RESUMO
Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.
Assuntos
Francisella tularensis , Francisella tularensis/genética , Francisella tularensis/química , Francisella tularensis/metabolismo , Lisina/metabolismo , Peptídeos/genética , Códon/genética , Sinais Direcionadores de Proteínas/genéticaRESUMO
Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding ß-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/ß hydrolase structure and a potential allosteric binding site within human APTs.
Assuntos
Domínio Catalítico , Ligação Proteica , Dobramento de Proteína , Tioléster Hidrolases , Humanos , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Ligantes , Modelos Moleculares , Sequência de Aminoácidos , Cinética , Sequência Conservada , Estabilidade Enzimática , Francisella tularensis/enzimologia , Francisella tularensis/metabolismo , Francisella tularensis/química , Cristalografia por Raios X , Especificidade por SubstratoRESUMO
Francisella tularensis is the causative agent for the potentially fatal disease tularemia. The lipoprotein Flpp3 has been identified as a virulence determinant of tularemia with no sequence homology outside the Francisella genus. We report a room temperature structure of Flpp3 determined by serial femtosecond crystallography that exists in a significantly different conformation than previously described by the NMR-determined structure. Furthermore, we investigated the conformational space and energy barriers between these two structures by molecular dynamics umbrella sampling and identified three low-energy intermediate states, transitions between which readily occur at room temperature. We have also begun to investigate organic compounds in silico that may act as inhibitors to Flpp3. This work paves the road to developing targeted therapeutics against tularemia and aides in our understanding of the disease mechanisms of tularemia.
Assuntos
Antibacterianos/química , Francisella tularensis , Lipoproteínas/química , Antibacterianos/farmacologia , Cristalografia por Raios X/métodos , Bases de Dados de Produtos Farmacêuticos , Avaliação Pré-Clínica de Medicamentos/métodos , Francisella tularensis/química , Francisella tularensis/patogenicidade , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lasers , Lipoproteínas/antagonistas & inibidores , Lipoproteínas/genética , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Tularemia/tratamento farmacológico , Fatores de Virulência/químicaRESUMO
The Lon protease selectively degrades abnormal proteins or certain normal proteins in response to environmental and cellular conditions in many prokaryotic and eukaryotic organisms. However, the mechanism(s) behind the substrate selection of normal proteins remains largely unknown. In this study, we identified 10 new substrates of F. tularensis Lon from a total of 21 candidate substrates identified in our previous work, the largest number of novel Lon substrates from a single study. Cross-species degradation of these and other known Lon substrates revealed that human Lon is unable to degrade many bacterial Lon substrates, suggestive of a "organism-adapted" substrate selection mechanism for the natural Lon variants. However, individually replacing the N, A, and P domains of human Lon with the counterparts of bacterial Lon did not enable the human protease to degrade the same bacterial Lon substrates. This result showed that the "organism-adapted" substrate selection depends on multiple domains of the Lon proteases. Further in vitro proteolysis and mass spectrometry analysis revealed a similar substrate cleavage pattern between the bacterial and human Lon variants, which was exemplified by predominant representation of leucine, alanine, and other hydrophobic amino acids at the P(-1) site within the substrates. These observations suggest that the Lon proteases select their substrates at least in part by fine structural matching with the proteins in the same organisms.
Assuntos
Proteases Dependentes de ATP/química , Proteínas de Bactérias/química , Francisella tularensis/enzimologia , Proteínas Mitocondriais/química , Protease La/química , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Francisella tularensis/química , Francisella tularensis/genética , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Protease La/genética , Protease La/metabolismo , Domínios Proteicos , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Francisella tularensisis, the causative agent of tularemia has been classified as a category A bioterrorism agent. Here, we present the crystal structure of apo and adenine bound form of the adenine phosphoribosyltransferase (APRT) from Francisella tularensis. APRT is an enzyme involved in the salvage of adenine (a 6-aminopurine), converting it to AMP. The purine salvage pathway relies on two essential and distinct enzymes to convert 6-aminopurine and 6-oxopurines into corresponding nucleotides. The mechanism by which these enzymes differentiate different purines is not clearly understood. Analysis of the structures of apo and adenine-bound APRT from F. tularensis, together with all other available structures of APRTs, suggests that (a) the base-binding loop is stabilized by a cluster of aromatic and conformation-restricting proline residues, and (b) an N-H···N hydrogen bond between the base-binding loop and the N1 atom of adenine is the key interaction that differentiates adenine from 6-oxopurines. These observations were corroborated by bioinformatics analysis of ~ 4000 sequences of APRTs (with 80% identity cutoff), which confirmed that the residues conferring rigidity to the base-binding loop are highly conserved. Furthermore, an F23A mutation on the base-binding loop severely affects the efficiency of the enzyme. We extended our analysis to the structure and sequences of APRTs from the Trypanosomatidae family with a destabilizing insertion on the base-binding loop and propose the mechanism by which these evolutionarily divergent enzymes achieve base specificity. Our results suggest that the base-binding loop not only confers appropriate affinity but also provides defined specificity for adenine. ENZYME: EC 2.4.2.7 DATABASE: Structural data are available in Protein Data Bank (PDB) under the accession numbers 5YW2 and 5YW5.
Assuntos
Adenina Fosforribosiltransferase/química , Adenina/química , Monofosfato de Adenosina/química , Apoproteínas/química , Proteínas de Bactérias/química , Francisella tularensis/enzimologia , Adenina/metabolismo , Adenina Fosforribosiltransferase/genética , Adenina Fosforribosiltransferase/metabolismo , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Francisella tularensis/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ligação de Hidrogênio , Cinética , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TermodinâmicaRESUMO
Label-free shotgun proteomics is routinely used to assess proteomes. However, extracting relevant information from the massive amounts of generated data remains difficult. This tutorial provides a strong foundation on analysis of quantitative proteomics data. We provide key statistical concepts that help researchers to design proteomics experiments and we showcase how to analyze quantitative proteomics data using our recent free and open-source R package MSqRob, which was developed to implement the peptide-level robust ridge regression method for relative protein quantification described by Goeminne et al. MSqRob can handle virtually any experimental proteomics design and outputs proteins ordered by statistical significance. Moreover, its graphical user interface and interactive diagnostic plots provide easy inspection and also detection of anomalies in the data and flaws in the data analysis, allowing deeper assessment of the validity of results and a critical review of the experimental design. Our tutorial discusses interactive preprocessing, data analysis and visualization of label-free MS-based quantitative proteomics experiments with simple and more complex designs. We provide well-documented scripts to run analyses in bash mode on GitHub, enabling the integration of MSqRob in automated pipelines on cluster environments (https://github.com/statOmics/MSqRob). SIGNIFICANCE: The concepts outlined in this tutorial aid in designing better experiments and analyzing the resulting data more appropriately. The two case studies using the MSqRob graphical user interface will contribute to a wider adaptation of advanced peptide-based models, resulting in higher quality data analysis workflows and more reproducible results in the proteomics community. We also provide well-documented scripts for experienced users that aim at automating MSqRob on cluster environments.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Software , Proteínas de Bactérias/análise , Biologia Computacional , Francisella tularensis/química , Humanos , Peptídeos/análiseRESUMO
Francisella tularensis is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies.IMPORTANCE The low infectious dose, the high potential mortality/morbidity rates, and the ability to be disseminated as an aerosol make Francisella tularensis a potential agent for bioterrorism. These characteristics led the Centers for Disease Control (CDC) to classify F. tularensis as a Tier 1 pathogen. Currently, there is no vaccine approved for general use in the United States.
Assuntos
Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Proteínas de Membrana/imunologia , Tularemia/prevenção & controle , Vacinas de Subunidades Antigênicas/imunologia , Adjuvantes Imunológicos , Animais , Modelos Animais de Doenças , Francisella tularensis/química , Francisella tularensis/patogenicidade , Ácido Láctico , Macrófagos/imunologia , Macrófagos/microbiologia , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas , Poli I-C/imunologia , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteômica , Tularemia/imunologia , Vacinação , Vacinas Atenuadas/imunologia , Vacinas de Subunidades Antigênicas/genéticaRESUMO
Francisella tularensis is a potent human pathogen that invades and survives in macrophage and epithelial cells. Two identical proteins, FTT_0924 from F. tularensis subsp. tularensis and FTL_1286 from F. tularensis subsp. holarctica LVS, have previously been identified as playing a role in protection of the bacteria from osmotic shock and its survival in macrophages. FTT_0924 has been shown to localize to the inner membrane, with its C-terminus exposed to the periplasm. Here, crystal structures of the F. novicida homologue FTN_0802, which we call FvfA, in two crystal forms are reported at 1.8â Å resolution. FvfA differs from FTT_0924 and FTL_1286 by a single amino acid. FvfA has a DUF1471 fold that closely resembles the Escherichia coli outer membrane lipoprotein RscF, a component of a phosphorelay pathway involved in protecting bacteria from outer membrane perturbation. The structural and functional similarities and differences between these proteins and their implications for F. tularensis pathogenesis are discussed.
Assuntos
Proteínas de Bactérias/química , Francisella tularensis/química , Fatores de Virulência/química , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Alinhamento de Sequência , Tularemia/microbiologiaRESUMO
Structural modification of lipid A, the lipid anchor of LPS, is one of the strategies used by Gram-negative bacteria to evade host innate immunity. Francisella tularensis is a human pathogen that infects and replicates within phagocytic cells. It produces an atypical lipid A, whose structure precludes an efficient recognition by both innate immune players, TLR4 and cationic antimicrobial peptides. Interestingly, a recent report indicates that the lipid A of Francisella (LVS vaccinal strain) undergoes polar modifications when bacteria are grown in human macrophages as compared to in broth. To characterize the structural modifications of lipid A that may be induced intracellularly, Francisella novicida, a surrogate strain for the highly virulent F. tularensis, was submitted to different stress conditions mimicking the harsh environment encountered in the macrophages. To analyze lipid A directly from intact bacteria without any chemical treatment or purification steps, we used a rapid and sensitive MALDI-TOF mass spectrometry approach. Among the many conditions tested, only bacteria exposure to acidic pHs (from 6 to 5) induced a change in lipid A structure. These changes were characterized by an increase in the relative abundance of molecular species bearing an additional hexose unit on the diglucosamine backbone, similar to species present when bacteria are grown under reduced environmental temperature. This lipid A glyco-form, which is observed in trace amounts in normal in vitro growth conditions at 37 °C, may contribute to the intracellular parasitism of macrophages by Francisella.
Assuntos
Francisella tularensis , Lipídeo A , Espectrometria de Massas , Estresse Fisiológico , Francisella tularensis/química , Francisella tularensis/metabolismo , Concentração de Íons de Hidrogênio , Lipídeo A/química , Lipídeo A/metabolismoRESUMO
Numerous studies have demonstrated that administration of antigen (Ag)-pulsed dendritic cells (DCs) is an effective strategy for enhancing immunity to tumors and infectious disease organisms. However, the generation and/or isolation of DCs can require substantial time and expense. Therefore, using inactivated F. tularensis (iFt) Ag as a model immunogen, we first sought to determine if DCs could be replaced with peripheral blood mononuclear cells (PBMCs) during the ex-vivo pulse phase and still provide protection against Ft infection. Follow up studies were then conducted using the S. pneumoniae (Sp) vaccine Prevnar ®13 as the Ag in the pulse phase followed by immunization and Sp challenge. In both cases, we demonstrate that PBMCs can be used in place of DCs when pulsing with iFt and/or Prevnar ®13 ex vivo and re-administering the Ag-pulsed PBMCs as a vaccine. In addition, utilization of the i.n. route for Ag-pulsed PBMC administration is superior to use of the i.v. route in the case of Sp immunization, as well as when compared to direct injection of Prevnar ®13 vaccine i.m. or i.n. Furthermore, this PBMC-based vaccine strategy provides a more marked and enduring protective immune response and is also capable of serving as a multi-organism vaccine platform. The potential for this ex-vivo vaccine strategy to provide a simpler, less time consuming, and less expensive approach to DC-based vaccines and vaccination in general is also discussed.
Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/farmacologia , Vacinas Bacterianas/administração & dosagem , Leucócitos Mononucleares/efeitos dos fármacos , Infecções Pneumocócicas/prevenção & controle , Tularemia/prevenção & controle , Administração Intranasal , Transferência Adotiva , Animais , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Francisella tularensis/química , Francisella tularensis/imunologia , Injeções Intramusculares , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Camundongos , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/mortalidade , Cultura Primária de Células , Streptococcus pneumoniae/química , Streptococcus pneumoniae/imunologia , Análise de Sobrevida , Tularemia/imunologia , Tularemia/microbiologia , Tularemia/mortalidadeRESUMO
Caseinolytic proteases are large oligomeric assemblies responsible for maintaining protein homeostasis in bacteria and in so doing influence a wide range of biological processes. The functional assembly involves three chaperones together with the oligomeric caseinolytic protease catalytic subunit P (ClpP). This protease represents a potential target for therapeutic intervention in pathogenic bacteria. Here, we detail an efficient protocol for production of recombinant ClpP from Francisella tularensis, and the structural characterization of three crystal forms which grow under similar conditions. One crystal form reveals a compressed state of the ClpP tetradecamer and two forms an open state. A comparison of the two types of structure infers that differences at the enzyme active site result from a conformational change involving a highly localized disorder-order transition of a ß-strand α-helix combination. This transition occurs at a subunit-subunit interface. Our study may now underpin future efforts in a structure-based approach to target ClpP for inhibitor or activator development. Proteins 2016; 85:188-194. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Bactérias/química , Endopeptidase Clp/química , Francisella tularensis/química , Subunidades Proteicas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Francisella tularensis/enzimologia , Expressão Gênica , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The Gram-negative bacterium Francisella tularensis is the etiological agent of the zoonotic disease tularemia. Its life cycle is characterized by an ability to survive within phagocytic cells through phagosomal escape and replication in the cytosol, ultimately causing inflammasome activation and host cell death. Required for these processes is the Francisella Pathogenicity Island (FPI), which encodes a Type VI secretion system (T6SS) that is active during intracellular infection. In this study, we analyzed the role of the FPI-component IglE, a lipoprotein which we previously have shown to be secreted in a T6SS-dependent manner. We demonstrate that in F. tularensis LVS, IglE is an outer membrane protein. Upon infection of J774 cells, an ΔiglE mutant failed to escape from phagosomes, and subsequently, to multiply and cause cytopathogenicity. Moreover, ΔiglE was unable to activate the inflammasome, to inhibit LPS-stimulated secretion of TNF-α, and showed marked attenuation in the mouse model. In F. novicida, IglE was required for in vitro secretion of IglC and VgrG. A mutagenesis-based approach involving frameshift mutations and alanine substitution mutations within the first â¼ 38 residues of IglE revealed that drastic changes in the sequence of the extreme N-terminus (residues 2-6) were well tolerated and, intriguingly, caused hyper-secretion of IglE during intracellular infection, while even subtle mutations further downstream lead to impaired protein function. Taken together, this study highlights the importance of IglE in F. tularensis pathogenicity, and the contribution of the N-terminus for all of the above mentioned processes.
Assuntos
Aminoácidos/química , Proteínas da Membrana Bacteriana Externa/química , Francisella tularensis/genética , Francisella tularensis/patogenicidade , Lipoproteínas/química , Mutagênese , Sistemas de Secreção Tipo VI , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Francisella tularensis/química , Ilhas Genômicas/genética , Inflamassomos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Macrófagos/microbiologia , Camundongos , Fagossomos/microbiologia , Tularemia/microbiologia , Sistemas de Secreção Tipo VI/genética , Fatores de Virulência/metabolismoRESUMO
Francisella tularensis is the causative agent of tularemia. Although major contributors and the main mechanism of the virulence are well known, some of the molecular details are still missing. Proteomics studies regarding F. tularensis have provided snapshot pictures of the organism grown under different culture conditions to understand the mechanism of virulence. In general, such studies were carried out with standard strains e.g., LVS and did not involve comparisons of F. tularensis isolates from either clinical or environmental sources. In this study, we performed two-dimensional gel electrophoresis (2DE)-based proteomic analysis and compared the protein profiles of the F. tularensis subsp. holarctica strains isolated from the clinical and the environmental samples. Regulations were detected in 14 spots when twofold regulation criteria were applied. The regulated protein spots were subjected to MALDI-TOF/TOF analysis and identified. Classification of the identified proteins based on metabolic functions revealed that the translation machinery was the most varying metabolic processes among the isolates. Using normalized protein spot intensities, PCA analysis was also performed. The results indicated that the strain isolated from water source was different then the strains isolated from the patients. Most interestingly, the isolates were strikingly distinguishable from the standard NCTC 10857 strain.
Assuntos
Microbiologia Ambiental , Francisella tularensis/química , Francisella tularensis/isolamento & purificação , Proteoma/análise , Tularemia/microbiologia , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Francisella tularensis is a highly virulent pathogen of humans and other mammals. Moreover, F. tularensis has been designated a category A biothreat agent, and there is growing interest in the development of a protective vaccine. In the present study, we determine the in vitro and in vivo immune responses of a subunit vaccine composed of recombinant peptides Tul4 and FopA from epitopes of the F. tularensis outer membrane proteins. The recombinant peptides with adjuvant CpG induced robust immunophenotypic change of dendritic cell (DC) maturation and secretion of inflammatory cytokines (IL-6, IL-12). In addition, the matured DCs enabled ex vivo proliferation of naive splenocytes in a mixed lymphocyte reaction. Lastly, we determined the in vivo immune response by assessment of antibody production in C57BL/ 6 mice. Total IgG levels were produced after immunization and peaked in 6 weeks, and moreover, Tul4-specific IgG was confirmed in the mice receiving peptides with or without CpG. Based on these results, we concluded that the recombinant peptides Tul4 and FopA have immunogenicity and could be a safe subunit vaccine candidate approach against F. tularensis.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Francisella tularensis/imunologia , Lipoproteínas/imunologia , Vacinas Sintéticas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Células Cultivadas , Francisella tularensis/química , Francisella tularensis/genética , Interleucinas/análise , Lipoproteínas/química , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Tularemia , Vacinas Sintéticas/químicaRESUMO
Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Francisella tularensis/imunologia , Análise em Microsséries , Proteoma/análise , Testes Sorológicos/métodos , Tularemia/diagnóstico , Adulto , Antígenos de Bactérias/análise , Francisella tularensis/química , Humanos , Espanha , Estados UnidosRESUMO
FipB, an essential virulence factor in the highly virulent Schu S4 strain of F. tularensis subsp. tularensis, shares sequence similarity with Disulfide Bond formation (Dsb) proteins, which can have oxidoreductase, isomerase, or chaperone activity. To further explore FipB's role in virulence potential substrates were identified by co-purification and 2D gel electrophoresis, followed by protein sequencing using mass spectrometry. A total of 119 potential substrates were identified. Proteins with predicted enzymatic activity were prevalent, and there were 19 proteins that had been previously identified as impacting virulence. Among the potential substrates were IglC, IglB, and PdpB, three components of the Francisella Type Six Secretion System (T6SS), which is also essential for virulence. T6SS are widespread in Gram-negative pathogens, but have not been reported to be dependent on Dsb-like proteins for assembly or function. The presented results suggest that FipB affects IglB and IglC substrates differently. In a fipB mutant there were differences in free sulfhydryl accessibility of IglC, but not IglB, when compared to wild-type bacteria. However, for both proteins FipB appears to act as a chaperone that facilitates proper folding and conformation. Understanding the role FipB plays the assembly and structure in this T6SS may reveal critical aspects of assembly that are common and novel among this widely distributed class of secretion systems.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Francisella tularensis/patogenicidade , Sistemas de Secreção Tipo VI/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Linhagem Celular , Francisella tularensis/química , Francisella tularensis/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Análise de Sequência de Proteína , Sistemas de Secreção Tipo VI/química , Sistemas de Secreção Tipo VI/genética , Virulência/genética , Fatores de Virulência/químicaRESUMO
Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institute of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica due to RD1 subspecies-specific PCR result. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories.
Assuntos
Francisella tularensis/química , Tularemia/diagnóstico , Análise Custo-Benefício , Francisella tularensis/genética , Humanos , Reação em Cadeia da Polimerase/economia , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia , Tularemia/microbiologia , TurquiaRESUMO
UNLABELLED: Mammalian lipopolysaccharide (LPS) binding proteins (LBPs) occur mainly in extracellular fluids and promote LPS delivery to specific host cell receptors. The function of LBPs has been studied principally in the context of host defense; the possible role of LBPs in nonpathogenic host-microbe interactions has not been well characterized. Using the Euprymna scolopes-Vibrio fischeri model, we analyzed the structure and function of an LBP family protein, E. scolopes LBP1 (EsLBP1), and provide evidence for its role in triggering a symbiont-induced host developmental program. Previous studies showed that, during initial host colonization, the LPS of V. fischeri synergizes with peptidoglycan (PGN) monomer to induce morphogenesis of epithelial tissues of the host animal. Computationally modeled EsLBP1 shares some but not all structural features of mammalian LBPs that are thought important for LPS binding. Similar to human LBP, recombinant EsLBP1 expressed in insect cells bound V. fischeri LPS and Neisseria meningitidis lipooligosaccharide (LOS) with nanomolar or greater affinity but bound Francisella tularensis LPS only weakly and did not bind PGN monomer. Unlike human LBP, EsLBP1 did not bind N. meningitidis LOS:CD14 complexes. The eslbp1 transcript was upregulated ~22-fold by V. fischeri at 24 h postinoculation. Surprisingly, this upregulation was not induced by exposure to LPS but, rather, to the PGN monomer alone. Hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) and immunocytochemistry (ICC) localized eslbp1 transcript and protein in crypt epithelia, where V. fischeri induces morphogenesis. The data presented here provide a window into the evolution of LBPs and the scope of their roles in animal symbioses. IMPORTANCE: Mammalian lipopolysaccharide (LPS)-binding protein (LBP) is implicated in conveying LPS to host cells and potentiating its signaling activity. In certain disease states, such as obesity, the overproduction of this protein has been a reliable biomarker of chronic inflammation. Here, we describe a symbiosis-induced invertebrate LBP whose tertiary structure and LPS-binding characteristics are similar to those of mammalian LBPs; however, the primary structure of this distantly related squid protein (EsLBP1) differs in key residues previously believed to be essential for LPS binding, suggesting that an alternative strategy exists. Surprisingly, symbiotic expression of eslbp1 is induced by peptidoglycan derivatives, not LPS, a pattern converse to that of RegIIIγ, an important mammalian immunity protein that binds peptidoglycan but whose gene expression is induced by LPS. Finally, EsLBP1 occurs along the apical surfaces of all the host's epithelia, suggesting that it was recruited from a general defensive role to one that mediates specific interactions with its symbiont.
Assuntos
Proteínas de Fase Aguda/química , Proteínas de Fase Aguda/metabolismo , Aliivibrio fischeri/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Decapodiformes/crescimento & desenvolvimento , Decapodiformes/microbiologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Simbiose , Proteínas de Fase Aguda/genética , Aliivibrio fischeri/química , Animais , Proteínas de Transporte/genética , Decapodiformes/fisiologia , Francisella tularensis/química , Perfilação da Expressão Gênica , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/genética , Neisseria meningitidis/química , Ligação Proteica , Transcrição GênicaRESUMO
To validate a method for predicting the binding affinities of FabI inhibitors, three implicit solvent methods, MM-PBSA, MM-GBSA, and QM/MM-GBSA were carefully compared using 16 benzimidazole inhibitors in complex with Francisella tularensis FabI. The data suggests that the prediction results are sensitive to radii sets, GB methods, QM Hamiltonians, sampling protocols, and simulation length, if only one simulation trajectory is used for each ligand. In this case, QM/MM-GBSA using 6 ns MD simulation trajectories together with GB(neck2) , PM3, and the mbondi2 radii set, generate the closest agreement with experimental values (r(2) = 0.88). However, if the three implicit solvent methods are averaged from six 1 ns MD simulations for each ligand (called "multiple independent sampling"), the prediction results are relatively insensitive to all the tested parameters. Moreover, MM/GBSA together with GB(HCT) and mbondi, using 600 frames extracted evenly from six 0.25 ns MD simulations, can also provide accurate prediction to experimental values (r(2) = 0.84). Therefore, the multiple independent sampling method can be more efficient than a single, long simulation method. Since future scaffold expansions may significantly change the benzimidazole's physiochemical properties (charges, etc.) and possibly binding modes, which may affect the sensitivities of various parameters, the relatively insensitive "multiple independent sampling method" may avoid the need of an entirely new validation study. Moreover, due to large fluctuating entropy values, (QM/)MM-P(G)BSA were limited to inhibitors' relative affinity prediction, but not the absolute affinity. The developed protocol will support an ongoing benzimidazole lead optimization program.
Assuntos
Benzimidazóis/química , Benzimidazóis/farmacologia , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Francisella tularensis/enzimologia , Desenho de Fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Entropia , Francisella tularensis/química , Humanos , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Tularemia/tratamento farmacológico , Tularemia/microbiologiaRESUMO
Francisella tularensis is a human zoonotic pathogen and the causative agent of tularemia, a severe infectious disease. Given the extreme infectivity of F. tularensis and its potential to be used as a biological warfare agent, a fast and sensitive detection method is highly desirable. Herein, we construct a novel detection platform composed of two units: (1) Magnetic beads conjugated with multiple capturing antibodies against F. tularensis for its simple and rapid separation and (2) Genetically-engineered apoferritin protein constructs conjugated with multiple quantum dots and a detection antibody against F. tularensis for the amplification of signal. We demonstrate a 10-fold increase in the sensitivity relative to traditional lateral flow devices that utilize enzyme-based detection methods. We ultimately envision the use of our novel nanoprobe detection platform in future applications that require the highly-sensitive on-site detection of high-risk pathogens.