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1.
Trends Parasitol ; 37(2): 165-175, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33502317

RESUMO

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, an infection that manifests as colitis and, in some cases, liver abscess. A better understanding of host protective factors is key to developing an effective remedy. Recently, significant advances have been made in understanding the mechanisms of MUC2 production by goblet cells upon amebic infection, regulation of antimicrobial peptide production by Paneth cells, the interaction of commensal microbiota with immune stimulation, and host genetics in conferring protection from amebiasis. In addition to host pathways that may serve as potential therapeutic targets, significant progress has also been made with respect to development of a vaccine against amebiasis. Here, we aim to highlight the current understanding and knowledge gaps critically.


Assuntos
Entamebíase/imunologia , Interações Hospedeiro-Parasita/imunologia , Entamoeba histolytica , Entamebíase/genética , Entamebíase/parasitologia , Entamebíase/prevenção & controle , Células Caliciformes/imunologia , Células Caliciformes/parasitologia , Humanos , Mucina-2/imunologia , Celulas de Paneth/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Vacinas Protozoárias
2.
BMC Immunol ; 20(1): 48, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842739

RESUMO

BACKGROUND: Yersinia pestis, the etiological pathogen of plague, is capable of repressing the immune response of white blood cells to evade phagocytosis. The V-antigen (LcrV) was found to be involved in this process by binding to human Toll-like Receptor 2 (TLR2). The detailed mechanism behind this LcrV and TLR2 mediated immune response repression, however, is yet to be fully elucidated due to the lack of structural information. RESULTS: In this work, with protein structure modelling, we were able to construct a structure model of the heterotetramer of Y. pestis LcrV and human TLR2. Molecular dynamics simulation suggests the stability of this structure in aquatic environment. The LcrV model has a dumbbell-like structure with two globule domains (G1 at N-terminus and G2 away from membrane) connected with a coiled-coil linker (CCL) domain. The two horseshoe-shape TLR2 subunits form a V-shape structure, are not in direct contact with each other, and are held together by the LcrV homodimer. In this structure model, both the G1 and CCL domains are involved in the formation of LcrV homodimer, while all three domains are involved in LcrV-TLR2 binding. A mechanistic model was proposed based on this heterotetrameric structure model: The LcrV homodimer separates the TLR2 subunits to inhibit the dimerization of TLR2 and subsequent signal transfer for immune response; while LcrV could also inhibit the formation of heterodimers of TLR2 with other TLRs, and leads to immune response repression. CONCLUSIONS: A heterotetrameric structure of Y. pestis LcrV and human TLR2 was modelled in this work. Analysis of this modelled structure showed its stability in aquatic environments and the role of LcrV domains and residues in protein-protein interaction. A mechanistic model for the role of LcrV in Y. pestis pathogenesis is raised based on this heterotetrameric structure model. This work provides a hypothesis of LcrV function, with which further experimental validation may elucidate the role of LcrV in human immune response repression.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Complexos Multiproteicos/química , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/metabolismo , Domínio Catalítico , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Molecular , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Relação Estrutura-Atividade
3.
Mol Immunol ; 116: 98-105, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31634816

RESUMO

Pseudomonas aeruginosa is a common nosocomial pathogen in burn patients, and rapidly acquires antibiotic resistance; thus, developing an effective therapeutic approach is the most promising strategy for combating infection. Type III secretion system (T3SS) translocates bacterial toxins into the cytosol of the targeted eukaryotic cells, which plays important roles in the virulence of P. aeruginosa infections in both acute pneumonia and burn wound models. The PcrV protein, a T3SS translocating protein, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targeting P. aeruginosa. In the present study, we evaluated the protective efficacy of chicken egg yolk antibodies (IgY) raised against recombinant PcrV (r-PcrV) in both acute pneumonia and burn wound models. R-PcrV protein was generated by expressing the pcrV gene (cloned in pET-28a vector) in E. coli BL-21. Anti-PcrV IgY was obtained by immunization of hen. Anti-PcrV IgY induced greater protection in P. aeruginosamurine acute pneumonia and burn wound models than control IgY (C-IgY) and PBS groups. Anti-PcrV IgY improved opsonophagocytic killing and inhibition of bacterial invasion of host cells. Taken together, our data provide evidence that anti-PcrV IgY can be a promising therapeutic candidate for combating P. aeruginosa infections.


Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Queimaduras/imunologia , Imunoglobulinas/imunologia , Pneumonia/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Queimaduras/microbiologia , Galinhas/imunologia , Galinhas/microbiologia , Modelos Animais de Doenças , Feminino , Imunização/métodos , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia/microbiologia , Vacinação/métodos , Virulência/imunologia
4.
PLoS Negl Trop Dis ; 13(8): e0007644, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31430284

RESUMO

Bacillus anthracis and Yersinia pestis are zoonotic bacteria capable of causing severe and sometimes fatal infections in animals and humans. Although considered as diseases of antiquity in industrialized countries due to animal and public health improvements, they remain endemic in vast regions of the world disproportionally affecting the poor. These pathogens also remain a serious threat if deployed in biological warfare. A single vaccine capable of stimulating rapid protection against both pathogens would be an extremely advantageous public health tool. We produced multiple-antigen fusion proteins (MaF1 and MaF2) containing protective regions from B. anthracis protective antigen (PA) and lethal factor (LF), and from Y. pestis V antigen (LcrV) and fraction 1 (F1) capsule. The MaF2 sequence was also expressed from a plasmid construct (pDNA-MaF2). Immunogenicity and protective efficacy were investigated in mice following homologous and heterologous prime-boost immunization. Antibody responses were determined by ELISA and anthrax toxin neutralization assay. Vaccine efficacy was determined against lethal challenge with either anthrax toxin or Y. pestis. Both constructs elicited LcrV and LF-specific serum IgG, and MaF2 elicited toxin-neutralizing antibodies. Immunizations with MaF2 conferred 100% and 88% protection against Y. pestis and anthrax toxin, respectively. In contrast, pDNA-MaF2 conferred only 63% protection against Y. pestis and no protection against anthrax toxin challenge. pDNA-MaF2-prime MaF2-boost induced 75% protection against Y. pestis and 25% protection against anthrax toxin. Protection was increased by the molecular adjuvant CARDif. In conclusion, MaF2 is a promising multi-antigen vaccine candidate against anthrax and plague that warrants further investigation.


Assuntos
Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Vacinas Bacterianas/imunologia , Peste/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Yersinia pestis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Vacinas Bacterianas/isolamento & purificação , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos BALB C , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Recombinantes de Fusão/genética , Análise de Sobrevida , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Yersinia pestis/genética
5.
Front Immunol ; 10: 1830, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428104

RESUMO

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.


Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Lactococcus lactis/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Yersinia pestis/imunologia , Yersinia pseudotuberculosis/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Proteção Cruzada/imunologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vacinação/métodos , Virulência/imunologia , Yersiniose/imunologia
6.
Infect Immun ; 87(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31331960

RESUMO

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.


Assuntos
Anticorpos Antibacterianos/biossíntese , Imunoglobulina A/biossíntese , Vacina contra a Peste/administração & dosagem , Peste/prevenção & controle , Proteínas Recombinantes de Fusão/administração & dosagem , Yersinia pestis/efeitos dos fármacos , Infecções por Yersinia pseudotuberculosis/prevenção & controle , Yersinia pseudotuberculosis/efeitos dos fármacos , Administração Oral , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Proteção Cruzada , Feminino , Expressão Gênica , Humanos , Imunização , Interferon gama/genética , Interferon gama/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Masculino , Camundongos , Peste/imunologia , Peste/microbiologia , Peste/mortalidade , Vacina contra a Peste/biossíntese , Vacina contra a Peste/genética , Vacina contra a Peste/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sobrevida , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Vacinas Sintéticas , Yersinia pestis/imunologia , Yersinia pestis/patogenicidade , Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/patogenicidade , Infecções por Yersinia pseudotuberculosis/imunologia , Infecções por Yersinia pseudotuberculosis/microbiologia , Infecções por Yersinia pseudotuberculosis/mortalidade
7.
Front Immunol ; 10: 781, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31068928

RESUMO

Pseudomonas aeruginosa (PA) is a major cause of nosocomial infections, which remain an unsolved problem in the clinic despite conventional antibiotic treatment. A PA vaccine could be both an effective and economical strategy to address this issue. Many studies have shown that PcrV, a structural protein of the type 3 secretion system (T3SS) from PA, is an ideal target for immune prevention and therapy. However, difficulties in the production of high-quality PcrV likely hinder its further application in the vaccine industry. Thus, we hypothesized that an optimized PcrV derivative with a rational design could be produced. In this study, the full-length PcrV was divided into four domains with the guidance of its structure, and the Nter domain (Met1-Lys127) and H12 domain (Leu251-Ile294) were found to be immunodominant. Subsequently, Nter and H12 were combined with a flexible linker to generate an artificial PcrV derivative (PcrVNH). PcrVNH was successfully produced in E. coli and behaved as a homogenous monomer. Moreover, immunization with PcrVNH elicited a multifactorial immune response and conferred broad protection in an acute PA pneumonia model and was equally effective to full-length PcrV. In addition, passive immunization with anti-PcrVNH antibodies alone also showed significant protection, at least based on inhibition of the T3SS and mediation of opsonophagocytic killing activities. These results provide an additional example for the rational design of antigens and suggest that PcrVNH is a promising vaccine candidate for the control of PA infection.


Assuntos
Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Vacinas de Subunidades/imunologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Epitopos Imunodominantes/imunologia , Camundongos , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/imunologia , Infecções por Pseudomonas/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
8.
Cell Commun Signal ; 17(1): 32, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979375

RESUMO

BACKGROUND: A major challenge in the development of effective cancer immunotherapy is the ability of tumors and their microenvironment to suppress immune cells through immunosuppressive cells such as myeloid -derived suppressor cells and regulatory T cells. We previously demonstrated that Plasmodium infection promotes innate and adaptive immunity against cancer in a murine Lewis lung cancer model but its effects on immunosuppressive cells in the tumor microenvironment are unknown. METHODS: Whole Tumors and tumor-derived sorted cells from tumor-bearing mice treated with or without plasmodium infected red blood cells were harvested 17 days post tumor implantation and analyzed using QPCR, western blotting, flow cytometry, and functional assays. Differences between groups were analyzed for statistical significance using Student's t-test. RESULTS: Here we found that Plasmodium infection significantly reduced the proportions of MDSCs and Tregs in the lung tumor tissues of the treated mice by downregulating their recruiting molecules and blocking cellular activation pathways. Importantly, CD8+ T cells isolated from the tumors of Plasmodium-treated mice exhibited significantly higher levels of granzyme B and perforin and remarkably lower levels of PD-1. CONCLUSION: We reveal for the first time, the effects of Plasmodium infection on the expansion and activation of MDSCs and Tregs with a consequent elevation of CD8+T cell-mediated cytotoxicity within the tumor microenvironment and hold great promise for the development of effective immunotherapeutic strategies.


Assuntos
Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/terapia , Imunossupressão/métodos , Malária/imunologia , Células Supressoras Mieloides/imunologia , Plasmodium yoelii/imunologia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Feminino , Granzimas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Citotóxicas Formadoras de Poros/imunologia , Receptor de Morte Celular Programada 1/imunologia
9.
mSphere ; 4(2)2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996108

RESUMO

Pseudomonas aeruginosa is a common Gram-negative opportunistic pathogen that is intrinsically resistant to a wide range of antibiotics. The development of a broadly protective vaccine against P. aeruginosa remains a major challenge. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express P. aeruginosa antigens. A fusion between the S. enterica type III secretion effector protein SseJ and the P. aeruginosa antigen PcrV expressed under the control of the sseA promoter was translocated by Salmonella into host cells in vitro and elicited the generation of specific antibodies in mice. Mice immunized with attenuated Salmonella expressing this fusion had reduced bacterial loads in the spleens and lungs and lower serum levels of proinflammatory cytokines than control mice after P. aeruginosa infection. Importantly, immunized mice also showed significantly enhanced survival in this model. These results suggest that type III secretion effectors of S. enterica are appropriate carriers in the design of a live vaccine to prevent infections caused by P. aeruginosa IMPORTANCE The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen that causes infections in cystic fibrosis and hospitalized patients. Therapeutic treatments are limited due to the emergence and spread of new antibiotic-resistant strains. In this context, the development of a vaccine is a priority. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express and deliver the Pseudomonas antigen PcrV. This vaccine induced the generation of specific antibodies in mice and protected them from lethal infections with P. aeruginosa This is an important step toward the development of an effective vaccine for the prevention of infections caused by P. aeruginosa in humans.


Assuntos
Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Salmonella/administração & dosagem , Sistemas de Secreção Tipo III/genética , Animais , Anticorpos Antibacterianos/sangue , Carga Bacteriana , Citocinas/sangue , Feminino , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa , Baço/microbiologia , Sistemas de Secreção Tipo III/imunologia , Vacinas Atenuadas/administração & dosagem
10.
Commun Biol ; 2: 59, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30775460

RESUMO

Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. However, functional studies on these ALPs remain in their infancy. ßγ-CAT is the first example of a secreted pore-forming protein that functions to modulate the endolysosome pathway via endocytosis and pore formation on endolysosomes. However, the specific cell surface molecules mediating the action of ßγ-CAT remain elusive. Here, the actions of ßγ-CAT were largely attenuated by either addition or elimination of acidic glycosphingolipids (AGSLs). Further study revealed that the ALP and trefoil factor (TFF) subunits of ßγ-CAT bind to gangliosides and sulfatides, respectively. Additionally, disruption of lipid rafts largely impaired the actions of ßγ-CAT. Finally, the ability of ßγ-CAT to clear pathogens was attenuated in AGSL-eliminated frogs. These findings revealed a previously unknown double binding pattern of an animal-secreted ALP in complex with TFF that initiates ALP-induced endolysosomal pathway regulation, ultimately leading to effective antimicrobial responses.


Assuntos
Glicoesfingolipídeos Acídicos/química , Proteínas de Anfíbios/imunologia , Toxinas Bacterianas/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Lisossomos/imunologia , Complexos Multiproteicos/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Fator Trefoil-3/imunologia , Glicoesfingolipídeos Acídicos/antagonistas & inibidores , Glicoesfingolipídeos Acídicos/biossíntese , Aeromonas hydrophila/crescimento & desenvolvimento , Aeromonas hydrophila/patogenicidade , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Anuros , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Ceramidas/antagonistas & inibidores , Ceramidas/biossíntese , Ceramidas/química , Cerebrosídeos/antagonistas & inibidores , Cerebrosídeos/biossíntese , Cerebrosídeos/química , Gangliosídeos/antagonistas & inibidores , Gangliosídeos/biossíntese , Gangliosídeos/química , Expressão Gênica , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Interleucina-1beta/biossíntese , Lisossomos/efeitos dos fármacos , Lisossomos/microbiologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/microbiologia , Meperidina/análogos & derivados , Meperidina/farmacologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Esfingosina/antagonistas & inibidores , Esfingosina/biossíntese , Esfingosina/química , Células THP-1 , Fator Trefoil-3/genética , Fator Trefoil-3/metabolismo
11.
J Immunol ; 202(7): 2005-2016, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30787109

RESUMO

Yersinia enterocolitica causes a severe enteric infection in infants and young children. There is no vaccine approved for use in humans. We investigated the immunogenicity and protective capacity of Yersinia YopB, a conserved type III secretion system protein, alone or combined with LcrV in adult mice immunized intranasally. YopB or LcrV (5 µg) administered with the Escherichia coli double mutant heat-labile toxin (dmLT) adjuvant afforded modest (10-30%) protection against lethal Y. enterocolitica oral infection. The combination of YopB and LcrV (5 µg each) dramatically improved vaccine efficacy (70-80%). Additionally, it afforded complete protection against Y. pestis pulmonary infection. Immunization with YopB/LcrV+dmLT resulted in Ag-specific serum IgG, systemic and mucosal Ab-secreting cells, as well as IFN-γ, TNF-α, IL-2, IL-6, IL-17A, and KC production by spleen cells. Serum Abs elicited by YopB/LcrV+dmLT had enhanced bactericidal and opsonophagocytic killing activity. After Y. enterocolitica challenge, YopB/LcrV+dmLT-vaccinated mice exhibited intact intestinal tissue, active germinal centers in mesenteric lymph nodes, IgG+ and IgA+ plasmablasts in the lamina propria, and Abs in intestinal fluid. On the contrary, complete tissue destruction and abscesses were seen in placebo recipients that succumbed to infection. Mice immunized as infants with YopB+dmLT or LcrV+dmLT achieved 60% protection against lethal Y. enterocolitica infection, and vaccine efficacy increased to 90-100% when they received YopB/LcrV+dmLT. YopB+dmLT also afforded substantial (60%) protection when administered intradermally to infant mice. YopB/LcrV+dmLT is a promising subunit vaccine candidate with the potential to elicit broad protection against Yersinia spp.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Yersiniose/prevenção & controle , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Subunidades/imunologia
12.
Int J Biol Macromol ; 122: 1062-1070, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30218736

RESUMO

The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective protein that is considered as a vaccine component for humans. LcrV mediates the delivery of Yop toxins into host cells and upregulates TLR2-dependent IL-10 production. Although LcrV can interact with the receptor-bound human interferon-γ (hIFN-γ), the significance of these interactions in plague pathogenesis is not known. In this study, we determined the parameters of specific interactions of LcrV and LcrV68-326 with primary human thymocytes and Jurkat T-leukemia cells in the presence of receptor-bound hIFN-γ. Although the C-terminal region of hIFN-γ contains a GRRA138-141 site needed for high-affinity binding of LcrV and LcrV68-326, in the hIFN-γ homodimer, these GRRA138-141 target sites becomes accessible for targeting by LcrV or LcrV68-326 only after immobilization of the hIFN-γ homodimer on the hIFN-γ receptors of thymocytes or Jurkat T-cells. The interaction of LcrV or LcrV68-326 with receptor-bound hIFN-γ on the thymocytes or Jurkat T-cells caused apoptosis of both cell types, which can be completely blocked by the addition of monoclonal antibodies specific to the LEEL32-35 and DEEI203-206 sites of LcrV. The ability of LcrV to utilize hIFN-γ is insidious and may account in part for the severe symptoms of plague in humans.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Apoptose , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/química , Humanos , Lactente , Células Jurkat , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/química , Ligação Proteica , Conformação Proteica
13.
Nat Commun ; 9(1): 4805, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30442932

RESUMO

CD8 T cells protect the liver against viral infection, but can also cause severe liver damage that may even lead to organ failure. Given the lack of mechanistic insights and specific treatment options in patients with acute fulminant hepatitis, we develop a mouse model reflecting a severe acute virus-induced CD8 T cell-mediated hepatitis. Here we show that antigen-specific CD8 T cells induce liver damage in a perforin-dependent manner, yet liver failure is not caused by effector responses targeting virus-infected hepatocytes alone. Additionally, CD8 T cell mediated elimination of cross-presenting liver sinusoidal endothelial cells causes endothelial damage that leads to a dramatically impaired sinusoidal perfusion and indirectly to hepatocyte death. With the identification of perforin-mediated killing as a critical pathophysiologic mechanism of liver failure and the protective function of a new class of perforin inhibitor, our study opens new potential therapeutic angles for fulminant viral hepatitis.


Assuntos
Células Endoteliais/efeitos dos fármacos , Hepatite Viral Animal/tratamento farmacológico , Fígado/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inibidores , Substâncias Protetoras/farmacologia , Sulfonamidas/farmacologia , Adenoviridae/genética , Adenoviridae/imunologia , Adenoviridae/patogenicidade , Animais , Anticorpos/administração & dosagem , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/genética , Antígenos CD40/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Capilares/efeitos dos fármacos , Capilares/virologia , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/virologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatite Viral Animal/imunologia , Hepatite Viral Animal/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Fígado/irrigação sanguínea , Fígado/patologia , Fígado/virologia , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Poli I-C/administração & dosagem , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia
14.
Microbiol Immunol ; 62(12): 774-785, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30378708

RESUMO

An effective vaccine against Pseudomonas aeruginosa would be hugely beneficial to people who are susceptible to the serious infections it can cause. Vaccination against PcrV of the P. aeruginosa type III secretion system is a potential prophylactic strategy for improving the incidence and prognosis of P. aeruginosa pneumonia. Here, the effect of nasal PcrV adjuvanted with CpG oligodeoxynucleotide (CpG) was compared with a nasal PcrV/aluminum hydroxide gel (alum) vaccine. Seven groups of mice were vaccinated intranasally with one of the following: 1, PcrV-CpG; 2, PcrV-alum; 3, PcrV alone; 4, CpG alone; 5, alum alone; 6 and 7, saline control. Fifty days after the first immunization, anti-PcrV IgG, IgA and IgG isotype titers were measured; significant increases in these titers were detected only in the PcrV-CpG vaccinated mice. The vaccinated mice were then intratracheally infected with a lethal dose of P. aeruginosa and their body temperatures and survival monitored for 24 hr, edema, bacteria, myeloperoxidase activity and lung histology also being evaluated at 24 hr post-infection. It was found that 73% of the PcrV-CpG-vaccinated mice survived, whereas fewer than 30% of the mice vaccinated with PcrV-alum or adjuvant alone survived. Lung edema and other inflammation-related variables were less severe in the PcrV-CpG group. The significant increase in PcrV-specific IgA titers detected following PcrV-CpG vaccination is probably a component of the disease protection mechanism. Overall, our data show that intranasal PcrV-CpG vaccination has potential efficacy for clinical application against P. aeruginosa pneumonia.


Assuntos
Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Oligodesoxirribonucleotídeos/imunologia , Pneumonia/prevenção & controle , Proteínas Citotóxicas Formadoras de Poros/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/efeitos dos fármacos , Vacinação , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Temperatura Corporal , Modelos Animais de Doenças , Edema , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Oligodesoxirribonucleotídeos/genética , Peroxidase/análise , Proteínas Citotóxicas Formadoras de Poros/genética , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Vacinas contra Pseudomonas/administração & dosagem , Pseudomonas aeruginosa/patogenicidade , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Taxa de Sobrevida , Sistemas de Secreção Tipo III/imunologia
15.
Molecules ; 23(10)2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-30287801

RESUMO

A group of stable, water-soluble and membrane-bound proteins constitute the pore forming toxins (PFTs) in cnidarians. They interact with membranes to physically alter the membrane structure and permeability, resulting in the formation of pores. These lesions on the plasma membrane causes an imbalance of cellular ionic gradients, resulting in swelling of the cell and eventually its rupture. Of all cnidarian PFTs, actinoporins are by far the best studied subgroup with established knowledge of their molecular structure and their mode of pore-forming action. However, the current view of necrotic action by actinoporins may not be the only mechanism that induces cell death since there is increasing evidence showing that pore-forming toxins can induce either necrosis or apoptosis in a cell-type, receptor and dose-dependent manner. In this review, we focus on the response of the cellular immune system to the cnidarian pore-forming toxins and the signaling pathways that might be involved in these cellular responses. Since PFTs represent potential candidates for targeted toxin therapy for the treatment of numerous cancers, we also address the challenge to overcoming the immunogenicity of these toxins when used as therapeutics.


Assuntos
Imunidade Inata/efeitos dos fármacos , Necrose/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Toxinas Biológicas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Membrana Celular/química , Cnidários/química , Cnidários/imunologia , Humanos , Necrose/tratamento farmacológico , Necrose/patologia , Proteínas Citotóxicas Formadoras de Poros/química , Transdução de Sinais/efeitos dos fármacos , Toxinas Biológicas/química
16.
mBio ; 9(5)2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30327445

RESUMO

Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, are two of the deadliest pathogenic bacteria that have been used as biological warfare agents. Although Biothrax is a licensed vaccine against anthrax, no Food and Drug Administration-approved vaccine exists for plague. Here, we report the development of a dual anthrax-plague nanoparticle vaccine employing bacteriophage (phage) T4 as a platform. Using an in vitro assembly system, the 120- by 86-nm heads (capsids) of phage T4 were arrayed with anthrax and plague antigens fused to the small outer capsid protein Soc (9 kDa). The antigens included the anthrax protective antigen (PA) (83 kDa) and the mutated (mut) capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system from Y. pestis (F1mutV) (56 kDa). These viral nanoparticles elicited robust anthrax- and plague-specific immune responses and provided complete protection against inhalational anthrax and/or pneumonic plague in three animal challenge models, namely, mice, rats, and rabbits. Protection was demonstrated even when the animals were simultaneously challenged with lethal doses of both anthrax lethal toxin and Y. pestis CO92 bacteria. Unlike the traditional subunit vaccines, the phage T4 vaccine uses a highly stable nanoparticle scaffold, provides multivalency, requires no adjuvant, and elicits broad T-helper 1 and 2 immune responses that are essential for complete clearance of bacteria during infection. Therefore, phage T4 is a unique nanoparticle platform to formulate multivalent vaccines against high-risk pathogens for national preparedness against potential bioterror attacks and emerging infections.IMPORTANCE Following the deadly anthrax attacks of 2001, the Centers for Disease Control and Prevention (CDC) determined that Bacillus anthracis and Yersinia pestis that cause anthrax and plague, respectively, are two Tier 1 select agents that pose the greatest threat to the national security of the United States. Both cause rapid death, in 3 to 6 days, of exposed individuals. We engineered a virus nanoparticle vaccine using bacteriophage T4 by incorporating key antigens of both B. anthracis and Y. pestis into one formulation. Two doses of this vaccine provided complete protection against both inhalational anthrax and pneumonic plague in animal models. This dual anthrax-plague vaccine is a strong candidate for stockpiling against a potential bioterror attack involving either one or both of these biothreat agents. Further, our results establish the T4 nanoparticle as a novel platform to develop multivalent vaccines against pathogens of high public health significance.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Bacteriófago T4 , Vacina contra a Peste/imunologia , Peste/prevenção & controle , Infecções Respiratórias/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Bacillus anthracis , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Proteínas do Capsídeo/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas , Proteínas Citotóxicas Formadoras de Poros/imunologia , Coelhos , Ratos , Células Th1/imunologia , Células Th2/imunologia , Yersinia pestis
17.
J Immunol ; 201(9): 2710-2720, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30249808

RESUMO

Perforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study, we used Salmonella enterica serovar Typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2's bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of Ags contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface Ag was found to be independent of Perforin-2. Consistent with our in vitro results, a protease-sensitive, periplasmic superoxide dismutase (SodCII) contributed to the virulence of S. Typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate, our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria, facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial cell.


Assuntos
Proteínas Citotóxicas Formadoras de Poros/imunologia , Salmonelose Animal/imunologia , Animais , Parede Celular , Camundongos , Camundongos Knockout , Fagocitose/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Salmonelose Animal/metabolismo , Salmonella typhimurium
18.
Front Immunol ; 9: 1688, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30100903

RESUMO

Bacterial infectious diseases are a leading cause of death. Pore-forming toxins (PFTs) are important virulence factors of Gram-positive pathogens, which disrupt the plasma membrane of host cells and can lead to cell death. Yet, host defense and cell membrane repair mechanisms have been identified: i.e., PFTs can be eliminated from membranes as microvesicles, thus limiting the extent of cell damage. Released into an inflammatory environment, these host-derived PFTs-carrying microvesicles encounter innate immune cells as first-line defenders. This study investigated the impact of microvesicle- or liposome-sequestered PFTs on human macrophage polarization in vitro. We show that microvesicle-sequestered PFTs are phagocytosed by macrophages and induce their polarization into a novel CD14+MHCIIlowCD86low phenotype. Macrophages polarized in this way exhibit an enhanced response to Gram-positive bacterial ligands and a blunted response to Gram-negative ligands. Liposomes, which were recently shown to sequester PFTs and so protect mice from lethal bacterial infections, show the same effect on macrophage polarization in analogy to host-derived microvesicles. This novel type of polarized macrophage exhibits an enhanced response to Gram-positive bacterial ligands. The specific recognition of their cargo might be of advantage in the efficiency of targeted bacterial clearance.


Assuntos
Toxinas Bacterianas/imunologia , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/imunologia , Transdução de Sinais , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunidade , Imunomodulação , Imunofenotipagem , Modelos Biológicos , Monócitos/imunologia , Monócitos/metabolismo , Fenótipo
19.
Clin Immunol ; 194: 34-42, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29940333

RESUMO

To test the relative roles of perforin (pfp) vs. FasL in CTL control of autoreactive B cell expansion, we used the parent-into-F1 model of murine graft-vs.-host disease in which donor CD8 CTL prevent lupus like disease by eliminating activated autoreactive B cells. F1 mice receiving either pfp or FasL defective donor T cells exhibited an intermediate short-term phenotype. Pairing of purified normal CD4 T cells with either pfp or FasL defective CD8 T cell subsets resulted in impaired host B cell elimination and mild lupus like disease that was roughly equivalent in the two experimental groups. Thus, in addition to major roles in tumor and intracellular pathogen control, pfp mediated CD8 CTL killing plays a significant role in controlling autoreactive B cell expansion and lupus downregulation that is comparable to that mediated by FasL killing. Importantly, both pathways are required for optimal elimination of activated autoreactive B cells.


Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteína Ligante Fas/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Doença Enxerto-Hospedeiro/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
20.
Sci Rep ; 8(1): 5846, 2018 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-29643440

RESUMO

Pore-forming toxins are the most common virulence factor in pathogenic bacteria. They lead to membrane permeabilization and cell death. Herein, we show that respiratory epithelial cells (REC) undergoing bacterial pore-forming toxin (PFT)-induced necroptosis simultaneously experienced caspase activation independently of RIPK3. MLKL deficient REC treated with a pan-caspase inhibitor were protected in an additive manner against PFT-induced death. Subsequently, cleaved versions of caspases-2, -4 and -10 were detected within REC undergoing necroptosis by immunoblots and monoclonal antibody staining. Caspase activation was observed in lung samples from mice and non-human primates experiencing Gram-negative and Gram-positive bacterial pneumonia, respectively. During apoptosis, caspase activation normally leads to cell shrinkage, nuclear condensation, and immunoquiescent death. In contrast, caspase activity during PFT-induced necroptosis increased the release of alarmins to the extracellular milieu. Caspase-mediated alarmin release was found sufficient to activate resting macrophages, leading to Interleukin-6 production. In a mouse model of Gram-negative pneumonia, deletion of caspases -2 and -11, the mouse orthologue of caspase-4, reduced pulmonary inflammation, immune cell infiltration and lung damage. Thus, our study describes a previously unrecognized role for caspase activation in parallel to necroptosis, and indicates that their activity plays a critical pro-inflammatory role during bacterial pneumonia.


Assuntos
Alarminas/metabolismo , Toxinas Bacterianas/metabolismo , Caspases/metabolismo , Pneumonia Bacteriana/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Células A549 , Alarminas/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Toxinas Bacterianas/imunologia , Inibidores de Caspase/farmacologia , Caspases/genética , Caspases/imunologia , Membrana Celular/ultraestrutura , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Necrose/imunologia , Papio , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/patologia , Proteínas Citotóxicas Formadoras de Poros/imunologia
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