RESUMO
To identify Yersinia pestis genes involved in the microbe's resistance to cationic antimicrobial peptides, the strategy of random transposon mutagenesis with a Tn5 minitransposon was used, and the library was screened for detecting polymyxin B (PMB) susceptible mutants. The mutation responsible for PMB-sensitive phenotype and the lipopolysaccharide (LPS) structure were characterized for the Y. pestis strain KM218-A3. In this strain the mini-Tn5 was located in an open reading frame with the product homologous to the E. coli protein GmhB (82% identity) functioning as d-glycero-d-manno-heptose-1,7-diphosphate phosphatase. ESI FT ICR mass spectrometry of anions was used to study the structure of the unmodified LPS of Y. pestis KM218-A3, and molecules were revealed with the full-size LPS core or with two types of an incomplete core: consisting of Kdo-Kdo or Ko-Kdo disaccharides and Hep-(Kdo)-Kdo or Hep-(Ko)-Kdo trisaccharides. The performed complementation confirmed that the defect in the biological properties of the mutant strain was caused by inactivation of the gmhB gene. These findings indicated that the gmhB gene product of Y. pestis is essential for production of wild-type LPS resistant to antimicrobial peptides and serum.
Assuntos
Elementos de DNA Transponíveis/genética , Yersinia pestis/metabolismo , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Carboidratos , Farmacorresistência Bacteriana/genética , Lipopolissacarídeos/análise , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Testes de Sensibilidade Microbiana , Mutagênese , Polimixina B/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Yersinia pestis/efeitos dos fármacos , Yersinia pestis/genéticaRESUMO
Experimental serological tests were developed to determine anti-tularensis antibodies in humans in immunochromatography formats (LF-test LPS Ft15) and enzyme immunoassay (ELISA LPS Ft15) using as an antigen highly purified LPS F. tularensis 15 NIIEG. Analysis was conducted of the sensitivity and specificity of the developed tests and commercial tularemia antigen «RNGA-Tul-AG¼ (production Stavropol research anti-plague Institute) in comparison with the commercial reference antigen, registered in the Russian Federation for the quantitative determination of human IgG tularemia - «ELISA classic Francisella tularensis IgG¼ SERION, Germany (IgG SERION ELISA). A study of human blood serum vaccinated against tularemia showed that the sensitivity and specificity of detection of anti-tularemia antibodies by «ELISA LPS Ft15¼ were 97.7 and 100%, compared with «ELISA IgG series¼. When determining antitularemia antibodies with the diagnosis «LF-test LPS Ft15¼, these parameters were compared to «ELISA IgG series¼ 94.3 and 100%. The sensitivity and specificity of «RNGA-Tul-AG¼ made compared to the «IgG ELISA, SERION¼ of 59.1% and 80%.
Assuntos
Tularemia , Anticorpos Antibacterianos , Ensaio de Imunoadsorção Enzimática , Humanos , Federação Russa , Testes SorológicosRESUMO
The live vaccine based on the Francisella tularensis subsp. holarctica vaccine strain 15 NIIEG line is used in Russia against tularemia. This vaccine is highly effective, but fairly unstable. Therefore, development of stable live tularemia vaccine with minimal side effect is rather urgent. The method of allel removal in the F. tularensis vaccine strain was used to remove one copy of the iglC gene, which is required to provide intracellular production of the vaccine strain, as well as removal of the recA gene. The latter is crucial for homological recombination. pGM5 suicide vector based on pHV33 bireplicon plasmid was constructed to provide replacement of intact F. tularensis chromosome segments by modified segments. Modified chromosome segments contain F. Tularensis DNA fragment without iglC structural gene segment 545 p. b. (in pGMΔiglC plasmid), as well as DNA fragment containing no recA structural gene segment 1060 p.b. (pGMΔrecA plasmid). The constructed 15/23-1ΔrecA mutant, in contrast to the vaccine strain 15, was capable of reproducing in the macrophage-like cells J774A.1 line, whereas the efficiency of the reproduction was 8-10 times less. BALB/c mouse responded to immunization by the 15/23-1ΔrecA strain by smaller weight decrease (-2%) as compared to the strain 15 (-14%). Bacteria of the 15/23-1ΔrecA strain were virtually incapable of germinating from the BALB/c murine spleen 14 days after invasion, whereas bacteria of the strain 15 were found in the murine organs even after 21 days. The F. tularensis 15/23-1ΔrecA strain having smaller reaction ability can be used as a basis for construction of stable live safe tularemia vaccine.
Assuntos
Proteínas de Bactérias , Genes Bacterianos/imunologia , Vetores Genéticos , Recombinases Rec A , Tularemia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Linhagem Celular , Francisella tularensis/genética , Francisella tularensis/imunologia , Francisella tularensis/metabolismo , Camundongos , Recombinases Rec A/genética , Recombinases Rec A/imunologia , Recombinases Rec A/metabolismo , Tularemia/genética , Tularemia/imunologia , Tularemia/metabolismo , Tularemia/prevenção & controleRESUMO
The immunomodulating effect of the components of an Ixodes persulcatus (Ixodidae) tick salivary gland extract (SGE) on BALB/c mice lymphocytes was evaluated. SGE of partially engorged ticks at a concentration of 50 microg/ml causes the maximum suppression ofT- and B-lymphocyte subpopulations. SGE of hungry ticks at the same concentration induces the suppression of only CD69+ T cells and TLR-2+ B cells, but produces no suppressive effect on CD69+ B lymphocytes, TLR-2+ T lymphocytes, and TLR-4+ T and B lymphocytes. SGE shows different effects on the synthesis of IFN-gamma and IL-4 by T helper cells. SGE of hungry ticks stimulated the increase of IFN-gamma and IL-4 synthesis by 4.7 and 2.6 times, respectively, as compared to the control. The findings may be of value in studying the pathogenesis of transmissible infections and in designing the vaccines based on tick gland components.
Assuntos
Linfócitos B/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Ixodes/química , Glândulas Salivares/química , Linfócitos T/efeitos dos fármacos , Extratos de Tecidos/farmacologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Células Cultivadas , Expressão Gênica , Fatores Imunológicos/imunologia , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-10/biossíntese , Interleucina-10/imunologia , Interleucina-4/biossíntese , Interleucina-4/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/citologia , Linfócitos T/imunologia , Extratos de Tecidos/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
In silico analysis of available bacterial genomes revealed the phylogenetic proximity levels of enzymes responsible for biosynthesis of lipopolysaccharide (LPS) of Yersinia pestis, the cause of plague, to homologous proteins of closely related Yersinia spp. and some other bacteria (Serratia proteamaculans, Erwinia carotovora, Burkholderia dolosa, Photorhabdus luminescens and others). Isogenic Y. pestis mutants with single or double mutations in 14 genes of LPS biosynthetic pathways were constructed by site-directed mutagenesis on the base of the virulent strain 231 and its attenuated derivative. Using high-resolution electrospray ionization mass spectrometry, the full LPS structures were elucidated in each mutant, and the sequence of monosaccharide transfers in the assembly of the LPS core was inferred. Truncation of the core decreased significantly the resistance of bacteria to normal human serum and polymyxin B, the latter probably as a result of a less efficient incorporation of 4-amino-4-deoxyarabinose into lipid A. Impairing of LPS biosynthesis resulted also in reduction of LPS-dependent enzymatic activities of plasminogen activator and elevation of LD(50) and average survival time in mice and guinea pigs infected with experimental plague. Unraveling correlations between biological properties of bacteria and particular LPS structures may help a better understanding of pathogenesis of plague and implication of appropriate genes as potential molecular targets for treatment of plague.
Assuntos
Genes Bacterianos/fisiologia , Lipopolissacarídeos/biossíntese , Yersinia pestis/enzimologia , Yersinia pestis/genética , Amino Açúcares/metabolismo , Animais , Atividade Bactericida do Sangue , Farmacorresistência Bacteriana , Feminino , Cobaias , Humanos , Lipídeo A/biossíntese , Masculino , Camundongos , Peste/microbiologia , Ativadores de Plasminogênio/metabolismo , Polimixina B/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Virulência , Yersinia pestis/efeitos dos fármacos , Yersinia pestis/patogenicidadeRESUMO
A knockout mutant with a deletion in a quorum sensing system gene qseC was generated from the vaccine strain Francisella tularensis 15 by site-directed mutagenesis. The variant with the inactivated gene qseC differed from the parental strain in growth rate on solid nutrient medium but had the same growth dynamics in liquid nutrient medium. The mutation abolished almost completely the resistance of the vaccine strain to normal rabbit serum and its ability to survive in macrophages; in addition, the strain lost the residual virulence. A significant phenotypic alteration was observed in the lipopolysaccharide of F. tularensis. Particularly, the mutant strain synthesized no noticeable amount of the lipopolysaccharide with the high-molecular-mass O-polysaccharide, presumably as a result of impairing biosynthesis of the repeating unit, namely, a loss of the ability to incorporate a formyl group, an N-acyl substituent of 4-amino-4,6-dideoxy-D-glucose.
Assuntos
Proteínas de Bactérias/genética , Francisella tularensis/genética , Lipopolissacarídeos/química , Percepção de Quorum/genética , Animais , Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Francisella tularensis/metabolismo , Técnicas de Inativação de Genes , Mutagênese Sítio-Dirigida , Antígenos O/química , Fenótipo , Coelhos , Espectrometria de Massas por Ionização por Electrospray , VirulênciaRESUMO
The endotoxic activity of the lipopolysaccharides (LPS) with defined chemical structure from Yersinia pestis strains of various subspecies differing in their epidemic potential was studied. The LPS of two strains of Y. pestis ssp. caucasica and ssp. altaica, whose structures have not been studied earlier, were analyzed by high-resolution mass spectrometry. In addition to reported structural changes, an increase in the degree of LPS phosphorylation was observed when strain I-2377 (ssp. altaica) was cultivated at an elevated temperature. A high tumor necrosis factor alpha(TNF-alpha)-inducing activity observed for LPS samples from Y. pestis cultures grown at 25 degrees C correlated with an increased degree of lipid A acylation, particularly, with the presence of the hexaacyl form of lipid A, which was absent from the LPS when bacteria were cultivated at 37 degrees C. No correlation was found between the lethal toxicity of the LPS in vivo and its ability to induce TNF-alpha production in vitro.
Assuntos
Lipopolissacarídeos/química , Lipopolissacarídeos/toxicidade , Yersinia pestis/química , Animais , Sequência de Carboidratos , Linhagem Celular , Feminino , Dose Letal Mediana , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Dados de Sequência Molecular , Temperatura , Fator de Necrose Tumoral alfa/metabolismoRESUMO
It was shown that spore germination of different Bacillus anthracis strains in macrophage-like cells J774A.1 depended on the genotype of the strains. The virulent B. anthracis strains contain plasmids pXO1 and pX02 responsible for the synthesis of a toxin and a capsule, respectively. The loss of one of the plasmids results in the reduction of strain virulence. It was shown that effective survival of germinating spores in macrophages occurred in the presence of plasmid pXO1 only. The spores of the B. anthracis strains ?Ames and STI-Rif deprived of plasmid pXO1 were least adapted to passing through the intracellular stage. The B. anthracis strains 81/1 and 71/12 (carrying plasmids pXO1 and pXO2 and synthesizing the toxin and capsule) less effectively survived in the cytoplasm of macrophages than the strain STI-1 which has only the plasmid pXO1. It was found that the rate of synthesis of the capsule consisting of polymer gamma-D-glutamic acid depended on the ability of bacterial cells to escape from macrophages. In the B. anthracis strains carrying plasmid pXO2, capsule synthesis by vegetative cells was activated within macrophages that promoted a rapid escape of the vegetative cells from the macrophages. On the contrary, most of capsule-free cells of the vaccine strain STI-1 remained inside macrophages during the whole period of observation. Thus, integrated regulation of two processes, namely synthesis of the toxin components participating in the transition of the germinating cell from phagosome into cytoplasm, and synthesis of the capsule whose presence promotes rapid escape of bacterial cells from macrophages by presently unknown mechanism play the key role in anthrax development at early stages.
Assuntos
Bacillus anthracis/genética , Bacillus anthracis/patogenicidade , Esporos Bacterianos/patogenicidade , Animais , Antraz/microbiologia , Vacinas contra Antraz/genética , Bacillus anthracis/fisiologia , Proteínas de Bactérias/metabolismo , Células Cultivadas , Macrófagos/microbiologia , Camundongos , Plasmídeos/genética , Ácido Poliglutâmico/metabolismo , Especificidade da Espécie , Esporos Bacterianos/citologia , Esporos Bacterianos/genética , Transativadores/metabolismoRESUMO
Antibiotic fosmidomycin will know as inhibitor of the nonmevalonate pathway of isoprenoid biosynthesis and as possible antimalarial drug, was shown to possess a certain protective effect on mice experimentally infected with tularemia, tiphus or coli-septicemia. Positive effect on mice with chronic form of tuberculosis was not observed when the animals were given 1 mg of fosmidomycin per capita twice a day. Under oxidative conditions an ESR signal of long living nitroxil free radicals were registered in the water solution of fosmidomycin. The radicals are supposed to be involved in the therapeutic effect of the antibiotic.
Assuntos
Antibacterianos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Fosfomicina/análogos & derivados , Fosfomicina/uso terapêutico , Animais , Antibacterianos/administração & dosagem , Infecções Bacterianas/microbiologia , Infecções Bacterianas/mortalidade , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/mortalidade , Fosfomicina/administração & dosagem , Camundongos , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/microbiologia , Infecções por Salmonella/mortalidade , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/mortalidade , Tularemia/tratamento farmacológico , Tularemia/microbiologia , Tularemia/mortalidade , Tifo Epidêmico Transmitido por Piolhos/tratamento farmacológico , Tifo Epidêmico Transmitido por Piolhos/microbiologia , Tifo Epidêmico Transmitido por Piolhos/mortalidadeRESUMO
A strong immunomodulatory effect of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC) responsible for the survival of bacteria was shown on isolated macrophages and in experimental infections in mice (typhoid and tularemia). Derivatives of MEC were found by 1H-NMR spectroscopy under stress conditions in colorless mutants of the bacteria and isolated to be subsequently purified and used for modulation of the immune system of animals.
Assuntos
Adjuvantes Imunológicos/farmacologia , Eritritol/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Adjuvantes Imunológicos/química , Animais , Corynebacterium , Eritritol/análogos & derivados , Eritritol/química , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Tularemia/imunologia , Febre Tifoide/imunologiaRESUMO
Some aspects of homeostasis impairment in monkeys infected with Pseudomonas mallei were investigated. The levels of the hormonal shifts, complement components, beta 2-microglobulin and prostaglandins evident of the infection severity were estimated. The pathomorphological profiles of various forms of malleus in the primates were studied.