RESUMEN
Paper spray ionization mass spectrometry (PSI-MS) is a relatively new analytical technique allowing for rapid mass spectrometric analysis of biological samples with little or no sample preparation. The expeditious nature of the analysis and minimal requirement for sample preparation make PSI-MS a promising avenue for future clinical assays with one potential application in the identification of different types of bacteria. Although past PSI-MS studies have demonstrated the ability to distinguish between bacteria of different species and morphological classes, achieving within-species strain-level differentiation has never been performed. In this report, we demonstrate the first strain-level bacterial differentiation by PSI-MS with the mammalian intestinal bacterium Oxalobacter formigenes ( Oxf). This novel application holds promising clinical significance as it could be used to differentiate between pathogenic bacteria and their harmless, commensal relatives, saving time and money in clinical diagnostics. Both whole cells and cell lysates of Oxf strains HC1 and OxWR were analyzed using the Prosolia Velox 360TM PSI source coupled to a Thermo Scientific Q Exactive high-resolution mass spectrometer with a rapid 30 s analytical method. Multivariate statistical analysis followed by examination of significant features provided for and confirmed differentiation between Oxf HC1 and OxWR. We report a panel of strain-exclusive metabolites that could serve as potential strain-indicating biomarkers.
Asunto(s)
Espectrometría de Masas/métodos , Oxalobacter formigenes/química , Oxalobacter formigenes/metabolismo , Papel , Especificidad de la EspecieRESUMEN
OxlT, the oxalate transporter of Oxalobacter formigenes, is a member of the Major Facilitator Superfamily of transporters (MFS), one of the largest groups of membrane proteins with substantial relevance to solute transport physiology, pharmacology, and possible drug development. MFS proteins transport a wide range of substrates such as organic and inorganic anions, sugars, drugs, and neurotransmitters. This review succinctly summarizes experimental work on a model MFS protein, OxlT, beginning with its identification as an electrogenic oxalate/formate exchanger, its three-dimensional structure, and discussion of biochemical and biophysical data that have shed further light on its structure and function. We also discuss the structure and function of OxlT in relation to notable MFS carriers such as LacY and GlpT.
Asunto(s)
Proteínas Bacterianas , Proteínas de Transporte de Membrana , Modelos Biológicos , Modelos Moleculares , Oxalobacter formigenes , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transporte Biológico Activo/fisiología , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Ácido Oxálico/química , Ácido Oxálico/metabolismo , Oxalobacter formigenes/química , Oxalobacter formigenes/metabolismoRESUMEN
The L-arginine/agmatine antiporter AdiC is a key component of the arginine-dependent extreme acid resistance system of Escherichia coli. Phylogenetic analysis indicated that AdiC belongs to the amino acid/polyamine/organocation (APC) transporter superfamily having sequence identities of 15-17% to eukaryotic and human APC transporters. For functional and structural characterization, we cloned, overexpressed, and purified wild-type AdiC and the point mutant AdiC-W293L, which is unable to bind and consequently transport L-arginine. Purified detergent-solubilized AdiC particles were dimeric. Reconstitution experiments yielded two-dimensional crystals of AdiC-W293L diffracting beyond 6 angstroms resolution from which we determined the projection structure at 6.5 angstroms resolution. The projection map showed 10-12 density peaks per monomer and suggested mainly tilted helices with the exception of one distinct perpendicular membrane spanning alpha-helix. Comparison of AdiC-W293L with the projection map of the oxalate/formate antiporter from Oxalobacter formigenes, a member from the major facilitator superfamily, indicated different structures. Thus, two-dimensional crystals of AdiC-W293L yielded the first detailed view of a transport protein from the APC superfamily at sub-nanometer resolution.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos/química , Antiportadores/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Filogenia , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Básicos/genética , Animales , Antiportadores/genética , Cristalografía por Rayos X/métodos , Dimerización , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Oxalobacter formigenes/química , Oxalobacter formigenes/genética , Estructura Cuaternaria de Proteína/fisiologíaRESUMEN
An OxlT homology model suggests R272 and K355 in transmembrane helices 8 and 11, respectively, are critical to OxlT-mediated transport. We offer positive evidence supporting this idea by studying OxlT function after cysteine residues were separately introduced at these positions. Without further treatment, both mutant proteins had a null phenotype when they were reconstituted into proteoliposomes. By contrast, significant recovery of function occurred when proteoliposomes were treated with MTSEA (methanethiosulfonate ethylamine), a thiol-specific reagent that implants a positively charged amino group. In each case, there was a 2-fold increase in the Michaelis constant (K(M)) for oxalate self-exchange (from 80 to 160 microM), along with a 5-fold (K355C) or 100-fold (R272C) reduction in V(max) compared to that of the cysteine-less parental protein. Analysis by MALDI-TOF confirmed that MTSEA introduced the desired modification. We also examined substrate selectivity for the treated derivatives. While oxalate remained the preferred substrate, there was a shift in preference among other substrates so that the normal rank order (oxalate > malonate > formate) was altered to favor smaller substrates (oxalate > formate > malonate). This shift is consistent with the idea that the substrate-binding site is reduced in size via introduction of the SCH(2)CH(2)NH(3)(+) adduct, which generates a side chain that is approximately 1.85 A longer than that of lysine or arginine. These findings lead us to conclude that R272 and K355 are essential components of the OxlT substrate-binding site.
Asunto(s)
Antiportadores/química , Proteínas Bacterianas/química , Formiatos/química , Oxalatos/química , Oxalobacter formigenes/química , Proteolípidos/química , Antiportadores/genética , Antiportadores/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico/fisiología , Formiatos/metabolismo , Oxalatos/metabolismo , Oxalobacter formigenes/genética , Oxalobacter formigenes/metabolismo , Proteolípidos/metabolismo , Homología Estructural de ProteínaRESUMEN
The major facilitator superfamily (MFS) represents the largest collection of evolutionarily related members within the class of membrane 'carrier' proteins. OxlT, a representative example of the MFS, is an oxalate-transporting membrane protein in Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional crystals of OxlT, we have determined the projection structure of this membrane transporter. The projection map at 6 A resolution indicates the presence of 12 transmembrane helices in each monomer of OxlT, with one set of six helices related to the other set by an approximate internal two-fold axis. The projection map reveals the existence of a central cavity, which we propose to be part of the pathway of oxalate transport. By combining information from the projection map with related biochemical data, we present probable models for the architectural arrangement of transmembrane helices in this protein superfamily.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Proteínas de la Membrana/química , Proteínas de Transporte de Membrana , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Oxalobacter formigenes/química , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His(9)-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni2+ linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.