RESUMEN
Membrane proteins constitute about 20% of the human proteome and play crucial roles in cellular functions. However, a complete understanding of their structure and function is limited by their hydrophobic nature, which poses significant challenges in purification and stabilization. Detergents, essential in the isolation process, risk destabilizing or altering the proteins' native conformations, thus affecting stability and functionality. This study leverages single-particle cryo-electron microscopy to elucidate the structural nuances of membrane proteins, focusing on the SLAC1 bacterial homolog from Haemophilus influenzae (HiTehA) purified with diverse detergents, including n-dodecyl ß-D-maltopyranoside (DDM), glycodiosgenin (GDN), ß-D-octyl-glucoside (OG), and lauryl maltose neopentyl glycol (LMNG). This research not only contributes to the understanding of membrane protein structures but also addresses detergent effects on protein purification. By showcasing that the overall structural integrity of the channel is preserved, our study underscores the intricate interplay between proteins and detergents, offering insightful implications for drug design and membrane biology.
Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , Detergentes , Haemophilus influenzae , Microscopía por Crioelectrón/métodos , Haemophilus influenzae/ultraestructura , Haemophilus influenzae/química , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Detergentes/química , Microscopía Electrónica de Transmisión/métodos , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructura , Proteínas de la Membrana/metabolismoRESUMEN
Nontypeable Haemophilus influenzae (NTHi) are clinically important Gram-negative bacteria that are responsible for various human mucosal diseases, including otitis media (OM). Recurrent OM caused by NTHi is common, and infections that recur less than 2 weeks following antimicrobial therapy are largely attributable to the recurrence of the same strain of bacteria. Toxin-antitoxin (TA) modules encoded by bacteria enable rapid responses to environmental stresses and are thought to facilitate growth arrest, persistence, and tolerance to antibiotics. The vapBC-1 locus of NTHi encodes a type II TA system, comprising the ribonuclease toxin VapC1 and its cognate antitoxin VapB1. The activity of VapC1 has been linked to the survival of NTHi during antibiotic treatment both in vivo and ex vivo. Therefore, inhibitors of VapC1 might serve as adjuvants to antibiotics, preventing NTHi from entering growth arrest and surviving; however, none have been reported to date. A truncated VapB1 peptide from a crystal structure of the VapBC-1 complex was used to generate pharmacophore queries to facilitate a scaffold hopping approach for the identification of small-molecule VapC1 inhibitors. The National Center for Advancing Translational Sciences small-molecule library was virtually screened using the shape-based method rapid overlay of chemical structures (ROCS), and the top-ranking hits were docked into the VapB1 binding pocket of VapC1. Two hundred virtual screening hits with the best docking scores were selected and tested in a biochemical VapC1 activity assay, which confirmed eight compounds as VapC1 inhibitors. An additional 60 compounds were selected with structural similarities to the confirmed VapC1 inhibitors, of which 20 inhibited VapC1 activity. Intracellular target engagement of five inhibitors was indicated by the destabilization of VapC1 within bacterial cells from a cellular thermal shift assay; however, no impact on bacterial growth was observed. Thus, this virtual screening and scaffold hopping approach enabled the discovery of VapC1 ribonuclease inhibitors that might serve as starting points for preclinical development.
Asunto(s)
Antitoxinas , Toxinas Bacterianas , Antitoxinas/química , Proteínas Bacterianas/química , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Haemophilus influenzae/química , Haemophilus influenzae/metabolismo , Humanos , Ribonucleasas/metabolismoRESUMEN
An innovative label-free DNA genosensing assay based on a direct hybridization followed by DPASV in the presence of [Fe(CN)6]4-/3- was developed for recognizing the H. influenza genome in human plasma samples. To attain this objective, Zn-based MOF was synthesized and combined with carboxymethyl cellulose (CMC), which were immobilized on the surface of Au electrode and AuNPs were immobilized on the Zn-based MOF/CMC/Au-modified electrode surface. The genosensing bio-assay provides high specificity, sensitivity, and good performance for the determination of L-fuculokinase gene from the Haemophilus influenza genome. Various characterization techniques were applied including Fe-SEM, EDS, FT-IR, and XRD for investigation of morphological features and particle size. Under optimal conditions LOD and LOQ were 1.48 fM and 3.23 fM, respectively. Moreover, a wide linear range was obtained ranging from 0.1 pM-10 nM for t-DNA. The recoveries and RSDs were 98.4-103% and 2.2-3.2, respectively. The fabricated biosensing assay presented high selective ability of one, two, and three-base mismatched sequences. In addition, negative control of the genosensing assay for investigation of the selectivity was performed by the t-DNAs of Salmonella typhimurium and Shigella flexneri bacteria. Likewise, reproducibility and repeatability of the related bio-assay were investigated. It is to be noted that the organized genosensing bio-assay can be straightforwardly reused and regenerated to assess the hybridization process.
Asunto(s)
Técnicas Biosensibles/métodos , ADN Bacteriano/sangre , Técnicas Electroquímicas/métodos , Haemophilus influenzae/química , Estructuras Metalorgánicas/química , ADN Bacteriano/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Oro/química , Haemophilus influenzae/enzimología , Humanos , Límite de Detección , Nanopartículas del Metal/química , Hibridación de Ácido Nucleico , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Reproducibilidad de los Resultados , Zinc/químicaRESUMEN
Surface expression of the common vertebrate sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) by commensal and pathogenic microbes appears structurally to represent "molecular mimicry" of host sialoglycans, facilitating multiple mechanisms of host immune evasion. In contrast, ketodeoxynonulosonic acid (Kdn) is a more ancestral Sia also present in prokaryotic glycoconjugates that are structurally quite distinct from vertebrate sialoglycans. We detected human antibodies against Kdn-terminated glycans, and sialoglycan microarray studies found these anti-Kdn antibodies to be directed against Kdn-sialoglycans structurally similar to those on human cell surface Neu5Ac-sialoglycans. Anti-Kdn-glycan antibodies appear during infancy in a pattern similar to those generated following incorporation of the nonhuman Sia N-glycolylneuraminic acid (Neu5Gc) onto the surface of nontypeable Haemophilus influenzae (NTHi), a human commensal and opportunistic pathogen. NTHi grown in the presence of free Kdn took up and incorporated the Sia into its lipooligosaccharide (LOS). Surface display of the Kdn within NTHi LOS blunted several virulence attributes of the pathogen, including Neu5Ac-mediated resistance to complement and whole blood killing, complement C3 deposition, IgM binding, and engagement of Siglec-9. Upper airway administration of Kdn reduced NTHi infection in human-like Cmah null (Neu5Gc-deficient) mice that express a Neu5Ac-rich sialome. We propose a mechanism for the induction of anti-Kdn antibodies in humans, suggesting that Kdn could be a natural and/or therapeutic "Trojan horse" that impairs colonization and virulence phenotypes of free Neu5Ac-assimilating human pathogens.IMPORTANCE All cells in vertebrates are coated with a dense array of glycans often capped with sugars called sialic acids. Sialic acids have many functions, including serving as a signal for recognition of "self" cells by the immune system, thereby guiding an appropriate immune response against foreign "nonself" and/or damaged cells. Several pathogenic bacteria have evolved mechanisms to cloak themselves with sialic acids and evade immune responses. Here we explore a type of sialic acid called "Kdn" (ketodeoxynonulosonic acid) that has not received much attention in the past and compare and contrast how it interacts with the immune system. Our results show potential for the use of Kdn as a natural intervention against pathogenic bacteria that take up and coat themselves with external sialic acid from the environment.
Asunto(s)
Antígenos CD/inmunología , Infecciones por Haemophilus/inmunología , Haemophilus influenzae/inmunología , Interacciones Huésped-Patógeno/inmunología , Ácido N-Acetilneuramínico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Ácidos Siálicos/inmunología , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Antígenos CD/metabolismo , Transporte Biológico , Complemento C3/inmunología , Complemento C3/metabolismo , Femenino , Glicoconjugados/química , Glicoconjugados/inmunología , Infecciones por Haemophilus/genética , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/química , Interacciones Huésped-Patógeno/genética , Humanos , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Ratones , Ratones Endogámicos C57BL , Imitación Molecular/genética , Imitación Molecular/inmunología , Ácido N-Acetilneuramínico/inmunología , Unión Proteica , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Ácidos Siálicos/química , Azúcares Ácidos/química , Azúcares Ácidos/inmunologíaRESUMEN
Joining the global fight against Tuberculosis, the world's most deadly infectious disease, herein we present the design and synthesis of novel isatin-nicotinohydrazide hybrids (5a-m and 9a-c) as promising anti-tubercular and antibacterial agents. The anti-tubercular activity of the target hybrids was evaluated against drug-susceptible M. tuberculosis strain (ATCC 27294) where hybrids 5d, 5g and 5h were found to be as potent as INH with MIC = 0.24 µg/mL, also the activity was evaluated against Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823) where compounds 5g and 5h showed excellent activity (MIC = 3.9 µg/mL). Moreover, the target hybrids were examined against six bronchitis causing-bacteria. Most derivatives exhibited excellent antibacterial activity. K. pneumonia emerged as the most sensitive strain with MIC range: 0.49-7.81 µg/mL. Furthermore, a molecular docking study has proposed DprE1 as a probable enzymatic target for herein reported isatin-nicotinohydrazide hybrids, and explored the binding interactions within the vicinity of DprE1 active site.
Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antibacterianos/síntesis química , Proteínas Bacterianas/antagonistas & inhibidores , Farmacorresistencia Bacteriana/efectos de los fármacos , Hidrazinas/química , Isatina/química , Mycobacterium tuberculosis/enzimología , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Bordetella pertussis/química , Bordetella pertussis/enzimología , Bordetella pertussis/aislamiento & purificación , Bronquitis/tratamiento farmacológico , Bronquitis/microbiología , Diseño de Fármacos , Farmacorresistencia Bacteriana/genética , Haemophilus influenzae/química , Haemophilus influenzae/enzimología , Haemophilus influenzae/aislamiento & purificación , Isoniazida/farmacología , Klebsiella pneumoniae/química , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Moraxella catarrhalis/química , Moraxella catarrhalis/enzimología , Moraxella catarrhalis/aislamiento & purificación , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/aislamiento & purificación , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Streptococcus pneumoniae/química , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/aislamiento & purificación , Estreptomicina/farmacología , Relación Estructura-Actividad , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Bacterial type II toxin-antitoxin (TA) modules encode a toxic protein that downregulates metabolism and a specific antitoxin that binds and inhibits the toxin during normal growth. In non-typeable Haemophilus influenzae, a common cause of infections in humans, the vapXD locus was found to constitute a functional TA module and contribute to pathogenicity; however, the mode of action of VapD and the mechanism of inhibition by the VapX antitoxin remain unknown. Here, we report the structure of the intact H. influenzae VapXD complex, revealing an unusual 2:1 TA molecular stoichiometry where a Cas2-like homodimer of VapD binds a single VapX antitoxin. VapX consists of an oligonucleotide/oligosaccharide-binding domain that docks into an asymmetrical cavity on the toxin dimer. Structures of isolated VapD further reveal how a symmetrical toxin homodimer adapts to interacting with an asymmetrical antitoxin and suggest how a primordial TA system evolved to become part of CRISPR-Cas immunity systems.
Asunto(s)
Proteínas Bacterianas/química , Toxinas Bacterianas/química , Glicoproteínas de Membrana/química , Sistemas Toxina-Antitoxina , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Sitios de Unión , Haemophilus influenzae/química , Haemophilus influenzae/enzimología , Glicoproteínas de Membrana/metabolismo , Simulación del Acoplamiento Molecular , Dominios ProteicosRESUMEN
Peptides mimicking antigenic epitopes targeted by antibodies can be powerful tools to be used as antigen surrogates for the specific diagnosis and treatment of autoimmune diseases. Obtaining structural insights about the nature of peptide-antibody interaction in complex mixtures such as sera is a critical goal. In multiple sclerosis (MS), we previously demonstrated that the N-linked ß-d-glucopyranosyl moieties (N-Glc) containing epitopes in nontypeable Haemophilus influenzae adhesin C-terminal portion HMW1(1205-1526) were essential for high-affinity antibody binding in a subpopulation of MS patients. With the aim of developing peptide probes and assessing their binding properties to antibodies from sera of representative patients, we performed the systematic analysis of synthetic peptides based on HMW1(1347-1354) fragment bearing one or two N-Glc respectively on Asn-1349 and/or Asn-1352. The N-glucosylated nonapeptides efficiently bind to IgG antibodies, displaying IC50 in the range 10-8 -10-10 M by competitive indirect enzyme-linked immunosorbent assay (ELISA) in three representative MS patient sera. We selected the di-N-glucosylated adhesin peptide Ac-KAN (Glc)VTLN (Glc)TT-NH2 as the shortest sequence able to inhibit high-avidity interaction with N-Glc targeting IgM antibodies. Nuclear magnetic resonance (NMR)- and circular dichroism (CD)-based characterization showed that the binding properties of these antigens could not be ascribed to structural differences induced by the presence of up to two N-glucosyl moieties. Therefore, the antibody binding is not easily correlated to the position of the sugar or to a determined conformation in water.
Asunto(s)
Adhesinas Bacterianas/inmunología , Antígenos/inmunología , Esclerosis Múltiple/inmunología , Péptidos/inmunología , Adhesinas Bacterianas/química , Glicosilación , Haemophilus influenzae/química , Humanos , Modelos Moleculares , Péptidos/síntesis química , Péptidos/químicaRESUMEN
Characterisation of protein function based solely on homology searches may overlook functions under specific environmental conditions, or the possibility of a protein having multiple roles. In this study we investigated the role of YtfB, a protein originally identified in a genome-wide screen to cause inhibition of cell division, and has demonstrated to localise to the Escherichia coli division site with some degree of glycan specificity. Interestingly, YtfB also shows homology to the virulence factor OapA from Haemophilus influenzae, which is important for adherence to epithelial cells, indicating the potential of additional function(s) for YtfB. Here we show that E. coli YtfB binds to N'acetylglucosamine and mannobiose glycans with high affinity. The loss of ytfB results in a reduction in the ability of the uropathogenic E. coli strain UTI89 to adhere to human kidney cells, but not to bladder cells, suggesting a specific role in the initial adherence stage of ascending urinary tract infections. Taken together, our results suggest a role for YtfB in adhesion to specific eukaryotic cells, which may be additional, or complementary, to its role in cell division. This study highlights the importance of understanding the possible multiple functions of proteins based on homology, which may be specific to different environmental conditions.
Asunto(s)
Adhesión Bacteriana/genética , Proteínas de Ciclo Celular/genética , División Celular/genética , Proteínas de Escherichia coli/genética , Escherichia coli Uropatógena/genética , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Secuencia de Carbohidratos , Adhesión Celular , Proteínas de Ciclo Celular/deficiencia , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Expresión Génica , Células HEK293 , Haemophilus influenzae/química , Haemophilus influenzae/metabolismo , Humanos , Mananos/química , Mananos/metabolismo , Filogenia , Polisacáridos/química , Polisacáridos/metabolismo , Unión Proteica , Infecciones Urinarias/microbiología , Infecciones Urinarias/patología , Escherichia coli Uropatógena/clasificación , Escherichia coli Uropatógena/citología , Escherichia coli Uropatógena/patogenicidad , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Mycobacterium smegmatis is a good model for studying the physiology and pathogenesis of Mycobacterium tuberculosis due to its genetic similarity. As methionine biosynthesis exists only in microorganisms, the enzymes involved in methionine biosynthesis can be a potential target for novel antibiotics. Homoserine O-acetyltransferase from M. smegmatis (MsHAT) catalyzes the transfer of acetyl-group from acetyl-CoA to homoserine. To investigate the molecular mechanism of MsHAT, we determined its crystal structure in apo-form and in complex with either CoA or homoserine and revealed the substrate binding mode of MsHAT. A structural comparison of MsHAT with other HATs suggests that the conformation of the α5 to α6 region might influence the shape of the dimer. In addition, the active site entrance shows an open or closed conformation and might determine the substrate binding affinity of HATs.
Asunto(s)
Acetilcoenzima A/química , Acetiltransferasas/química , Apoproteínas/química , Proteínas Bacterianas/química , Homoserina/química , Mycobacterium smegmatis/química , Acetilcoenzima A/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Haemophilus influenzae/química , Haemophilus influenzae/enzimología , Haemophilus influenzae/genética , Homoserina/metabolismo , Cinética , Leptospira interrogans/química , Leptospira interrogans/enzimología , Leptospira interrogans/genética , Modelos Moleculares , Mycobacteriaceae/química , Mycobacteriaceae/enzimología , Mycobacteriaceae/genética , Mycobacterium abscessus/química , Mycobacterium abscessus/enzimología , Mycobacterium abscessus/genética , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The bacterial periplasmic protein LpoA is an outer membrane lipoprotein and an activator for the cross-linking activity of PBP1A, a bifunctional peptidoglycan synthase. Previous structures of the amino-terminal (N) domain of LpoA showed it to consist entirely of helices and loops, with at least four tetratricopeptide-like repeats. Although the previously determined orthorhombic crystal structure of the N domain of Haemophilus influenzae LpoA showed a typical curved structure with a concave groove, an NMR structure of the same domain from Escherichia coli was relatively flat. Here, a crystal structure of the N domain of E. coli LpoA was determined to a resolution of 2.1â Å and was found to be more similar to the H. influenzae crystal structure than to the E. coli NMR structure. To provide a quantitative description for these comparisons, the various structures were superimposed pairwise by fitting the first half of each structure to its pairwise partner and then calculating the rotation axis that would optimally superimpose the second half. Differences in both the magnitude of the rotation and the direction of the rotation axis were observed between different pairs of structures. A 1.35â Å resolution structure of a monoclinic crystal form of the N domain of H. influenzae LpoA was also determined. In this structure, the subdomains rotate 10° relative to those in the original orthorhombic H. influenzae crystal structure to further narrow the groove between the subdomains. To accommodate this, a bound chloride ion (in place of sulfate) allowed the closer approach of a helix that forms one side of the groove.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Cloruros/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Haemophilus influenzae/química , Lipoproteínas/química , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de ProteínaRESUMEN
Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. The vapBC (virulence-associated protein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth of Escherichia coli, and pathogenesis ex vivo were tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1 strain complemented in cis with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the vapBC-1 native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues ex vivoIMPORTANCE Herein the crystal structure of the VapBC-1 complex from nontypeable Haemophilus influenzae (NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity in E. coli, but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin, VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissues ex vivo Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to that of the in cis wild-type toxin under the same conditions.
Asunto(s)
Proteínas Bacterianas/química , Toxinas Bacterianas/química , Haemophilus influenzae/genética , Sistemas Toxina-Antitoxina , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Cristalización , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Haemophilus influenzae/química , Haemophilus influenzae/patogenicidad , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Operón , Regiones Promotoras Genéticas , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Because of the serious neurologic consequences of iron deficiency and iron excess in the brain, interest in the iron status of the central nervous system has increased significantly in the past decade. While iron plays an important role in many physiological processes, its accumulation may lead to diseases such as Huntington's, Parkinson's, and Alzheimer's. Therefore, it is important to develop methodologies that can monitor the presence of iron in a selective and sensitive manner. In this paper, we first showed the synthesis and characterization of the iron-binding protein (FBP) from Haemophilus influenzae, specific for ferrous ions. Subsequently, we employed this protein in our nanopipette platform and utilized it in functionalized nanoprobes to monitor the presence of ferrous ions. A suite of characterization techniques: absorbance spectroscopy, dynamic light scattering, and small-angle X-ray scattering were used for FBP. The functionalized Fe-nanoprobe calibrated in ferrous chloride enabled detection from 0.05 to 10 µM, and the specificity of the modified iron probe was evaluated by using various metal ion solutions.
Asunto(s)
Dispersión Dinámica de Luz/instrumentación , Haemophilus influenzae/metabolismo , Proteínas de Unión a Hierro/metabolismo , Hierro/metabolismo , Nanotecnología/instrumentación , Dispersión del Ángulo Pequeño , Dispersión Dinámica de Luz/métodos , Haemophilus influenzae/química , Hierro/análisis , Proteínas de Unión a Hierro/análisis , Nanotecnología/métodosRESUMEN
In nontypeable Haemophilus influenzae (NTHi), the oligopeptide-binding protein (OppA) serves as the substrate-binding protein (SBP) of the oligopeptide transport system responsible for the import of peptides. We solved the crystal structure of nthiOppA in complex with hydrophobic peptides of various sizes. Our novel hexapeptide complex demonstrates the flexibility of the nthiOppA-binding cavity to expand and accommodate the longer peptide while maintaining similar protein-peptide interactions of smaller peptide complexes. In addition to acquiring peptides from the host environment, as a heme auxotroph NTHi utilizes host hemoproteins as a source of essential iron. OppA is a member of the Cluster C SBP family, and unlike other SBP families, some members recognize two distinctly different substrates. DppA (dipeptide), MppA (murein tripeptide), and SapA (antimicrobial peptides) are Cluster C proteins known to also transport heme. We observed nthiOppA shares this heme-binding characteristic and established heme specificity and affinity by surface plasmon resonance (SPR) of the four Cluster C proteins in NTHi. Ligand-docking studies predicted a distinct heme-specific cleft in the binding pocket, and using SPR competition assays, we observed that heme does not directly compete with peptide in the substrate-binding pocket. Additionally, we identified that the individual nthiOppA domains differentially contribute to substrate binding, with one domain playing a dominant role in heme binding and the other in peptide binding. Our results demonstrate the multisubstrate specificity of nthiOppA and the role of NTHi Cluster C proteins in the heme-uptake pathway for this pathogen.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Haemophilus influenzae/química , Hemo/química , Lipoproteínas/química , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas Portadoras/genética , Cristalografía por Rayos X , Haemophilus influenzae/genética , Hemo/genética , Lipoproteínas/genéticaRESUMEN
Iron is an essential micronutrient for most living species. In mammals, hemoglobin (Hb) stores more than two thirds of the body's iron content. In the bloodstream, haptoglobin (Hp) and hemopexin (Hpx) sequester free Hb or heme. Pathogenic microorganisms usually acquire iron from their hosts and have evolved complex systems of iron piracy to circumvent nutritional immunity. Herein, we performed an evolutionary analysis of genes coding for mammalian heme-binding proteins and heme-scavengers in pathogen species. The underlying hypothesis is that these molecules are engaged in a molecular arms race. We show that positive selection drove the evolution of mammalian Hb and Hpx. Positively selected sites in Hb are located at the interaction surface with Neisseria meningitidis heme scavenger HpuA and with Staphylococcus aureus iron-regulated surface determinant B (IsdB). In turn, positively selected sites in HpuA and IsdB are located in the flexible protein regions that contact Hb. A residue in Hb (S45H) was also selected on the Caprinae branch. This site stabilizes the interaction with Trypanosoma brucei hemoglobin-haptoglobin (HbHp) receptor (TbHpHbR), a molecule that also mediates trypanosome lytic factor (TLF) entry. In TbHpHbR, positive selection drove the evolution of a variant (L210S) which allows evasion from TLF but reduces affinity for HbHp. Finally, selected sites in Hpx are located at the interaction surface with the Haemophilus influenzae hemophore HxuA, which in turn displays fast evolving sites at the Hpx-binding interface. These results shed light into host-pathogens conflicts and establish the importance of nutritional immunity as an evolutionary force.
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Proteínas Bacterianas/química , Proteínas de Transporte de Catión/química , Haptoglobinas/química , Hemopexina/química , Hierro/química , Proteínas Protozoarias/química , Receptores de Superficie Celular/química , Animales , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Haemophilus influenzae/química , Haemophilus influenzae/metabolismo , Haptoglobinas/metabolismo , Hemopexina/metabolismo , Humanos , Hierro/metabolismo , Neisseria meningitidis/química , Neisseria meningitidis/metabolismo , Proteínas Protozoarias/metabolismo , Receptores de Superficie Celular/metabolismo , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/metabolismoRESUMEN
Phosphocholine-modified bacterial cell wall components are virulence factors enabling immune evasion and permanent colonization of the mammalian host, by mechanisms that are poorly understood. Recently, we demonstrated that free phosphocholine (PC) and PC-modified lipooligosaccharides (PC-LOS) from Haemophilus influenzae, an opportunistic pathogen of the upper and lower airways, function as unconventional nicotinic agonists and efficiently inhibit the ATP-induced release of monocytic IL-1ß. We hypothesize that H. influenzae PC-LOS exert similar effects on pulmonary epithelial cells and on the complex lung tissue. The human lung carcinoma-derived epithelial cell lines A549 and Calu-3 were primed with lipopolysaccharide from Escherichia coli followed by stimulation with ATP in the presence or absence of PC or PC-LOS or LOS devoid of PC. The involvement of nicotinic acetylcholine receptors was tested using specific antagonists. We demonstrate that PC and PC-LOS efficiently inhibit ATP-mediated IL-1ß release by A549 and Calu-3 cells via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Primed precision-cut lung slices behaved similarly. We conclude that H. influenzae hijacked an endogenous anti-inflammatory cholinergic control mechanism of the lung to evade innate immune responses of the host. These findings may pave the way towards a host-centered antibiotic treatment of chronic airway infections with H. influenzae.
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Adenosina Trifosfato/farmacología , Células Epiteliales/metabolismo , Haemophilus influenzae/química , Interleucina-1beta/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/farmacología , Pulmón/citología , Fosforilcolina/química , Células A549 , Animales , Células Epiteliales/efectos de los fármacos , Humanos , Ratones , Nicotina/farmacología , Receptores Nicotínicos/metabolismoRESUMEN
Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in individuals of all ages. The lipooligosaccharide (LOS) of NTHi has evolved a complex structure that can be attributed to a multiplicity of glycosyltransferases, the random switching of glycosyltransferase gene expression via phase variation, and the complex structure of its core region with multiple glycoform branch points. This article adds to that complexity by describing a multifunctional enzyme (LsgB) which optimally functions when the species is grown on a solid surface and which can add either a ketodeoxyoctanoate (KDO) or an N-acetylneuramic acid (Neu5Ac) moiety to a terminal N-acetyllactosamine structure of LOS. Our studies show that expression of lsgB is reduced four- to sixfold when NTHi is grown in broth. The substrate that the enzyme utilizes is dependent upon the concentration of free Neu5Ac (between 1 and 10 µg/ml) in the environment. In environments in which Neu5Ac is below that level, the enzyme utilizes endogenous CMP-KDO as the substrate. Our studies show that during in vivo growth in an NTHi biofilm, the KDO moiety is expressed by the organism. Monoclonal antibody 6E4, which binds KDO, is bactericidal for NTHi strains that express the KDO epitope at high levels. In a survey of 33 NTHi strains isolated from healthy and diseased individuals, the antibody was bactericidal (>90% kill) for 12 strains (36%). These studies open up the possibility of using a KDO-based glycoconjugate vaccine as part of a multicomponent vaccine against NTHi.IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in middle ear infections in children, sinusitis in adults, and acute bronchitis in individuals with chronic obstructive lung disease. The organism is very well adapted to the human host environment, and this has hindered successful development of an effective vaccine. In this article, we describe a mechanism by which the bacteria decorates its surface lipooligosaccharide with a sugar unique to Gram-negative bacteria, ketodeoxyoctanoate (KDO). This sugar decoration is present during active infection and we have shown that an antibody directed against this sugar can result in killing of the organism. These data demonstrate that the lipooligosaccharide ketodeoxyoctanoate epitope may be a novel NTHi-specific candidate vaccine antigen.
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Anticuerpos Antibacterianos/inmunología , Caprilatos/inmunología , Haemophilus influenzae/química , Haemophilus influenzae/inmunología , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Haemophilus influenzae/enzimología , Haemophilus influenzae/metabolismo , Lipopolisacáridos/metabolismo , Viabilidad Microbiana , Ácido N-Acetilneuramínico/metabolismoRESUMEN
Nontypeable Haemophilus influenzae (NTHi), one of the most common acute otitis media (OM) pathogens, is postulated to promote middle-ear epithelial remodeling in the progression of OM from acute to chronic. The goal of this study was to examine early quantitative proteomic secretome effects of NTHi lysate exposure in a human middle-ear epithelial cell (HMEEC) line. NTHi lysates were used to stimulate HMEEC, and conditional quantitative stable isotope labeling with amino acids in cell culture of cell secretions was performed. Mass spectrometry analysis identified 766 proteins across samples. Of interest, several heterogeneous nuclear ribonucleoproteins (hnRNPs) were regulated by NTHi lysate treatment, especially hnRNP A2B1 and hnRNP Q, known to be implicated in microRNA (miRNA) packaging in exosomes. After purification, the presence of exosomes in HMEEC secretions was characterized by dynamic light scattering (<100 nm), transmission electron microscopy, and CD63/heat shock protein 70 positivity. hnRNP A2B1 and hnRNP Q were confirmed to be found in exosomes by Western blot and proteomic analysis. Finally, exosomal miRNA content comprised 110 unique miRNAs, with 5 found to be statistically induced by NTHi lysate (miR-378a-3p + miR-378i, miR-200a-3p, miR-378g, miR30d-5p, and miR-222-3p), all known to target innate immunity genes. This study demonstrates that NTHi lysates promote release of miRNA-laden exosomes from middle-ear epithelium in vitro. -Val, S., Krueger, A., Poley, M., Cohen, A., Brown, K., Panigrahi, A., Preciado, D. Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle-ear epithelial cells.
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Oído Medio/citología , Células Epiteliales/metabolismo , Exosomas/metabolismo , Haemophilus influenzae/patogenicidad , Ribonucleoproteínas/metabolismo , Extractos Celulares/farmacología , Línea Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Exocitosis , Haemophilus influenzae/química , Humanos , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
In many Gram-negative bacteria, the peptidoglycan synthase PBP1A requires the outer membrane lipoprotein LpoA for constructing a functional peptidoglycan required for bacterial viability. Previously, we have shown that the C-terminal domain of Haemophilus influenzae LpoA (HiLpoA) has a highly conserved, putative substrate-binding cleft between two α/ß lobes. Here, we report a 2.0 Å resolution crystal structure of the HiLpoA N-terminal domain. Two subdomains contain tetratricopeptide-like motifs that form a concave groove, but their relative orientation differs by â¼45° from that observed in an NMR structure of the Escherichia coli LpoA N domain. We also determined three 2.0-2.8 Å resolution crystal structures containing four independent full-length HiLpoA molecules. In contrast to an elongated model previously suggested for E. coli LpoA, each HiLpoA formed a U-shaped structure with a different C-domain orientation. This resulted from both N-domain twisting and rotation of the C domain (up to 30°) at the end of the relatively immobile interdomain linker. Moreover, a previously predicted hinge between the lobes of the LpoA C domain exhibited variations of up to 12°. Small-angle X-ray scattering data revealed excellent agreement with a model calculated by normal mode analysis from one of the full-length HiLpoA molecules but even better agreement with an ensemble of this molecule and two of the partially extended normal mode analysis-predicted models. The different LpoA structures helped explain how an outer membrane-anchored LpoA can either withdraw from or extend toward the inner membrane-bound PBP1A through peptidoglycan gaps and hence regulate the synthesis of peptidoglycan necessary for bacterial viability.
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Proteínas de la Membrana Bacteriana Externa/química , Activadores de Enzimas/química , Haemophilus influenzae/química , Proteínas de Unión a las Penicilinas , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cristalografía por Rayos X , Activadores de Enzimas/metabolismo , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Resonancia Magnética Nuclear Biomolecular , Dominios ProteicosRESUMEN
The Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel `hairpin-like' architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3â Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the `hairpin-like' hypothesis.
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Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Haemophilus influenzae/química , Adhesinas Bacterianas/metabolismo , Secuencia de Aminoácidos , Adhesión Bacteriana , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Haemophilus influenzae/metabolismo , Modelos Moleculares , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de ProteínaRESUMEN
Fatty acid biosynthesis is essential to bacterial growth in Gram-negative pathogens. Several small molecules identified through a combination of high-throughput and fragment screening were cocrystallized with FabH (ß-ketoacyl-acyl carrier protein synthase III) from Escherichia coli and Streptococcus pneumoniae. Structure-based drug design was used to merge several scaffolds to provide a new class of inhibitors. After optimization for Gram-negative enzyme inhibitory potency, several compounds demonstrated antimicrobial activity against an efflux-negative strain of Haemophilus influenzae. Mutants resistant to these compounds had mutations in the FabH gene near the catalytic triad, validating FabH as a target for antimicrobial drug discovery.