RESUMO
The apprehension of needles related to injection site pain, risk of transmitting bloodborne pathogens, and effective mass immunization have led to the development of a needle-free injection system (NFIS). Here, we evaluated the efficacy of the NFIS and needle injection system (NIS) for the delivery and immunogenicity of DNA vaccine candidate ZyCoV-D in rhesus macaques against SARS-CoV-2 infection. Briefly, 20 rhesus macaques were divided into 5 groups (4 animals each), that is, I (1 mg dose by NIS), II (2 mg dose by NIS), III (1 mg dose by NFIS), IV (2 mg dose by NFIS) and V (phosphate-buffer saline [PBS]). The macaques were immunized with the vaccine candidates/PBS intradermally on Days 0, 28, and 56. Subsequently, the animals were challenged with live SARS-CoV-2 after 15 weeks of the first immunization. Blood, nasal swab, throat swab, and bronchoalveolar lavage fluid specimens were collected on 0, 1, 3, 5, and 7 days post infection from each animal to determine immune response and viral clearance. Among all the five groups, 2 mg dose by NFIS elicited significant titers of IgG and neutralizing antibody after immunization with enhancement in their titers postvirus challenge. Besides this, it also induced increased lymphocyte proliferation and cytokine response. The minimal viral load post-SARS-CoV-2 challenge and significant immune response in the immunized animals demonstrated the efficiency of NFIS in delivering 2 mg ZyCoV-D vaccine candidate.
Assuntos
COVID-19 , Vacinas de DNA , Vacinas Virais , Animais , SARS-CoV-2 , Macaca mulatta , Anticorpos Neutralizantes , Anticorpos Antivirais , Imunogenicidade da VacinaRESUMO
In vitro methods for quantification of immunodominant glycoprotein in the rabies vaccine formulations serve as good alternative to the cumbersome and variable mice potency assay as a batch release test for the vaccine. The present study presents the development of a sandwich ELISA with optimal concentrations of a high affinity recombinant diabody (D06) and a specific monoclonal antibody (M5B4) against rabies glycoprotein for its quantification in the vaccine formulations. The glycoprotein estimate correlated linearly (r2 = 0.8) to the in vivo potency estimate for the vaccine formulations. This ELISA promises a good forecast of the mice potency values and thereby can serve as a simple, yet effective batch release test for the rabies vaccines replacing the in vivo assay.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas/análise , Vacina Antirrábica/química , Animais , Glicoproteínas/imunologia , Humanos , Camundongos , Vacina Antirrábica/imunologiaRESUMO
Specific antibody responses were assessed in pigs immunized with the Taenia solium vaccine TSOL18. Anti-TSOL18 responses were compared 2 weeks after secondary immunization, where the interval between primary and secondary immunization was 4, 8, 12, 16 or 20 weeks. All animals responded to the vaccine and there was no diminution in antibody responses in animals receiving their second injection after an interval up to 20 weeks. Pigs receiving vaccinations at an interval of 12 weeks developed significantly increased antibody responses compared with animals receiving immunizations 4 weeks apart (P = 0.046). The ability to deliver TSOL18 vaccination effectively where the revaccination schedule can be delayed for up to 12-16 weeks in pigs increases the options available for designing T. solium control interventions that incorporate TSOL18 vaccination.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Imunização Secundária/veterinária , Doenças dos Suínos/prevenção & controle , Taenia solium/imunologia , Teníase/veterinária , Vacinas/imunologia , Animais , Anticorpos Anti-Helmínticos/biossíntese , Feminino , Esquemas de Imunização , Imunização Secundária/normas , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Injeções Intramusculares/veterinária , Masculino , Suínos , Doenças dos Suínos/imunologia , Teníase/imunologia , Teníase/prevenção & controle , Fatores de Tempo , Vacinas/administração & dosagemRESUMO
Japanese Encephalitis (JE) is a mosquito borne arboviral infection caused by Japanese Encephalitis Virus (JEV). It is a major cause of viral encephalitis in Asian countries including India. In the present study, we have used a Tymovirus [i.e. Physalis Mottle Virus (PhMV) coat protein (CP)], which forms virus like particles (VLPs) as a template to display immunodominant epitopes of JEV envelope (E) protein. The immunodominant epitopes of JEV were inserted at the N-terminus of the wild type PhMV CP, and these constructs were cloned and expressed in Escherichia coli. The chimeric proteins were purified from the inclusion bodies and evaluated for VLP formation. The purified protein was identified by Western blotting and VLP formation was studied and confirmed by transmission electron microscopy and dynamic light scattering. Finally, the immunogenicity was studied in mice. Our results indicate that the chimeric protein with JEV epitopes assembled efficiently to form VLPs generating neutralizing antibodies. Hence, we report the purified chimeric VLP would be a potent vaccine candidate, which needs to be evaluated in a mouse challenge model.
Assuntos
Proteínas do Capsídeo/metabolismo , Epitopos Imunodominantes/metabolismo , Corpos de Inclusão Viral/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tymovirus/genética , Proteínas do Envelope Viral/metabolismo , Animais , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Corpos de Inclusão Viral/química , Corpos de Inclusão Viral/imunologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Redobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
OBJECTIVE: To evaluate the persistence of antibodies three years after primary vaccination with typhoid conjugate vaccine (TCV) of either Cadila Healthcare Ltd. (Cadila-TCV) or Bharat Biotech International Ltd. (Bharat-TCV) administered in a previous phase II/III study, and to study the booster dose response to Cadila-TCV. METHODS: This was an open-label, phase IV extension study conducted in tertiary care and multispecialty hospitals in India. 112 subjects (Cadila-TCV-57, Bharat-TCV-55) who had participated in previous study were enrolled. Of these, eligible subjects received a single-dose of Cadila-TCV and were followed-up for 28 days post-booster. Primary outcome was persistence of antibodies 3 years after primary vaccination and seroconversion (≥4-fold rise in antibody titre from baseline) 28 days post-booster. Safety was based on reported adverse events (AEs) post-booster. RESULTS: The baseline GMT reported in the current study was significantly higher than pre-vaccination GMT reported in the previous study. 89/112 (79.5%) subjects had antibody titer ≥10 IU/mL at baseline; eligible subjects (n=17) who had baseline antibody titre <10 IU/mL were administered booster dose. All the vaccinated subjects showed seroconversion post-booster. The GMTs reported at 10 days and 28 days post-booster were significantly higher as compared to GMTs reported after primary vaccination in previous study. 4 (23.5%) vaccinated subjects reported 9 AEs; all were solicited and of mild/moderate intensity. CONCLUSIONS: There was a significant persistence of immunogenicity after primary vaccination with both the TCVs, and robust immune response after booster vaccination with Cadila-TCV.
Assuntos
Febre Tifoide , Vacinas Tíficas-Paratíficas , Humanos , Imunização Secundária , Soroconversão , Febre Tifoide/prevenção & controle , Vacinas Tíficas-Paratíficas/efeitos adversos , Vacinação , Vacinas Conjugadas/efeitos adversosRESUMO
BACKGROUND: ZyCoV-D is a DNA vaccine candidate, which comprises a plasmid DNA carrying spike-S gene of SARS-CoV-2 virus along with gene coding for signal peptide. The spike(S) region includes the receptor-binding domain (RBD), which binds to the human angiotensin converting Enzyme (ACE)-2 receptor and mediates the entry of virus inside the cell. METHODS: We conducted a single-center, open-label, non-randomized, Phase 1 trial in India between July 2020 and October 2020. Healthy adults aged between 18 and 55 years were sequentially enrolled and allocated to one of four treatment arms in a dose escalation manner. Three doses of vaccine were administered 28 days apart and each subject was followed up for 28 days post third dose to evaluate safety and immunogenicity. FINDINGS: Out of 126 individuals screened for eligibility. Forty-eight subjects (mean age 34·9 years) were enrolled and vaccinated in the Phase 1 study Overall, 12/48 (25%) subjects reported at least one AE (i.e. combined solicited and unsolicited) during the study. There were no deaths or serious adverse events reported in Phase 1 of the study. The proportion of subjects who seroconverted based on IgG titers on day 84 was 4/11 (36·36%), 4/12 (33·33%), 10/10 (100·00%) and 8/10 (80·00%) in the treatment Arm 1 (1 mg: Needle), Arm 2 (1 mg: NFIS), Arm 3 (2 mg: Needle) and Arm 4 (2 mg: NFIS), respectively. INTERPRETATION: ZyCoV-D vaccine is found to be safe, well-tolerated and immunogenic in the Phase 1 trial. Our findings suggest that the DNA vaccine warrants further investigation.
RESUMO
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We developed a candidate vaccine ZyCoV-D comprising of a DNA plasmid vector carrying the gene encoding the spike protein (S) of the SARS-CoV-2 virus. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500 µg dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also elicited Th-1 response as evidenced by elevated IFN-γ levels.
Assuntos
COVID-19 , Vacinas de DNA , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Formação de Anticorpos , Vacinas contra COVID-19 , China , Escherichia coli , Cobaias , Humanos , Camundongos , Modelos Animais , Coelhos , Ratos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Interleukin-2 (IL-2), a potent cytokine has been used in anti-cancer therapy for over a decade now. IL-2, originally identified as a growth factor for T lymphocytes is a 15 kDa hydrophobic glycoprotein that induces the activation, clonal proliferation and differentiation of T and B-lymphocytes and enhances the cytotoxicity of monocytes and natural killer (NK) cells. Here, we report a simple method for the cloning, high-level expression and purification of IL-2 protein, which can be easily extended to other bioactive therapeutic proteins. The IL-2 gene was amplified from human spleen cDNA and cloned in a prokaryotic (E. coli) expression system. An optimal expression of the IL-2 protein was determined by varying the expression conditions like temperature, inducer concentration and duration of induction. The protein was expressed as inclusion bodies and a panel of reagents including detergents, urea and guanidine hydrochloride were used to solubilize it. After solubilization, the protein was renatured and subjected to a single step gel-filtration chromatography to yield immunobioactive IL-2 protein with > 99% purity.
Assuntos
Escherichia coli/genética , Interleucina-2/genética , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-2/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Anthrax toxin produced by Bacillus anthracis is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF into the cytosol. EF is a calmodulin-dependent adenylate cyclase that causes edema whereas LF is a zinc metalloprotease and leads to necrosis of macrophages. It is also important to note that the exact mechanism of LF action is still unclear. With this view in mind, in the present study, we investigated a proteome wide effect of anthrax lethal toxin (LT) on mouse macrophage cells (J774A.1). Proteome analysis of LT-treated and control macrophages revealed 41 differentially expressed protein spots, among which phosphoglycerate kinase I, enolase I, ATP synthase (beta subunit), tubulin beta2, gamma-actin, Hsp70, 14-3-3 zeta protein and tyrosine/tryptophan-3-monooxygenase were found to be down-regulated, while T-complex protein-1, vimentin, ERp29 and GRP78 were found to be up-regulated in the LT-treated macrophages. Analysis of up- and down-regulated proteins revealed that primarily the stress response and energy generation proteins play an important role in the LT-mediated macrophage cell death.
Assuntos
Antígenos de Bactérias/toxicidade , Bacillus anthracis/química , Toxinas Bacterianas/toxicidade , Ativação Enzimática/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteoma/análise , Trifosfato de Adenosina/metabolismo , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Citoesqueleto/metabolismo , Regulação para Baixo , Edema/imunologia , Edema/metabolismo , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática/fisiologia , Proteínas de Choque Térmico/metabolismo , Dose Letal Mediana , Macrófagos/metabolismo , Metaloendopeptidases/metabolismo , Camundongos , Proteoma/metabolismo , Regulação para CimaRESUMO
Protective antigen (PA) is the central receptor binding component of anthrax toxin, which translocates catalytic components of the toxin into the cytosol of mammalian cells. Ever since the crystal structure of PA was solved, there have been speculations regarding the possible role of calcium ions present in domain I of the protein. We have carried out a systematic study to elucidate the effect of calcium removal on the structural stability of PA using various optical spectroscopic techniques, limited proteolysis and mutational analysis. Urea denaturation studies clearly suggest that the unfolding pathway of the protein follows a non-two state transition with apo-PA being an intermediate species, whereas the folding pathway shows that calcium ions may be critical for the initial protein assembly.
Assuntos
Antígenos de Bactérias/química , Bacillus anthracis/metabolismo , Toxinas Bacterianas/química , Cálcio/química , Substituição de Aminoácidos , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/farmacologia , Apoproteínas/química , Apoproteínas/genética , Bacillus anthracis/química , Bacillus anthracis/imunologia , Toxinas Bacterianas/genética , Cálcio/metabolismo , Linhagem Celular , Dicroísmo Circular , Immunoblotting , Dose Letal Mediana , Camundongos , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Espectrometria de Fluorescência , Tripsina/química , Ureia/químicaRESUMO
In the post-genomic era the concept of personalized medicine and molecular medicine emphasizes the utility of the proteomics approach. Proteomics is the global analysis of cellular proteins and complements the genomics approach. Proteins, in principle do all the work of the cell and ultimately dictate all biological processes and the cellular fate. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing and bioinformatics to identify and characterize proteins. This review aims at providing the various approaches and pitfalls associated with this technique and gives a brief overview of the utility of this approach in the area of biomedical research.
Assuntos
Proteômica/métodos , Algoritmos , Aminoácidos/química , Animais , Proteínas de Bactérias/química , Biologia Computacional/métodos , Eletroforese em Gel Bidimensional/métodos , Genoma , Genoma Bacteriano , Genômica , Humanos , Espectrometria de Massas/métodos , Neoplasias/metabolismo , ProteomaRESUMO
Vaccination is believed to be the most effective method for the prevention of infectious diseases. Thus it is imperative to develop cost effective and scalable process for the production of vaccines so as to make them affordable for mass use. In this study, performance of a novel disposable iCELLis fixed bed bioreactor system was investigated for the production of some viral vaccines like Rabies, Hepatitis-A and Chikungunya vaccines in comparison to conventional systems like the commercially available packed bed system and roller bottle system. Vero and MRC-5 cell substrates were evaluated for growth parameters in all the three systems maintaining similar seeding density, multiplicity of infection (MOI) and media components. It was observed that Vero cells showed similar growth in all the three bioreactors whereas MRC-5 cells showed better growth in iCELLis Nano system and roller bottle system. Subsequently, the virus infection and antigen production studies also revealed that for Hepatitis-A and Chikungunya iCELLis Nano bioreactor system was better to the commercial packed bed bioreactor and roller bottle systems. Although for rabies antigen production commercially available packed bed bioreactor system was found to be better. This study shows that different bioreactor platforms may be employed for viral vaccine production and iCELLis Nano is one of such new convenient and a stable platform for production of human viral vaccines.
Assuntos
Espectrometria de Massas/métodos , Proteínas/química , Animais , Bioquímica/métodos , Elétrons , Desenho de Equipamento , Análise de Fourier , Humanos , Íons , Lasers , Magnetismo , Modelos Químicos , Peptídeos/química , Pressão , Contagem de Cintilação , Análise de Sequência de DNA , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Plasmodium falciparum triose-phosphate isomerase, a homodimeric enzyme, contains four cysteine residues at positions 13, 126, 196, and 217 per subunit. Among these, Cys-13 is present at the dimer interface and is replaced by methionine in the corresponding human enzyme. We have investigated the effect of sulfhydryl labeling on the parasite enzyme, with a view toward developing selective covalent inhibitors by targeting the interface cysteine residue. Differential labeling of the cysteine residues by iodoacetic acid and iodoacetamide has been followed by electrospray ionization mass spectrometry and positions of the labels determined by analysis of tryptic fragments. The rates of labeling follows the order Cys-196 > Cys-13 Cys-217/Cys-126, which correlates well with surface accessibility calculations based on the enzyme crystal structure. Iodoacetic acid labeling leads to a soluble, largely inactive enzyme, whereas IAM labeling leads to precipitation. Carboxyl methylation of Cys-13 results in formation of monomeric species detectable by gel filtration. Studies with an engineered C13D mutant permitted elucidation of the effects of introducing a negative charge at the interface. The C13D mutant exhibits a reduced stability to denaturants and 7-fold reduction in the enzymatic activity even under the concentrations in which dimeric species are observed.
Assuntos
Cisteína/química , Plasmodium falciparum/enzimologia , Triose-Fosfato Isomerase/isolamento & purificação , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Mapeamento de Peptídeos , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Triose-Fosfato Isomerase/químicaRESUMO
A mutation at the dimer interface of Plasmodium falciparum triosephosphate isomerase (PfTIM) was created by mutating a tyrosine residue at position 74, at the subunit interface, to glycine. Tyr74 is a critical residue, forming a part of an aromatic cluster at the interface. The resultant mutant, Y74G, was found to have considerably reduced stability compared with the wild-type protein (TIMWT). The mutant was found to be much less stable to denaturing agents such as urea and guanidinium chloride. Fluorescence and circular dichroism studies revealed that the Y74G mutant and TIMWT have similar spectroscopic properties, suggestive of similar folded structures. Further, the Y74G mutant also exhibited a concentration-dependent loss of enzymatic activity over the range 0.1-10 microM. In contrast, the wild-type enzyme did not show a concentration dependence of activity in this range. Fluorescence quenching of intrinsic tryptophan emission was much more efficient in case of Y74G than TIMWT, suggestive of greater exposure of Trp11, which lies adjacent to the dimer interface. Analytical gel filtration studies revealed that in Y74G, monomeric and dimeric species are in dynamic equilibrium, with the former predominating at low protein concentration. Spectroscopic studies established that the monomeric form of the mutant is largely folded. Low concentrations of urea also drive the equilibrium towards the monomeric form. These studies suggest that the replacement of tyrosine with a small residue at the interface of triosephosphate isomerase weakens the subunit-subunit interactions, giving rise to structured, but enzymatically inactive, monomers at low protein concentration.
Assuntos
Plasmodium falciparum/enzimologia , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genética , Animais , Cromatografia/métodos , Dicroísmo Circular , Dimerização , Transferência de Energia , Estabilidade Enzimática/genética , Gliceraldeído 3-Fosfato/metabolismo , Guanidina/farmacologia , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação Puntual , Dobramento de Proteína , Subunidades Proteicas/genética , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Triose-Fosfato Isomerase/metabolismo , Ureia/farmacologiaRESUMO
Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active-site loop 6. Two single-tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild-type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blue-shifted emission (321 nm) and exhibits significant red-edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1-A crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue-shifted emission from a tryptophan in a polar environment in the protein. The wild-type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2-phosphoglycolic acid.
Assuntos
Plasmodium falciparum/enzimologia , Triose-Fosfato Isomerase/química , Triptofano/química , Animais , Dicroísmo Circular , Fluorescência , Glicolatos/metabolismo , Glicolatos/farmacologia , Guanidina/metabolismo , Espectrometria de Massas , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Espectrometria de Fluorescência , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/isolamento & purificação , Triptofano/metabolismo , Ureia/metabolismoRESUMO
Acidic pH plays an important role in the membrane insertion of protective antigen (PA) of anthrax toxin leading to the translocation of the catalytic moieties. The structural transitions occurring in PA as a consequence of change in pH were investigated by fluorescence and circular dichroism measurements. Our studies revealed the presence of two intermediates on-pathway of acid induced unfolding; one at pH 2.0 and other at pH 4-5. Intrinsic fluorescence measurements of these intermediates showed a red shift in the wavelength of emission maximum with a concomitant decrease in fluorescence intensity, indicative of the exposure of tryptophan residues to the bulk solvent. Furthermore, no significant change was seen in the secondary structure of PA at a pH of 2.0, as indicated by far UV-CD spectra. The low pH intermediate of PA was characterized using the hydrophobic dye, 8-anilino-1-naphthalenesulfonate, and was found to have properties similar to those of a molten globule state.
Assuntos
Antígenos de Bactérias/química , Toxinas Bacterianas/química , Naftalenossulfonato de Anilina/química , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
Nucleoside-diphosphate kinase of Mycobacterium tuberculosis (mNdK) is a secretory protein, but the rationale behind secreting an enzyme involved in the maintenance of cellular pool of nucleoside triphosphates is not clearly understood. To elucidate the biological significance of mNdK secretion, we expressed mNdK fused to green fluorescent protein in HeLa and COS-1 cells. Interestingly, mNdK was detected in the nuclei of HeLa and COS-1 cells. Incubation of mNdK with nuclei isolated from HeLa and COS-1 cells led to in situ damage of chromosomal DNA. Surface plasmon resonance studies demonstrated that mNdK binds supercoiled plasmid DNA lacking apurinic/apyrimidinic sites with a dissociation constant of 30 +/- 3.2 mum. Plasmid cleavage by mNdK was found to be dependent on the specific divalent metal ion and inhibited by a metal ion chelator. Moreover, the metal ion-dependent DNA cleavage by mNdK was mediated by superoxide radicals as detected by electron paramagnetic resonance. The cleavage reaction was inhibited under nitrogen atmosphere confirming the necessity of molecular oxygen for DNA cleavage. In view of the findings that mNdK is secreted by intracellular mycobacteria and damages the nuclear DNA, it can be postulated that mNdK may cause cell death that could help in the dissemination of the pathogen.