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2.
J Infect Dis ; 200(11): 1694-702, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19863438

RESUMO

BACKGROUND: Yersinia pestis, the causative agent of plague, showed a temperature-dependent change in lipid A composition, with a reduced degree of acylation when bacteria were grown at 37 degrees C (tetraacylated) versus ambient temperature (hexaacylated). METHODS: Human monocytes and monocyte-derived dendritic cells (DCs) were exposed to Y. pestis grown at 26 degrees C or 37 degrees C, to their corresponding lipopolysaccharides (LPS-26 degrees C or LPS-37 degrees C), and to ligands of different Toll-like receptors (TLRs), such as LPS from Escherichia coli (TLR4), lipoprotein (TLR2), polyinosinic-polycytidylic acid (poly-IC) (TLR9), and their combinations. Production of cytokines was measured, along with expression of surface markers of DC maturation. RESULTS: Y. pestis grown at 37 degrees C or LPS-37 degrees C induced much lower production of cytokines (such as tumor necrosis factor alpha and interleukins 1beta, 10, and 12) by DCs than did Y. pestis grown at 26 degrees C or LPS-26 degrees C. Expression of the surface markers HLA-DR, CD86, and CD40 by DCs was also reduced in response to treatment with LPS-37 degrees C compared with LPS-26 degrees C. Pretreatment of DCs with LPS-37 degrees C inhibited subsequent stimulation with LPS-26 degrees C, control LPS from E. coli, lipoprotein, or poly-IC. CONCLUSIONS: LPS-37 degrees C can inhibit stimulation of DCs not only via TLR4 signaling but also via TLR2 and TLR3. [corrected]


Assuntos
Células Dendríticas/imunologia , Lipopolissacarídeos/farmacologia , Receptores Toll-Like/antagonistas & inibidores , Yersinia pestis/imunologia , Acilação , Antígeno B7-2/metabolismo , Antígenos CD40/metabolismo , Células Cultivadas , Citocinas/biossíntese , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Escherichia coli/química , Antígenos HLA-DR/metabolismo , Humanos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Poli I-C/farmacologia , Transdução de Sinais , Temperatura , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Yersinia pestis/metabolismo
3.
Microb Pathog ; 47(5): 243-51, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19716410

RESUMO

Type VI secretion systems (T6SSs) have been identified recently in several Gram-negative organisms and have been shown to be associated with virulence in some bacterial pathogens. A T6SS of Yersinia pestis CO92 (locus YPO0499-YPO0516) was deleted followed by investigation of the phenotype of this mutation. We observed that this T6SS locus of Y. pestis was preferentially expressed at 26 degrees C in comparison to 37 degrees C suggesting a possible role in the flea cycle. However, we found that the deletion of T6SS locus YPO0499-YPO0516 in Y. pestis CO92 had no effect on the ability of this strain to infect the oriental rat flea, Xenopsylla cheopis. Nevertheless, this mutant displayed increased intracellular numbers in macrophage-like J774.A1 cells after 20 h post-infection for bacterial cells pre-grown at 26 degrees C indicating that expression of this T6SS locus limited intracellular replication in macrophages. In addition, deletion of the YPO0499-YPO0516 locus reduced the uptake by macrophages of the Y. pestis mutant pre-grown at 37 degrees C, suggesting that this T6SS locus has phagocytosis-promoting activity. Further study of the virulence of the T6SS mutant in murine bubonic and inhalation plague models revealed no attenuation in comparison with the parental CO92 strain.


Assuntos
Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/genética , Mutação , Peste/microbiologia , Sifonápteros/microbiologia , Yersinia pestis/genética , Yersinia pestis/patogenicidade , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Deleção de Sequência , Análise de Sobrevida , Temperatura
4.
Cell Microbiol ; 10(6): 1327-38, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18248629

RESUMO

Drosophila melanogaster is a widely used model organism for research on innate immunity and serves as an experimental model for infectious diseases. The aetiological agent of the zoonotic disease tularaemia, Francisella tularensis, can be transmitted by ticks and mosquitoes and Drosophila might be a useful, genetically amenable model host to elucidate the interactions between the bacterium and its arthropod vectors. We found that the live vaccine strain of F. tularensis was phagocytosed by Drosophila and multiplied in fly haemocytes in vitro and in vivo. Bacteria injected into flies resided both inside haemocytes and extracellularly in the open circulatory system. A continuous activation of the humoral immune response, i.e. production of antimicrobial peptides under control of the imd/Relish signalling pathway, was observed and it may have contributed to the relative resistance to F. tularensis as flies defective in the imd/Relish pathway died rapidly. Importantly, bacterial strains deficient for genes of the F. tularensis intracellular growth locus or the macrophage growth locus were attenuated in D. melanogaster. Our results demonstrate that D. melanogaster is a suitable model for the analysis of interactions between F. tularensis and its arthropod hosts and that it can also be used to identify F. tularensis virulence factors relevant for mammalian hosts.


Assuntos
Drosophila melanogaster/microbiologia , Francisella tularensis , Tularemia/metabolismo , Tularemia/microbiologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/farmacologia , Drosophila melanogaster/imunologia , Francisella tularensis/crescimento & desenvolvimento , Francisella tularensis/patogenicidade , Genes de Insetos/genética , Hemócitos/microbiologia , Imunidade Inata , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Transdução de Sinais , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência
5.
PLoS Negl Trop Dis ; 12(6): e0006511, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29889829

RESUMO

BACKGROUND: To establish correlates of human immunity to the live plague vaccine (LPV), we analyzed parameters of cellular and antibody response to the plasminogen activator Pla of Y. pestis. This outer membrane protease is an essential virulence factor that is steadily expressed by Y. pestis. METHODOLOGY/PRINCIPAL FINDINGS: PBMCs and sera were obtained from a cohort of naïve (n = 17) and LPV-vaccinated (n = 34) donors. Anti-Pla antibodies of different classes and IgG subclasses were determined by ELISA and immunoblotting. The analysis of antibody response was complicated with a strong reactivity of Pla with normal human sera. The linear Pla B-cell epitopes were mapped using a library of 15-mer overlapping peptides. Twelve peptides that reacted specifically with sera of vaccinated donors were found together with a major cross-reacting peptide IPNISPDSFTVAAST located at the N-terminus. PBMCs were stimulated with recombinant Pla followed by proliferative analysis and cytokine profiling. The T-cell recall response was pronounced in vaccinees less than a year post-immunization, and became Th17-polarized over time after many rounds of vaccination. CONCLUSIONS/SIGNIFICANCE: The Pla protein can serve as a biomarker of successful vaccination with LPV. The diagnostic use of Pla will require elimination of cross-reactive parts of the antigen.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Imunidade Celular , Imunidade Humoral , Vacina contra a Peste/imunologia , Ativadores de Plasminogênio/imunologia , Yersinia pestis/imunologia , Adulto , Idoso , Biomarcadores/sangue , Citocinas/biossíntese , Citocinas/imunologia , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Peste/sangue , Peste/imunologia , Peste/microbiologia , Peste/prevenção & controle , Células Th17/imunologia , Vacinação , Vacinas Vivas não Atenuadas/administração & dosagem , Vacinas Vivas não Atenuadas/imunologia , Fatores de Virulência
6.
Clin Vaccine Immunol ; 23(7): 586-600, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27170642

RESUMO

Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models.


Assuntos
Adenovírus Humanos/genética , Portadores de Fármacos , Vacina contra a Peste/imunologia , Peste/prevenção & controle , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Modelos Animais de Doenças , Feminino , Esquemas de Imunização , Injeções Intramusculares , Interferon gama/metabolismo , Macaca fascicularis , Masculino , Camundongos , Peste/patologia , Vacina contra a Peste/administração & dosagem , Vacina contra a Peste/genética , Análise de Sobrevida , Linfócitos T/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Replicação Viral , Yersinia pestis/genética , Yersinia pestis/imunologia
7.
Clin Vaccine Immunol ; 20(2): 227-38, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23239803

RESUMO

Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1(-) strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1(-) mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1(-) CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Imunização Passiva , Vacina contra a Peste/imunologia , Proteínas Recombinantes/imunologia , Yersinia pestis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Soros Imunes , Imunização , Levofloxacino , Camundongos , Ofloxacino/uso terapêutico , Peste/imunologia , Peste/microbiologia , Peste/prevenção & controle , Vacina contra a Peste/uso terapêutico , Ativadores de Plasminogênio/genética , Ativadores de Plasminogênio/imunologia , Ratos , Proteínas Recombinantes/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/uso terapêutico , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Yersinia pestis/genética
8.
J Med Microbiol ; 60(Pt 11): 1570-1583, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21700740

RESUMO

Francisella tularensis is a highly virulent intracellular bacterium capable of rapid multiplication in phagocytic cells. Previous studies have revealed that activation of F. tularensis-infected macrophages leads to control of infection and reactive nitrogen and oxygen species make important contributions to the bacterial killing. We investigated the effects of adding S-nitroso-acetyl-penicillamine (SNAP), which generates nitric oxide, or 3-morpholinosydnonimine hydrochloride, which indirectly leads to formation of peroxynitrite, to J774 murine macrophage-like cell cultures infected with F. tularensis LVS. Addition of SNAP led to significantly increased colocalization between LAMP-1 and bacteria, indicating containment of F. tularensis in the phagosome within 2 h, although no killing occurred within 4 h. A specific inhibitory effect on bacterial transcription was observed since the gene encoding the global regulator MglA was inhibited 50-100-fold. F. tularensis-infected J774 cells were incapable of secreting TNF-α in response to Escherichia coli LPS but addition of SNAP almost completely reversed the suppression. Similarly, infection with an MglA mutant did not inhibit LPS-induced TNF-α secretion of J774 cells. Strong staining of nitrotyrosine was observed in SNAP-treated bacteria, and MS identified nitration of two ribosomal 50S proteins, a CBS domain pair protein and bacterioferritin. The results demonstrated that addition of SNAP initially did not affect the viability of intracellular F. tularensis LVS but led to containment of the bacteria in the phagosome. Moreover, the treatment resulted in modification by nitration of several F. tularensis proteins.


Assuntos
Francisella tularensis/imunologia , Macrófagos/imunologia , Molsidomina/análogos & derivados , Doadores de Óxido Nítrico/farmacologia , S-Nitroso-N-Acetilpenicilamina/farmacologia , Tularemia/imunologia , Animais , Assialoglicoproteínas/genética , Assialoglicoproteínas/imunologia , Proteínas de Bactérias/imunologia , Western Blotting , Linhagem Celular , Sobrevivência Celular/imunologia , Francisella tularensis/genética , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Macrófagos/microbiologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Molsidomina/farmacologia , Ácido Peroxinitroso/imunologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tularemia/microbiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia
9.
Infect Genet Evol ; 10(1): 137-45, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19835996

RESUMO

The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium and is generally conserved between the epidemic strains of Yersinia pestis. We investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from 19 Y. pestis strains belonging to different phylogenetic groups (subspecies) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324-326. These major variations, together with other minor substitutions in amino acid sequences, allowed us to classify the LcrV alleles into five sequence types (A-E). We observed that the strains of different Y. pestis "subspecies" can have the same type of LcrV, including that conserved in epidemic strains, and different types of LcrV can exist within the same natural plague focus. Therefore, the phenomenon of "selective virulence" characteristic of the strains of the microtus biovar is unlikely to be the result of polymorphism of the V antigen. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in the oligomerization of LcrV.


Assuntos
Aminoácidos/genética , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Yersinia pestis/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/imunologia , Genes Bacterianos , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo , Yersinia pestis/imunologia , Yersinia pestis/patogenicidade
10.
Microb Pathog ; 34(6): 297-308, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12782482

RESUMO

The YopE cytotoxin of Yersinia is an essential virulence determinant that is translocated into the eukaryotic target cell via a plasmid-encoded type III secretion system. YopE possess a GTPase activating protein activity that in vitro has been shown to down regulate RhoA, Rac1, and Cdc42. Translocated YopE induces de-polymerisation of the actin microfilament structure in the eukaryotic cell which results in a rounding up of infected cells described as a cytotoxic effect. Here, we have investigated the importance of different regions of YopE for induction of cytotoxicity and in vitro GAP activity. Sequential removal of the N- and C-terminus of YopE identified the region between amino acids 90 and 215 to be necessary for induction of cytotoxicity. Internal deletions containing the essential arginine at position 144 resulted in a total loss of cytotoxic response. In-frame deletions flanking the arginine finger defined a region important for the cytotoxic effect to amino acids 166-183. Four triple-alanine substitution mutants in this region, YopE166-8A, 169-71A, 175-7A and 178-80A were still able to induce cytotoxicity on HeLa cells although they did not show any in vitro GAP activity towards RhoA, Rac1 or Cdc42. A substitution mutant in position 206-8A showed the same phenotype, ability to induce cytotoxic response but no in vitro GAP activity. We speculate that YopE may have additional unidentified targets within the eukaryotic cell.


Assuntos
Proteínas da Membrana Bacteriana Externa/toxicidade , Toxinas Bacterianas/toxicidade , Proteínas Ativadoras de GTPase/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Testes Imunológicos de Citotoxicidade , Citotoxinas/metabolismo , Deleção de Genes , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes/análise , Alinhamento de Sequência , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
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