 and human Vδ2+ T cells inhibited anti-microbial IFNγ production. Subversion was mediated by the adhesin YadA, injectisome component YopB, and translocated YopJ effector. A broad anti-pathogen gene signature and STAT4 phosphorylation levels were inhibited by translocated YopJ. Thus, Y. pseudotuberculosis attachment and translocation of YopJ directly into adaptive γδ T cells is a major mechanism of immune subversion in mice and humans. This study uncovered a conserved Y. pseudotuberculosis pathway that subverts adaptive γδ T cell function to promote pathogenicity.
Subject(s)
Bacterial Proteins/immunology , Immune Evasion/immunology , Interferon-gamma/biosynthesis , Intraepithelial Lymphocytes/immunology , Yersinia pseudotuberculosis Infections/immunology , Animals , Humans , Mice , Yersinia pseudotuberculosis/immunologyABSTRACT
Lipopolysaccharide (LPS), localized in the outer leaflet of the outer membrane, serves as the major surface component of the Gram-negative bacterial cell envelope responsible for the activation of the host's innate immune system. Variations of the LPS structure utilized by Gram-negative bacteria promote survival by providing resistance to components of the innate immune system and preventing recognition by TLR4. This review summarizes studies of the biosynthesis of Yersinia pseudotuberculosis complex LPSs, and the roles of their structural components in molecular mechanisms of yersiniae pathogenesis and immunogenesis.
Subject(s)
Host-Pathogen Interactions/immunology , Immunity, Innate/genetics , Lipopolysaccharides/chemistry , Yersinia pseudotuberculosis/chemistry , Host-Pathogen Interactions/genetics , Humans , Lipid A/genetics , Lipid A/immunology , Lipopolysaccharides/genetics , Lipopolysaccharides/immunology , Molecular Structure , Structure-Activity Relationship , Yersinia pseudotuberculosis/immunology , Yersinia pseudotuberculosis/pathogenicityABSTRACT
Prostaglandin E2 (PGE2) is an essential immunomodulatory lipid released by cells in response to infection with many bacteria, yet its function in macrophage-mediated bacterial clearance is poorly understood. Yersinia overall inhibits the inflammatory circuit, but its effect on PGE2 production is unknown. We hypothesized that one of the Yersinia effector proteins is responsible for the inhibition of PGE2 biosynthesis. We identified that yopB-deficient Y. enterocolitica and Y. pseudotuberculosis deficient in the secretion of virulence proteins via a type 3 secretion system (T3SS) failed to inhibit PGE2 biosynthesis in macrophages. Consistently, COX-2-mediated PGE2 biosynthesis is upregulated in cells treated with heat-killed or T3SS-deficient Y. pseudotuberculosis but diminished in the presence of a MAPK/ERK inhibitor. Mutants expressing catalytically inactive YopJ induce similar levels of PGE2 as heat-killed or ΔyopB Y. pseudotuberculosis, reversed by YopJ complementation. Shotgun proteomics discovered host pathways regulated in a YopJ-mediated manner, including pathways regulating PGE2 synthesis and oxidative phosphorylation. Consequently, this study identified that YopJ-mediated inhibition of MAPK signal transduction serves as a mechanism targeting PGE2, an alternative means of inflammasome inhibition by Yersinia. Finally, we showed that EP4 signaling supports macrophage function in clearing intracellular bacteria. In summary, our unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription, thereby enhancing the intracellular survival of yersiniae. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic molecule to improve the outcomes of specific bacterial infections. Since other pathogens encode YopJ homologs, this mechanism is expected to be present in other infections. IMPORTANCE PGE2 is a critical immunomodulatory lipid, but its role in bacterial infection and pathogen clearance is poorly understood. We previously demonstrated that PGE2 leads to macrophage polarization toward the M1 phenotype and stimulates inflammasome activation in infected macrophages. Finally, we also discovered that PGE2 improved the clearance of Y. enterocolitica. The fact that Y. enterocolitica hampers PGE2 secretion in a type 3 secretion system (T3SS)-dependent manner and because PGE2 appears to assist macrophage in the clearance of this bacterium indicates that targeting of the eicosanoid pathway by Yersinia might be an adaption used to counteract host defenses. Our study identified a mechanism used by Yersinia that obstructs PGE2 biosynthesis in human macrophages. We showed that Y. pseudotuberculosis interferes with PGE2 biosynthesis by using one of its T3SS effectors, YopJ. Specifically, YopJ targets the host COX-2 enzyme responsible for PGE2 biosynthesis, which happens in a MAPK/ER-dependent manner. Moreover, in a shotgun proteomics study, we also discovered other pathways that catalytically active YopJ targets in the infected macrophages. YopJ was revealed to play a role in limiting host LPS responses, including repression of EGR1 and JUN proteins, which control transcriptional activation of proinflammatory cytokine production such as interleukin-1ß. Since YopJ has homologs in other bacterial species, there are likely other pathogens that target and inhibit PGE2 biosynthesis. In summary, our study's unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic target.
Subject(s)
Bacterial Proteins/immunology , Cyclooxygenase 2/immunology , Dinoprostone/immunology , Extracellular Signal-Regulated MAP Kinases/immunology , Macrophages/immunology , Mitogen-Activated Protein Kinase Kinases/immunology , Yersinia pseudotuberculosis Infections/microbiology , Yersinia pseudotuberculosis/immunology , Animals , Bacterial Proteins/genetics , Cyclooxygenase 2/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , Host-Pathogen Interactions , Humans , Macrophage Activation , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase Kinases/genetics , Signal Transduction , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis Infections/immunologyABSTRACT
Primary infection of C57BL/6 mice with the bacterial pathogen Yersinia pseudotuberculosis elicits an unusually large H-2Kb-restricted CD8+ T cell response to the endogenous and protective bacterial epitope YopE69-77. To better understand the basis for this large response, the model OVA257-264 epitope was inserted into YopE in Y. pseudotuberculosis and antigen-specific CD8+ T cells in mice were characterized after foodborne infection with the resulting strain. The epitope YopE69-77 elicited significantly larger CD8+ T cell populations in the small intestine, mesenteric lymph nodes (MLNs), spleen, and liver between 7 and 30 days postinfection, despite residing in the same protein and having an affinity for H-2Kb similar to that of OVA257-264. YopE-specific CD8+ T cell precursors were â¼4.6 times as abundant as OVA-specific precursors in the MLNs, spleens, and other lymph nodes of naive mice, explaining the dominance of YopE69-77 over OVA257-264 at early infection times. However, other factors contributed to this dominance, as the ratio of YopE-specific to OVA-specific CD8+ T cells increased between 7 and 30 days postinfection. We also compared the YopE-specific and OVA-specific CD8+ T cells generated during infection for effector and memory phenotypes. Significantly higher percentages of YopE-specific cells were characterized as short-lived effectors, while higher percentages of OVA-specific cells were memory precursor effectors at day 30 postinfection in spleen and liver. Our results suggest that a large precursor number contributes to the dominance and effector and memory functions of CD8+ T cells generated in response to the protective YopE69-77 epitope during Y. pseudotuberculosis infection of C57BL/6 mice.
Subject(s)
Antigens, Bacterial/immunology , CD8-Positive T-Lymphocytes/immunology , Host-Pathogen Interactions/immunology , T-Cell Antigen Receptor Specificity , Yersinia pseudotuberculosis Infections/immunology , Yersinia pseudotuberculosis Infections/microbiology , Yersinia pseudotuberculosis/immunology , Animals , CD8-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Epitopes, T-Lymphocyte/immunology , Immunologic Memory , Mice , Mice, Inbred C57BL , Yersinia pseudotuberculosis Infections/transmissionABSTRACT
Adhesins facilitate bacterial colonization and invasion of host tissues and are considered virulence factors, but their impact on immune-mediated damage as a driver of pathogenesis remains unclear. Yersinia pseudotuberculosis encodes for a multivalent adhesion molecule (MAM), a mammalian cell entry (MCE) family protein and adhesin. MAMs are widespread in Gram-negative bacteria and enable enteric bacteria to colonize epithelial tissues. Their role in bacterial interactions with the host innate immune system and contribution to pathogenicity remains unclear. Here, we investigated howY. pseudotuberculosis MAM contributes to pathogenesis during infection of the Galleria mellonella insect model. We show that Y. pseudotuberculosis MAM is required for efficient bacterial binding and uptake by hemocytes, the host phagocytes. Y. pseudotuberculosis interactions with insect and mammalian phagocytes are determined by bacterial and host factors. Loss of MAM, and deficient microbe-phagocyte interaction, increased pathogenesis in G. mellonella. Diminished phagocyte association also led to increased bacterial clearance. Furthermore, Y. pseudotuberculosis that failed to engage phagocytes hyperactivated humoral immune responses, most notably melanin production. Despite clearing the pathogen, excessive melanization also increased phagocyte death and host mortality. Our findings provide a basis for further studies investigating how microbe- and host-factors integrate to drive pathogenesis in a tractable experimental system.
Subject(s)
Host-Pathogen Interactions , Larva/microbiology , Moths/microbiology , Phagocytes/microbiology , Phagocytes/pathology , Yersinia pseudotuberculosis/metabolism , Adhesins, Bacterial , Animals , Hemocytes , Moths/cytology , Phagocytes/immunology , Virulence Factors , Yersinia pseudotuberculosis/immunology , Yersinia pseudotuberculosis/pathogenicity , Yersinia pseudotuberculosis Infections/microbiologyABSTRACT
Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.
Subject(s)
Acyltransferases/immunology , Immune Evasion/immunology , Immunity, Innate/immunology , Lipid A/immunology , Yersinia pestis/immunology , Animals , Biological Evolution , Cell Line , Cell Line, Tumor , HEK293 Cells , Humans , Leukocytes, Mononuclear/immunology , Lipopolysaccharides/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Polymorphism, Single Nucleotide/immunology , THP-1 Cells/immunology , U937 Cells , Yersinia pseudotuberculosis/immunologyABSTRACT
Yersinia suppress neutrophil responses by using a type 3 secretion system (T3SS) to inject 6-7 Yersinia effector proteins (Yops) effectors into their cytoplasm. YopH is a tyrosine phosphatase that causes dephosphorylation of the adaptor protein SKAP2, among other targets in neutrophils. SKAP2 functions in reactive oxygen species (ROS) production, phagocytosis, and integrin-mediated migration by neutrophils. Here we identify essential neutrophil functions targeted by YopH, and investigate how the interaction between YopH and SKAP2 influence Yersinia pseudotuberculosis (Yptb) survival in tissues. The growth defect of a ΔyopH mutant was restored in mice defective in the NADPH oxidase complex, demonstrating that YopH is critical for protecting Yptb from ROS during infection. The growth of a ΔyopH mutant was partially restored in Skap2-deficient (Skap2KO) mice compared to wild-type (WT) mice, while induction of neutropenia further enhanced the growth of the ΔyopH mutant in both WT and Skap2KO mice. YopH inhibited both ROS production and degranulation triggered via integrin receptor, G-protein coupled receptor (GPCR), and Fcγ receptor (FcγR) stimulation. SKAP2 was required for integrin receptor and GPCR-mediated ROS production, but dispensable for degranulation under all conditions tested. YopH blocked SKAP2-independent FcγR-stimulated phosphorylation of the proximal signaling proteins Syk, SLP-76, and PLCγ2, and the more distal signaling protein ERK1/2, while only ERK1/2 phosphorylation was dependent on SKAP2 following integrin receptor activation. These findings reveal that YopH prevents activation of both SKAP2-dependent and -independent neutrophilic defenses, uncouple integrin- and GPCR-dependent ROS production from FcγR responses based on their SKAP2 dependency, and show that SKAP2 is not required for degranulation.
Subject(s)
Bacterial Outer Membrane Proteins/immunology , Intracellular Signaling Peptides and Proteins/immunology , Neutrophils/immunology , Protein Tyrosine Phosphatases/immunology , Signal Transduction/immunology , Yersinia pseudotuberculosis Infections/immunology , Yersinia pseudotuberculosis/immunology , Animals , Intracellular Signaling Peptides and Proteins/genetics , MAP Kinase Signaling System/genetics , MAP Kinase Signaling System/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Neutrophils/pathology , Reactive Oxygen Species/immunology , Signal Transduction/genetics , Yersinia pseudotuberculosis/pathogenicity , Yersinia pseudotuberculosis Infections/genetics , Yersinia pseudotuberculosis Infections/pathologyABSTRACT
We incorporated the ΔPfur::TT araC PBADfur deletion-insertion mutation on top of a previous Yersinia pseudotuberculosis mutant (Δasd ΔyopJ ΔyopK) to construct a new mutant designated as Yptb5, which manifests the arabinose-dependent regulated delayed fur (encoding ferric uptake regulator) shut-off. The Yptb5 strain was used to deliver an adjuvanted fusion protein, FliC180-LcrV. Levels of FliC180-LcrV synthesis were same in Yptb5 either harboring pSMV4, a p15A ori plasmid or pSMV8, a pSC101 ori plasmid containing the fliC180-lcrV fusion gene driven by Ptrc promoter. Tissue burdens of both Yptb5(pSMV4) and Yptb5(pSMV8) in mice had similar patterns. Mice vaccinated orally with 5 × 108 CFU of either Yptb5(pSMV4) or Yptb5(pSMV8) strain were primed high antibody titers with a balanced Th1/Th2 response, also developed potent T-cell responses with significant productions of IFN-γ, IL-17A and TNF-α. Immunization with each mutant strain conferred complete protection against pulmonary challenge with 5.5 × 103 CFU (55 LD50) of Y. pestis, but partial protection (50% survival) against 100 LD50 of Y. pestis. Our results demonstrate that arabinose-dependent regulated delayed fur shut-off is an effective strategy to develop live attenuated bacterial vaccines while retaining strong immunogenicity.
Subject(s)
Plague Vaccine , Plague , Yersinia pestis , Yersinia pseudotuberculosis , Animals , Antibodies, Bacterial , Antigens, Bacterial/genetics , Mice , Plague/prevention & control , Plague Vaccine/genetics , Pore Forming Cytotoxic Proteins , Vaccination , Vaccines, Attenuated , Yersinia pestis/genetics , Yersinia pestis/immunology , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis/immunologyABSTRACT
A single oral inoculation to mice of the live attenuated Yersinia pseudotuberculosis VTnF1 strain producing an F1 pseudocapsule protects against bubonic and pneumonic plague. However oral vaccination can fail in humans exposed to frequent intestinal infections. We evaluated in mice the efficacy of subcutaneous vaccine injection as an alternative way to induce protective immunity, while reducing the dose and avoiding strain release in nature. A single subcutaneous dose of up to 108 CFU induced dose-dependent antibody production. At the dose of 107 CFU, i.e. 10 times less than via the oral route, it caused a modest skin reaction and protected 100% against bubonic and 80% against pneumonic plague, caused by high doses of Yersinia pestis. Bacteria migrating to lymph nodes and spleen, but not feces, were rapidly eliminated. Thus, subcutaneous injection of VTnF1 would represent a good alternative when dissemination in nature and human intestinal responsiveness are limitations.
Subject(s)
Plague Vaccine/administration & dosage , Plague/prevention & control , Vaccination/methods , Animals , Dose-Response Relationship, Immunologic , Injections, Subcutaneous , Mice , Vaccines, Attenuated/administration & dosage , Yersinia pestis/immunology , Yersinia pseudotuberculosis/immunologyABSTRACT
Yersinioses caused by Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are significant concerns in human and veterinary health. The link between virulence and the potent LcrV antigen has prompted the latter's selection as a major component of anti-Yersinia vaccines. Here, we report that (i) the group of Yersinia species encompassing Y. pestis and Y. pseudotuberculosis produces at least five different clades of LcrV and (ii) vaccination of mice with an LcrV-secreting Lactococcus lactis only protected against Yersinia strains producing the same LcrV clade as that of used for vaccination. By vaccinating with engineered LcrVs and challenging mice with strains producing either type of LcrV or a LcrV mutated for regions of interest, we highlight key polymorphic residues responsible for the absence of cross-protection. Our results show that an anti-LcrV-based vaccine should contain multiple LcrV clades if protection against the widest possible array of Yersinia strains is sought.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Vaccines/immunology , Lactococcus lactis/immunology , Pore Forming Cytotoxic Proteins/immunology , Yersinia pestis/immunology , Yersinia pseudotuberculosis/immunology , Animals , Antibodies, Bacterial/immunology , Cross Protection/immunology , Disease Models, Animal , Female , Mice , Mice, Inbred BALB C , Vaccination/methods , Virulence/immunology , Yersinia Infections/immunologyABSTRACT
In this study, a novel recombinant attenuated Yersinia pseudotuberculosis PB1+ strain (χ10069) engineered with ΔyopK ΔyopJ Δasd triple mutations was used to deliver a Y. pestis fusion protein, YopE amino acid 1 to 138-LcrV (YopENt138-LcrV), to Swiss Webster mice as a protective antigen against infections by yersiniae. χ10069 bacteria harboring the pYA5199 plasmid constitutively synthesized the YopENt138-LcrV fusion protein and secreted it via the type 3 secretion system (T3SS) at 37°C under calcium-deprived conditions. The attenuated strain χ10069(pYA5199) was manifested by the establishment of controlled infection in different tissues without developing conspicuous signs of disease in histopathological analysis of microtome sections. A single-dose oral immunization of χ10069(pYA5199) induced strong serum antibody titers (log10 mean value, 4.2), secretory IgA in bronchoalveolar lavage (BAL) fluid from immunized mice, and Yersinia-specific CD4+ and CD8+ T cells producing high levels of tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin 2 (IL-2), as well as IL-17, in both lungs and spleens of immunized mice, conferring comprehensive Th1- and Th2-mediated immune responses and protection against bubonic and pneumonic plague challenges, with 80% and 90% survival, respectively. Mice immunized with χ10069(pYA5199) also exhibited complete protection against lethal oral infections by Yersinia enterocolitica WA and Y. pseudotuberculosis PB1+. These findings indicated that χ10069(pYA5199) as an oral vaccine induces protective immunity to prevent bubonic and pneumonic plague, as well as yersiniosis, in mice and would be a promising oral vaccine candidate for protection against plague and yersiniosis for human and veterinary applications.
Subject(s)
Antibodies, Bacterial/biosynthesis , Immunoglobulin A/biosynthesis , Plague Vaccine/administration & dosage , Plague/prevention & control , Recombinant Fusion Proteins/administration & dosage , Yersinia pestis/drug effects , Yersinia pseudotuberculosis Infections/prevention & control , Yersinia pseudotuberculosis/drug effects , Administration, Oral , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/microbiology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/microbiology , Cross Protection , Female , Gene Expression , Humans , Immunization , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-2/genetics , Interleukin-2/immunology , Lung/drug effects , Lung/immunology , Lung/microbiology , Male , Mice , Plague/immunology , Plague/microbiology , Plague/mortality , Plague Vaccine/biosynthesis , Plague Vaccine/genetics , Plague Vaccine/immunology , Plasmids/chemistry , Plasmids/metabolism , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Survival Analysis , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Vaccines, Synthetic , Yersinia pestis/immunology , Yersinia pestis/pathogenicity , Yersinia pseudotuberculosis/immunology , Yersinia pseudotuberculosis/pathogenicity , Yersinia pseudotuberculosis Infections/immunology , Yersinia pseudotuberculosis Infections/microbiology , Yersinia pseudotuberculosis Infections/mortalityABSTRACT
In vivo experiments showed that antibodies to OmpC and OmpF porins of Yersinia pseudotuberculosis increased thyroxine (T4) level in the blood of experimental animals. The mice were immunized with different antigens: recombinant OmpF porin in a soluble monomeric form, trimers of OmpC and OmpF porins isolated from the outer membrane, or antibodies to them. The level of thyroxine in the blood of mice immunized with OmpF and OmpC porins increased by 5.47 and 22.3 times, respectively; after immunization with antibodies to these proteins, blood thyroxine increased by 9.28 and 14.29 times. Immunization with recombinant OmpF porin induced no reliable increase in thyroxine level. Hence, the serum to recombinant OmpF porin contains no antibodies specific to conformational antigenic determinants that are present in the protein trimer and, according to our previous findings from molecular docking studies, determine cross-reactions between OmpF porin of Y. pseudotuberculosis and thyroidstimulating hormone receptor.
Subject(s)
Antigens, Bacterial/immunology , Hyperthyroidism/chemically induced , Porins/immunology , Yersinia pseudotuberculosis/chemistry , Animals , Antibodies, Bacterial/administration & dosage , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/chemistry , Female , Hyperthyroidism/immunology , Hyperthyroidism/metabolism , Immunization , Mice , Mice, Inbred BALB C , Porins/administration & dosage , Porins/chemistry , Protein Multimerization , Receptors, Thyrotropin/immunology , Receptors, Thyrotropin/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Thyroxine/biosynthesis , Yersinia pseudotuberculosis/immunologyABSTRACT
AIMS To assess the efficacy of an autogenous vaccine against Yersinia pseudotuberculosis III in preventing clinical disease and deaths due to yersiniosis in young Merino sheep, and to determine the effect of vaccination on the prevalence of faecal shedding of pathogenic Yersinia spp., daily liveweight gain, and development of antibodies to Yersinia spp. following vaccination and natural exposure. METHODS In six groups (three groups each from two farms) of young Merino sheep, 148-150 animals were systematically allocated to be vaccinated twice with an autogenous, formalin- killed bacterin vaccine containing Y. pseudotuberculosis serotype III or to remain non-vaccinated. All vaccinated and non-vaccinated sheep were run together in their original groups throughout the trial. Faecal and blood samples were collected, and liveweight measured, at the time of vaccination and subsequently over a 6-month period to determine faecal shedding of Y. enterocolitica and Y. pseudotuberculosis, seroprevalence of antibodies to Yersinia outer membrane proteins (YOP) and changes in liveweight. RESULTS None of the six trial groups experienced an outbreak of clinical yersiniosis during the study period. On Farm A, the prevalence of shedding of either or both Yersinia spp. was <40% on all but one sampling occasions. On Farm B the prevalence of shedding of both Yersinia spp. peaked at 98%, 96 days after vaccination. Mean liveweight and daily liveweight gain at the end of the study were similar in vaccinated and non-vaccinated groups on both farms (p>0.1), as was the prevalence of faecal shedding of Yersinia spp. (p>0.2), and the proportion of animals that became seropositive for antibodies to YOP following vaccination (p>0.1). CONCLUSIONS AND CLINICAL RELEVANCE This vaccine had, at most, limited effects on seroconversion and, under the conditions of this study, had no demonstrable impact on liveweight, mean daily liveweight gain or faecal shedding of Yersinia spp. Further studies are needed to determine the efficacy of this vaccine during outbreaks of yersiniosis or following experimental challenge with pathogenic Yersinia spp..
Subject(s)
Autovaccines/therapeutic use , Bacterial Shedding/drug effects , Sheep Diseases/microbiology , Sheep Diseases/prevention & control , Yersinia pseudotuberculosis Infections/veterinary , Yersinia pseudotuberculosis/immunology , Animals , Antibodies, Bacterial , Enzyme-Linked Immunosorbent Assay/veterinary , Feces/microbiology , Random Allocation , Sheep , Sheep Diseases/drug therapy , Vaccination , Victoria , Yersinia pseudotuberculosis/drug effects , Yersinia pseudotuberculosis Infections/drug therapy , Yersinia pseudotuberculosis Infections/prevention & controlABSTRACT
Immunization with the live-attenuated Yersinia pseudotuberculosis VTnF1 strain producing a Yersinia pestis F1 pseudocapsule efficiently protects mice against bubonic and pneumonic plague. In clinical trials, demonstration of a plague vaccine's efficacy in humans will not be feasible, and correlates of protection will be needed to bridge the immune response of protected animals to that of vaccinated humans. Using serum transfer and vaccination of antibody-deficient µMT mice, we established that both humoral and cellular responses elicited by VTnF1 independently conferred protection against bubonic plague. Thus, correlates were searched for in both responses, using blood only. Mice were vaccinated with increasing doses of VTnF1 to provide a range of immune responses and survival outcomes. The cellular response was evaluated using an in vitro IFNγ release assay, and IFNγ levels were significantly associated with protection, although some survivors were negative for IFNγ, so that IFNγ release is not a fully satisfactory correlate. Abundant serum IgG against the F1 capsule, Yop injectable toxins, and also non-F1 Y.pestis antigens were found, but none against the LcrV antigen. All readouts correlated to survival and to each other, confirming that vaccination triggered multiple protective mechanisms developing in parallel. Anti-F1 IgG was the most stringent correlate of protection, in both inbred BALB/c mice and outbred OF1 mice. This indicates that antibodies (Ab) to F1 play a dominant role for protection even in the presence of Ab to many other targets. Easy to measure, the anti-F1 IgG titer will be useful to evaluate the immune response in other animal species and in clinical trials.
Subject(s)
Antibodies, Bacterial/blood , Immunity, Cellular , Immunity, Humoral , Immunoglobulin G/blood , Plague Vaccine/immunology , Plague/immunology , Animals , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Female , Interferon-gamma/immunology , Mice, Inbred BALB C , Mice, Inbred C57BL , Plague/prevention & control , Vaccines, Attenuated/immunology , Yersinia pestis/immunology , Yersinia pseudotuberculosis/immunologyABSTRACT
Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling1. Recent studies indicate that the bacterial metabolite D-glycero-ß-D-manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol2-4, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-ß-D-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-κB. A CRISPR-Cas9 screen showed that activation of NF-κB by ADP-Hep involves an ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand-receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.
Subject(s)
Adenosine Diphosphate Sugars/immunology , Burkholderia cenocepacia , Cytosol , Immunity, Innate , Pathogen-Associated Molecular Pattern Molecules/immunology , Protein Kinases/metabolism , Yersinia pseudotuberculosis , Adenosine Diphosphate Sugars/metabolism , Animals , Burkholderia Infections/enzymology , Burkholderia Infections/immunology , Burkholderia Infections/pathology , Burkholderia cenocepacia/genetics , Burkholderia cenocepacia/immunology , Burkholderia cenocepacia/metabolism , CRISPR-Cas Systems , Crystallography, X-Ray , Cytokines/biosynthesis , Cytosol/enzymology , Cytosol/immunology , Disaccharides/metabolism , Enzyme Activation , Female , Gene Editing , Immunologic Factors/immunology , Immunologic Factors/metabolism , Immunomodulation , Inflammation/enzymology , Inflammation/immunology , Inflammation/pathology , Male , Mice , Mice, Inbred C57BL , Models, Molecular , NF-kappa B/metabolism , Pathogen-Associated Molecular Pattern Molecules/metabolism , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis/immunology , Yersinia pseudotuberculosis/metabolismABSTRACT
In a new paper, the protein InvD from Yersinia pseudotuberculosis, a zoonotic pathogen, is shown to assist late-stage invasion of intestinal epithelia. Remarkably, InvD acts by binding the Fab region of IgG or IgA. It straddles adjacent light-chain and heavy-chain variable domains, but its binding is different from that of antigens in that complementarity-determining regions do not participate. Structure determination revealed that its Fab-interacting domain adopts an immunoglobulin-like fold, fused to the preceding immunoglobulin-like domain and carried on a long stalk anchored to the bacterial outer membrane. Possible roles of this unusual host-pathogen interaction include avoidance of clearance from the intestine by secretory IgA.
Subject(s)
Adhesins, Bacterial/metabolism , Antibodies/metabolism , Immunoglobulin Fab Fragments/metabolism , Intestines/microbiology , Yersinia pseudotuberculosis Infections/microbiology , Yersinia pseudotuberculosis/pathogenicity , Adhesins, Bacterial/chemistry , Animals , Antibodies/immunology , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Variable Region , Intestines/immunology , Intestines/pathology , Yersinia pseudotuberculosis/immunology , Yersinia pseudotuberculosis Infections/metabolism , Yersinia pseudotuberculosis Infections/pathologyABSTRACT
Yersinia pseudotuberculosis is a Gram-negative bacterium and zoonotic pathogen responsible for a wide range of diseases, ranging from mild diarrhea, enterocolitis, lymphatic adenitis to persistent local inflammation. The Y. pseudotuberculosis invasin D (InvD) molecule belongs to the invasin (InvA)-type autotransporter proteins, but its structure and function remain unknown. In this study, we present the first crystal structure of InvD, analyzed its expression and function in a murine infection model, and identified its target molecule in the host. We found that InvD is induced at 37 °C and expressed in vivo 2-4 days after infection, indicating that InvD is a virulence factor. During infection, InvD was expressed in all parts of the intestinal tract, but not in deeper lymphoid tissues. The crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold that, apart from the canonical ß-sheets, comprises various modifications of and insertions into the Ig-core structure. We identified the Fab fragment of host-derived IgG/IgA antibodies as the target of the adhesion domain. Phage display panning and flow cytometry data further revealed that InvD exhibits a preferential binding specificity toward antibodies with VH3/VK1 variable domains and that it is specifically recruited to a subset of B cells. This finding suggests that InvD modulates Ig functions in the intestine and affects direct interactions with a subset of cell surface-exposed B-cell receptors. In summary, our results provide extensive insights into the structure of InvD and its specific interaction with the target molecule in the host.
Subject(s)
Adhesins, Bacterial/metabolism , Antibodies/metabolism , Immunoglobulin Fab Fragments/metabolism , Intestines/microbiology , Yersinia pseudotuberculosis Infections/microbiology , Yersinia pseudotuberculosis/pathogenicity , Adhesins, Bacterial/chemistry , Amino Acid Sequence , Animals , Antibodies/immunology , Bacterial Adhesion , Female , Immunoglobulin Fab Fragments/immunology , Intestines/immunology , Intestines/pathology , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Conformation , Sequence Homology , Virulence , Yersinia pseudotuberculosis/immunology , Yersinia pseudotuberculosis Infections/metabolism , Yersinia pseudotuberculosis Infections/pathologyABSTRACT
Murine Ly6Chi inflammatory monocytes (IMs) require CCR2 to leave the bone marrow and enter mesenteric lymph nodes (MLNs) and other organs in response to Yersinia pseudotuberculosis infection. We are investigating how IMs, which can differentiate into CD11c+ dendritic cells (DCs), contribute to innate and adaptive immunity to Y. pseudotuberculosis Previously, we obtained evidence that IMs are important for a dominant CD8+ T cell response to the epitope YopE69-77 and host survival using intravenous infections with attenuated Y. pseudotuberculosis Here we challenged CCR2+/+ or CCR2-/- mice orally with wild-type Y. pseudotuberculosis to investigate how IMs contribute to immune responses during intestinal infection. Unexpectedly, CCR2-/- mice did not have reduced survival but retained body weight better and their MLNs cleared Y. pseudotuberculosis faster and with reduced lymphadenopathy compared to controls. Enhanced bacterial clearance in CCR2-/- mice correlated with reduced numbers of IMs in spleens and increased numbers of neutrophils in livers. In situ imaging of MLNs and spleens from CCR2-GFP mice showed that green fluorescent protein-positive (GFP+) IMs accumulated at the periphery of neutrophil-rich Yersinia-containing pyogranulomas. GFP+ IMs colocalized with CD11c+ cells and YopE69-77-specific CD8+ T cells in MLNs, suggesting that IM-derived DCs prime adaptive responses in Yersinia pyogranulomas. Consistently, CCR2-/- mice had reduced numbers of splenic DCs, YopE69-77-specific CD8+ T cells, CD4+ T cells, and B cells in organs and lower levels of serum antibodies to Y. pseudotuberculosis antigens. Our data suggest that IMs differentiate into DCs in MLN pyogranulomas and direct adaptive responses in T cells at the expense of innate immunity during oral Y. pseudotuberculosis infection.
Subject(s)
Adaptive Immunity , Immunity, Innate , Monocytes/immunology , Mouth/microbiology , Receptors, CCR2/immunology , Yersinia pseudotuberculosis Infections/immunology , Yersinia pseudotuberculosis/immunology , Animals , B-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Female , Humans , Mice , Mice, Inbred C57BL , Receptors, CCR2/genetics , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis/physiology , Yersinia pseudotuberculosis Infections/genetics , Yersinia pseudotuberculosis Infections/microbiologyABSTRACT
White adipose tissue bridges body organs and plays a fundamental role in host metabolism. To what extent adipose tissue also contributes to immune surveillance and long-term protective defense remains largely unknown. Here, we have shown that at steady state, white adipose tissue contained abundant memory lymphocyte populations. After infection, white adipose tissue accumulated large numbers of pathogen-specific memory T cells, including tissue-resident cells. Memory T cells in white adipose tissue expressed a distinct metabolic profile, and white adipose tissue from previously infected mice was sufficient to protect uninfected mice from lethal pathogen challenge. Induction of recall responses within white adipose tissue was associated with the collapse of lipid metabolism in favor of antimicrobial responses. Our results suggest that white adipose tissue represents a memory T cell reservoir that provides potent and rapid effector memory responses, positioning this compartment as a potential major contributor to immunological memory.