RESUMO
Current practices for structural analysis of extremely large-molecular-weight polysaccharides via solution-state nuclear magnetic resonance (NMR) spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non-specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structural fragments that can complicate complete and accurate characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structural knowledge and optimal enzyme activity conditions that are not available to an analyst working with novel or unknown compounds. Herein, we describe an application of a permethylation strategy that allows the complete dissolution of intact polysaccharides for NMR structural characterization. This approach is utilized for NMR analysis of Xylella fastidiosa extracellular polysaccharide (EPS), which is essential for the virulence of the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year.
Assuntos
Xylella , Xylella/química , Xylella/metabolismo , Polissacarídeos/metabolismo , Espectroscopia de Ressonância MagnéticaRESUMO
Glycosyl composition and linkage analyses are important first steps toward understanding the structural diversity and biological importance of polysaccharides. Failure to fully solubilize samples prior to analysis results in the generation of incomplete and poor-quality composition and linkage data by gas chromatography-mass spectrometry (GC-MS). Acidic polysaccharides also do not give accurate linkage results, because they are poorly soluble in DMSO and tend to undergo ß-elimination during permethylation. Ionic liquids can solubilize polysaccharides, improving their derivatization and extraction for analysis. We show that water-insoluble polysaccharides become much more amenable to chemical analysis by first acetylating them in an ionic liquid. Once acetylated, these polysaccharides, having been deprived of their intermolecular hydrogen bonds, are hydrolyzed more readily for glycosyl composition analysis or methylated more efficiently for glycosyl linkage analysis. Acetylation in an ionic liquid greatly improves composition analysis of insoluble polysaccharides when compared to analysis without acetylation, enabling complete composition determination of normally recalcitrant polysaccharides. We also present a protocol for uronic acid linkage analysis that incorporates this preacetylation step. This protocol produces partially methylated alditol acetate derivatives in high yield with minimal ß-elimination and gives sensitive linkage results for acidic polysaccharides that more accurately reflect the structures being analyzed. We use important plant polysaccharides to show that the preacetylation step leads to superior results compared to traditional methodologies.
Assuntos
Líquidos Iônicos , Acetilação , Cromatografia Gasosa-Espectrometria de Massas , Processamento de Proteína Pós-Traducional , PolissacarídeosRESUMO
Clostridium perfringens is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against C. perfringens is currently available. Using an antiserum screen of mutants generated from a C. perfringens transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of C. perfringens HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(ß1â4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with C. perfringens JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.
Assuntos
Galinhas , Infecções por Clostridium , Clostridium perfringens , Lipopolissacarídeos , Doenças das Aves Domésticas , Ácidos Teicoicos , Animais , Galinhas/imunologia , Galinhas/microbiologia , Infecções por Clostridium/imunologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/química , Clostridium perfringens/imunologia , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/prevenção & controle , Ácidos Teicoicos/química , Ácidos Teicoicos/imunologia , Ácidos Teicoicos/farmacologiaRESUMO
Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model.
Assuntos
Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Sorogrupo , Streptococcus/classificação , Vacinas Conjugadas/imunologia , Administração Oral , Animais , Cápsulas Bacterianas/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/administração & dosagem , Polissacarídeos Bacterianos/imunologia , Streptococcus/imunologia , VirulênciaRESUMO
During the late phase of the HIV-1 replication cycle, the viral Gag polyproteins are targeted to the plasma membrane for assembly. The Gag-membrane interaction is mediated by binding of Gag's N-terminal myristoylated matrix (MA) domain to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The viral envelope (Env) glycoprotein is then recruited to the assembly sites and incorporated into budding particles. Evidence suggests that Env incorporation is mediated by interactions between Gag's MA domain and the cytoplasmic tail of the gp41 subunit of Env (gp41CT). MA trimerization appears to be an obligatory step for this interaction. Insufficient production of a recombinant MA trimer and unavailability of a biologically relevant membrane system have been barriers to detailed structural and biophysical characterization of the putative MA-gp41CT-membrane interactions. Here, we engineered a stable recombinant HIV-1 MA trimer construct by fusing a foldon domain (FD) of phage T4 fibritin to the MA C terminus. Results from NMR experiments confirmed that the FD attachment does not adversely alter the MA structure. Employing hydrogen-deuterium exchange MS, we identified an MA-MA interface in the MA trimer that is implicated in Gag assembly and Env incorporation. Utilizing lipid nanodiscs as a membrane mimetic, we show that the MA trimer binds to membranes 30-fold tighter than does the MA monomer and that incorporation of PI(4,5)P2 and phosphatidylserine enhances the binding of MA to nanodiscs. These findings advance our understanding of a fundamental mechanism in HIV-1 assembly and provide a template for investigating the interaction of MA with gp41CT.
Assuntos
HIV-1/metabolismo , Montagem de Vírus/fisiologia , Calorimetria , Membrana Celular/metabolismo , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Espectroscopia de Ressonância Magnética , Fosfatidilserinas/metabolismo , Ligação ProteicaRESUMO
Upon host infection, Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) into the cytosol of infected macrophages, leading to host cell death by necroptosis. TNT hydrolyzes NAD+ in the absence of any exogenous cofactor, thus classifying it as a ß-NAD+ glycohydrolase. However, TNT lacks sequence similarity with other NAD+ hydrolyzing enzymes and lacks the essential motifs involved in NAD+ binding and hydrolysis by these enzymes. In this study, we used NMR to examine the enzymatic activity of TNT and found that TNT hydrolyzes NADP+ as fast as NAD+ but does not cleave the corresponding reduced dinucleotides. This activity of TNT was not inhibited by ADP-ribose or nicotinamide, indicating low affinity of TNT for these reaction products. A selection assay for nontoxic TNT variants in Escherichia coli identified four of six residues in the predicted NAD+-binding pocket and four glycine residues that form a cradle directly below the NAD+-binding site, a conserved feature in the TNT protein family. Site-directed mutagenesis of residues near the predicted NAD+-binding site revealed that Phe727, Arg757, and Arg780 are essential for NAD+ hydrolysis by TNT. These results identify the NAD+-binding site of TNT. Our findings also show that TNT is an NAD+ glycohydrolase with properties distinct from those of other bacterial glycohydrolases. Because many of these residues are conserved within the TNT family, our findings provide insights into understanding the function of the >300 TNT homologs.
Assuntos
Toxinas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , NAD+ Nucleosidase/metabolismo , Sequência de Aminoácidos , Toxinas Bacterianas/química , Hidrólise , Espaço Intracelular/microbiologia , Modelos Moleculares , Mycobacterium tuberculosis/fisiologia , NAD/metabolismo , NADP/metabolismo , Conformação Proteica , Domínios ProteicosRESUMO
The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P2-binding site in the ASV MA domain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase Cδ (PH-PLC), two proteins that bind PI(4,5)P2, affects ASV Gag trafficking to the PM and budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P2-binding signal in ASV MA could target HIV-1 Gag to the PM when substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P2 is a strong determinant of ASV Gag targeting to the PM and budding.
Assuntos
Vírus do Sarcoma Aviário/química , Membrana Celular/metabolismo , Produtos do Gene gag/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Galinhas , Chlorocebus aethiops , Produtos do Gene gag/química , Produtos do Gene gag/genética , Humanos , Proteínas de Membrana/metabolismo , Mutação , Fosfolipase C delta/metabolismo , Ligação Proteica , Domínios Proteicos , Liberação de Vírus/fisiologiaRESUMO
For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA87) to lipids and liposomes. We report that MA87 binds to the polar head of phosphoinositides such as PI(4,5)P2 We found that MA87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA87-IP binding is governed by charge-charge interactions. Using a sensitive NMR-based liposome-binding assay, we show that binding of MA87 to liposomes is enhanced by incorporation of PI(4,5)P2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly.
Assuntos
Vírus do Sarcoma Aviário/química , Membrana Celular/metabolismo , Produtos do Gene gag/metabolismo , Montagem de Vírus/fisiologia , Acilação , Sítios de Ligação , Membrana Celular/química , Produtos do Gene gag/química , Fosfatos de Inositol/química , Fosfatos de Inositol/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Ligação Proteica , Domínios Proteicos , Eletricidade EstáticaRESUMO
In the last decade, strains of the genera Franconibacter and Siccibacter have been misclassified as first Enterobacter and later Cronobacter Because Cronobacter is a serious foodborne pathogen that affects premature neonates and elderly individuals, such misidentification may not only falsify epidemiological statistics but also lead to tests of powdered infant formula or other foods giving false results. Currently, the main ways of identifying Franconibacter and Siccibacter strains are by biochemical testing or by sequencing of the fusA gene as part of Cronobacter multilocus sequence typing (MLST), but in relation to these strains the former is generally highly difficult and unreliable while the latter remains expensive. To address this, we developed a fast, simple, and most importantly, reliable method for Franconibacter and Siccibacter identification based on intact-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Our method integrates the following steps: data preprocessing using mMass software; principal-component analysis (PCA) for the selection of mass spectrum fingerprints of Franconibacter and Siccibacter strains; optimization of the Biotyper database settings for the creation of main spectrum projections (MSPs). This methodology enabled us to create an in-house MALDI MS database that extends the current MALDI Biotyper database by including Franconibacter and Siccibacter strains. Finally, we verified our approach using seven previously unclassified strains, all of which were correctly identified, thereby validating our method.IMPORTANCE We show that the majority of methods currently used for the identification of Franconibacter and Siccibacter bacteria are not able to properly distinguish these strains from those of Cronobacter While sequencing of the fusA gene as part of Cronobacter MLST remains the most reliable such method, it is highly expensive and time-consuming. Here, we demonstrate a cost-effective and reliable alternative that correctly distinguishes between Franconibacter, Siccibacter, and Cronobacter bacteria and identifies Franconibacter and Siccibacter at the species level. Using intact-cell MALDI-TOF MS, we extend the current MALDI Biotyper database with 11 Franconibacter and Siccibacter MSPs. In addition, the use of our approach is likely to lead to a more reliable identification scheme for Franconibacter and Siccibacter strains and, consequently, a more trustworthy epidemiological picture of their involvement in disease.
Assuntos
Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/isolamento & purificação , Tipagem de Sequências Multilocus/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas de Bactérias/genética , Cronobacter/química , Cronobacter/classificação , Cronobacter/genética , Cronobacter/isolamento & purificação , Enterobacteriaceae/química , Enterobacteriaceae/classificação , Enterobacteriaceae/genética , Humanos , FilogeniaRESUMO
Bacteria from the genus Cronobacter are opportunistic foodborne pathogens that can cause severe infections. More rapid, cost-effective and reliable methods are still required for the species identification of Cronobacter spp. In this study, we present a novel PCR-RFLP-based method that uses a newly designed pair of primers for the PCR-amplification of a partial rpoB gene sequence (1635 bp). The amplified products of DNA from 80 Cronobacter strains were separately digested with three restriction endonucleases (Csp6I, HinP1I, MboI). Using the obtained restriction patterns, a PCR-RFLP identification system was created to enable differentiation between all seven currently-known Cronobacter species. The functionality of our method was successfully verified on real food samples. Moreover, the relationships between the Cronobacter species were determined via a phylogenetic tree created from the RFLP patterns.
Assuntos
Cronobacter/classificação , RNA Polimerases Dirigidas por DNA/genética , Microbiologia de Alimentos , Genes Bacterianos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Cronobacter/genética , Primers do DNA , DNA Bacteriano , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Contaminação de Alimentos/análise , Filogenia , Reação em Cadeia da Polimerase/economia , Análise de Sequência de DNARESUMO
Localization of the HIV type-1 (HIV-1) Gag protein on the plasma membrane (PM) for virus assembly is mediated by specific interactions between the N-terminal myristoylated matrix (MA) domain and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)]. The PM bilayer is highly asymmetric, and this asymmetry is considered crucial in cell function. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and contains minor populations of phosphatidylcholine (PC) and PI(4,5)P(2). There is strong evidence that efficient binding of HIV-1 Gag to membranes is sensitive not only to lipid composition and net negative charge, but also to the hydrophobic character of the acyl chains. Here, we show that PS, PE, and PC interact directly with MA via a region that is distinct from the PI(4,5)P(2) binding site. Our NMR data also show that the myristoyl group is readily exposed when MA is bound to micelles or bicelles. Strikingly, our structural data reveal a unique binding mode by which the 2'-acyl chain of PS, PE, and PC lipids is buried in a hydrophobic pocket whereas the 1'-acyl chain is exposed. Sphingomyelin, a major lipid localized exclusively on the outer layer of the PM, does not bind to MA. Our findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the 1'-acyl chains of PI(4,5)P(2) and PS/PE/PC and the myristoyl group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane.
Assuntos
Membrana Celular/metabolismo , HIV-1/metabolismo , Bicamadas Lipídicas/metabolismo , Ácido Mirístico/metabolismo , Fosfolipídeos/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Micelas , Modelos Biológicos , Modelos Moleculares , Ácido Mirístico/química , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Fosfolipídeos/química , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Subcellular distribution of calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM co-localizes and interacts with the HIV-1 Gag protein in the cytosol of infected cells. Although it has been shown that binding of Gag to CaM is mediated by the matrix (MA) domain, the structural details of this interaction are not known. We have recently shown that binding of CaM to MA induces a conformational change that triggers myristate exposure, and that the CaM-binding domain of MA is confined to a region spanning residues 8-43 (MA-(8-43)). Here, we present the NMR structure of CaM bound to MA-(8-43). Our data revealed that MA-(8-43), which contains a novel CaM-binding motif, binds to CaM in an antiparallel mode with the N-terminal helix (α1) anchored to the CaM C-terminal lobe, and the C-terminal helix (α2) of MA-(8-43) bound to the N-terminal lobe of CaM. The CaM protein preserves a semiextended conformation. Binding of MA-(8-43) to CaM is mediated by numerous hydrophobic interactions and stabilized by favorable electrostatic contacts. Our structural data are consistent with the findings that CaM induces unfolding of the MA protein to have access to helices α1 and α2. It is noteworthy that several MA residues involved in CaM binding have been previously implicated in membrane binding, envelope incorporation, and particle production. The present findings may ultimately help in identification of the functional role of CaM in HIV-1 replication.
Assuntos
Calmodulina/química , Calmodulina/metabolismo , Antígenos HIV/química , Antígenos HIV/metabolismo , HIV-1/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/química , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Cronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and PubMLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.
Assuntos
Cronobacter/química , Cronobacter/genética , Variação Genética , Antígenos O/análise , Antígenos O/genética , Cronobacter/classificação , Cronobacter/isolamento & purificação , Impressões Digitais de DNA , DNA Bacteriano/genética , Infecções por Enterobacteriaceae/microbiologia , Genótipo , Humanos , Família Multigênica , Tipagem de Sequências Multilocus , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , SorogrupoRESUMO
Current practices for structure analysis of extremely large molecular weight polysaccharides via solution-state NMR spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non-specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structure fragments that can complicate the complete characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structure information and optimal enzyme activity conditions that are not available to the analyst working with novel or unknown compounds. Herein, we describe the application of a permethylation strategy that allows the complete dissolution of the intact polysaccharides for NMR structure characterization. This approach is utilized for NMR analysis of Xylella fastidiosa EPS, which is essential for the virulence the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year.
RESUMO
IMPORTANCE: In this study, we identify a separate role for the Campylobacter jejuni l-fucose dehydrogenase in l-fucose chemotaxis and demonstrate that this mechanism is not only limited to C. jejuni but is also present in Burkholderia multivorans. We now hypothesize that l-fucose energy taxis may contribute to the reduction of l-fucose-metabolizing strains of C. jejuni from the gastrointestinal tract of breastfed infants, selecting for isolates with increased colonization potential.
RESUMO
O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Antígenos O , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Hidrólise , Antígenos O/químicaRESUMO
Kingella kingae is a leading cause of bone and joint infections and other invasive diseases in young children. A key K. kingae virulence determinant is a secreted exopolysaccharide that mediates resistance to serum complement and neutrophils and is required for full pathogenicity. The K. kingae exopolysaccharide is a galactofuranose homopolymer called galactan and is encoded by the pamABC genes in the pamABCDE locus. In this study, we sought to define the mechanism by which galactan is tethered on the bacterial surface, a prerequisite for mediating evasion of host immune mechanisms. We found that the pamD and pamE genes encode glycosyltransferases and are required for synthesis of an atypical lipopolysaccharide (LPS) O-antigen. The LPS O-antigen in turn is required for anchoring of galactan, a novel mechanism for association of an exopolysaccharide with the bacterial surface. IMPORTANCE Kingella kingae is an emerging pediatric pathogen and produces invasive disease by colonizing the oropharynx, invading the bloodstream, and disseminating to distant sites. This organism produces a uniquely multifunctional exopolysaccharide called galactan that is critical for virulence and promotes intravascular survival by mediating resistance to serum and neutrophils. In this study, we established that at least some galactan is anchored to the bacterial surface via a novel structural interaction with an atypical lipopolysaccharide O-antigen. Additionally, we demonstrated that the atypical O-antigen is synthesized by the products of the pamD and pamE genes, located downstream of the gene cluster responsible for galactan biosynthesis. This work addresses how the K. kingae exopolysaccharide can mediate innate immune resistance and advances understanding of bacterial exopolysaccharides and lipopolysaccharides.
Assuntos
Kingella kingae , Infecções por Neisseriaceae , Humanos , Criança , Pré-Escolar , Kingella kingae/química , Lipopolissacarídeos , Antígenos O/genética , Galactanos , Glicosiltransferases/genética , Infecções por Neisseriaceae/microbiologiaRESUMO
Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason-Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C- and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.
Assuntos
Membrana Celular/metabolismo , Membrana Celular/virologia , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Nucleares/fisiologia , Retroviridae/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Humanos , Vírus dos Macacos de Mason-Pfizer/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fenótipo , Estrutura Terciária de Proteína , Região do Complexo-t do GenomaRESUMO
We are exposed daily to many glycans from bacteria and food plants. Bacterial glycans are generally antigenic and elicit antibody responses. It is unclear if food glycans' sharing of antigens with bacterial glycans influences our immune responses to bacteria. We studied 14 different plant foods for cross-reactivity with monoclonal antibodies (MAbs) against 24 pneumococcal serotypes which commonly cause infections and are included in pneumococcal vaccines. Serotype 15B-specific MAb cross-reacts with fruit peels, and serotype 10A MAb cross-reacts with many natural and processed plant foods. The serotype 10A cross-reactive epitope is terminal 1,6-linked ß-galactose [ßGal(1-6)], present in the rhamno-galacturonan I (RG-I) domain of pectin. Despite wide consumption of pectin, the immune response to 10A is comparable to the responses to other serotypes. An antipectin antibody can opsonize serotype 10A pneumococci, and the shared ßGal(1-6) may be useful as a simple vaccine against 10A. Impact of food glycans should be considered in host-pathogen interactions and future vaccine designs.IMPORTANCE The impact of food consumption on vaccine responses is unknown. Streptococcus pneumoniae (the pneumococcus) is an important human pathogen, and its polysaccharide capsule is used as a vaccine. We show that capsule type 10A in a pneumococcal vaccine shares an antigenic epitope, ßGal(1-6), with pectin, which is in many plant foods and is widely consumed. Immune response to 10A is comparable to that seen with other capsule types, and pectin ingestion may have little impact on vaccine responses. However, antibody to pectin can kill serotype 10A pneumococci and this shared epitope may be considered in pneumococcal vaccine designs.
Assuntos
Antígenos de Bactérias/imunologia , Cápsulas Bacterianas/imunologia , Reações Cruzadas , Pectinas/imunologia , Streptococcus pneumoniae/imunologia , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Frutas , Humanos , Fagocitose , Sorogrupo , VerdurasRESUMO
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93-based mechanism-of-action studies and drug discovery efforts.