RESUMEN
Having multiple rounds of translation of the same mRNA creates dynamic complexities along with opportunities for regulation related to ribosome pausing and stalling at specific sequences. Yet, mechanisms controlling these critical processes and the principles guiding their evolution remain poorly understood. Through genetic, genomic, physiological, and biochemical approaches, we demonstrate that regulating ribosome pausing at specific amino acid sequences can produce ~2-fold changes in protein expression levels which strongly influence cell growth and therefore evolutionary fitness. We demonstrate, both in vivo and in vitro, that the ABC-F protein EttA directly controls the translation of mRNAs coding for a subset of enzymes in the tricarboxylic acid (TCA) cycle and its glyoxylate shunt, which modulates growth in some chemical environments. EttA also modulates expression of specific proteins involved in metabolically related physiological and stress-response pathways. These regulatory activities are mediated by EttA rescuing ribosomes paused at specific patterns of negatively charged residues within the first 30 amino acids of nascent proteins. We thus establish a unique global regulatory paradigm based on sequence-specific modulation of translational pausing.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas de Escherichia coli , Escherichia coli , Biosíntesis de Proteínas , Ribosomas , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Ciclo del Ácido Cítrico , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Glioxilatos/metabolismo , Ribosomas/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors that provide resistance against clinically important ribosome-targeting antibiotics which are proliferating among pathogens. Here, we combine genetic and structural approaches to determine the regulation of streptococcal ARE ABC-F gene msrD in response to macrolide exposure. We show that binding of cladinose-containing macrolides to the ribosome prompts insertion of the leader peptide MsrDL into a crevice of the ribosomal exit tunnel, which is conserved throughout bacteria and eukaryotes. This leads to a local rearrangement of the 23 S rRNA that prevents peptide bond formation and accommodation of release factors. The stalled ribosome obstructs the formation of a Rho-independent terminator structure that prevents msrD transcriptional attenuation. Erythromycin induction of msrD expression via MsrDL, is suppressed by ectopic expression of mrsD, but not by mutants which do not provide antibiotic resistance, showing correlation between MsrD function in antibiotic resistance and its action on this stalled complex.
Asunto(s)
Enfermedades del Nervio Abducens , Antibacterianos , Humanos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Macrólidos/farmacología , Acomodación OcularRESUMEN
Multiple paralogous ABCF ATPases are encoded in most genomes, but the physiological functions remain unknown for most of them. We herein compare the four Escherichia coli K12 ABCFs - EttA, Uup, YbiT, and YheS - using assays previously employed to demonstrate EttA gates the first step of polypeptide elongation on the ribosome dependent on ATP/ADP ratio. A Δ uup knockout, like Δ ettA , exhibits strongly reduced fitness when growth is restarted from long-term stationary phase, but neither Δ ybiT nor Δ yheS exhibits this phenotype. All four proteins nonetheless functionally interact with ribosomes based on in vitro translation and single-molecule fluorescence resonance energy transfer experiments employing variants harboring glutamate-to-glutamine active-site mutations (EQ 2 ) that trap them in the ATP-bound conformation. These variants all strongly stabilize the same global conformational state of a ribosomal elongation complex harboring deacylated tRNA Val in the P site. However, EQ 2 -Uup uniquely exchanges on/off the ribosome on a second timescale, while EQ 2 -YheS-bound ribosomes uniquely sample alternative global conformations. At sub-micromolar concentrations, EQ 2 -EttA and EQ 2 -YbiT fully inhibit in vitro translation of an mRNA encoding luciferase, while EQ 2 -Uup and EQ 2 -YheS only partially inhibit it at ~10-fold higher concentrations. Moreover, tripeptide synthesis reactions are not inhibited by EQ 2 -Uup or EQ 2 -YheS, while EQ 2 -YbiT inhibits synthesis of both peptide bonds and EQ 2 -EttA specifically traps ribosomes after synthesis of the first peptide bond. These results support the four E. coli ABCF paralogs all having different activities on translating ribosomes, and they suggest that there remains a substantial amount of functionally uncharacterized "dark matter" involved in mRNA translation.
RESUMEN
Energy-dependent translational throttle A (EttA) from Escherichia coli is a paradigmatic ABC-F protein that controls the first step in polypeptide elongation on the ribosome according to the cellular energy status. Biochemical and structural studies have established that ABC-F proteins generally function as translation factors that modulate the conformation of the peptidyl transferase center upon binding to the ribosomal tRNA exit site. These factors, present in both prokaryotes and eukaryotes but not in archaea, use related molecular mechanisms to modulate protein synthesis for heterogenous purposes, ranging from antibiotic resistance and rescue of stalled ribosomes to modulation of the mammalian immune response. Here, we review the canonical studies characterizing the phylogeny, regulation, ribosome interactions, and mechanisms of action of the bacterial ABC-F proteins, and discuss the implications of these studies for the molecular function of eukaryotic ABC-F proteins, including the three human family members.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Farmacorresistencia Bacteriana/inmunología , Proteínas de Escherichia coli/inmunología , Escherichia coli/inmunología , Biosíntesis de Proteínas/inmunología , Ribosomas/inmunología , Animales , HumanosAsunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Bacterias/química , Bacterias/genética , Proteínas Bacterianas/genética , Dominios ProteicosRESUMEN
The ATP binding cassette protein superfamily comprises ATPase enzymes which are, for the most part, involved in transmembrane transport. Within this superfamily however, some protein families have other functions unrelated to transport. One example is the ABC-F family, which comprises an extremely diverse set of cytoplasmic proteins. All of the proteins in the ABC-F family characterized to date act on the ribosome and are translation factors. Their common function is ATP-dependent modulation of the stereochemistry of the peptidyl transferase center (PTC) in the ribosome coupled to changes in its global conformation and P-site tRNA binding geometry. In this review, we give an overview of the function, structure, and theories for the mechanisms-of-action of microbial proteins in the ABC-F family, including those involved in mediating resistance to ribosome-binding antibiotics.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Farmacorresistencia Bacteriana Múltiple/fisiología , Escherichia coli/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Antibacterianos/metabolismo , Antibacterianos/farmacología , Transporte Biológico/fisiología , Cristalografía por Rayos X , Farmacorresistencia Bacteriana Múltiple/genética , Biosíntesis de Proteínas/genética , Conformación Proteica , Dominios Proteicos , Ribosomas/metabolismoRESUMEN
The development of synthetic biology calls for accurate understanding of the critical functions that allow construction and operation of a living cell. Besides coding for ubiquitous structures, minimal genomes encode a wealth of functions that dissipate energy in an unanticipated way. Analysis of these functions shows that they are meant to manage information under conditions when discrimination of substrates in a noisy background is preferred over a simple recognition process. We show here that many of these functions, including transporters and the ribosome construction machinery, behave as would behave a material implementation of the information-managing agent theorized by Maxwell almost 150 years ago and commonly known as Maxwell's demon (MxD). A core gene set encoding these functions belongs to the minimal genome required to allow the construction of an autonomous cell. These MxDs allow the cell to perform computations in an energy-efficient way that is vastly better than our contemporary computers.
Asunto(s)
Fenómenos Fisiológicos Celulares , Modelos Biológicos , Biología Sintética/métodosRESUMEN
Recombinant proteins are essential for biotechnology. Here we review some of the key points for improving the production of heterologous proteins, and what can be the future of the field.
Asunto(s)
Bacterias/genética , Bacterias/metabolismo , Biotecnología/métodos , Ingeniería Metabólica/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Biotecnología/tendencias , Ingeniería Metabólica/tendenciasRESUMEN
Many disease-causing mutations impair protein stability. Here, we explore a thermodynamic strategy to correct the disease-causing F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR). F508del destabilizes nucleotide-binding domain 1 (hNBD1) in hCFTR relative to an aggregation-prone intermediate. We developed a fluorescence self-quenching assay for compounds that prevent aggregation of hNBD1 by stabilizing its native conformation. Unexpectedly, we found that dTTP and nucleotide analogs with exocyclic methyl groups bind to hNBD1 more strongly than ATP and preserve electrophysiological function of full-length F508del-hCFTR channels at temperatures up to 37 °C. Furthermore, nucleotides that increase open-channel probability, which reflects stabilization of an interdomain interface to hNBD1, thermally protect full-length F508del-hCFTR even when they do not stabilize isolated hNBD1. Therefore, stabilization of hNBD1 itself or of one of its interdomain interfaces by a small molecule indirectly offsets the destabilizing effect of the F508del mutation on full-length hCFTR. These results indicate that high-affinity binding of a small molecule to a remote site can correct a disease-causing mutation. We propose that the strategies described here should be applicable to identifying small molecules to help manage other human diseases caused by mutations that destabilize native protein conformation.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Nucleótidos de Timina/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Enlace de Hidrógeno , Ligandos , Mutación , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estabilidad Proteica , Desplegamiento Proteico , TermodinámicaRESUMEN
Synonymous variations in protein-coding sequences alter protein expression dynamics, which has important implications for cellular physiology and evolutionary fitness, but disentangling the underlying molecular mechanisms remains challenging.
Asunto(s)
Evolución Biológica , Codón , Expresión Génica , Sistemas de Lectura AbiertaRESUMEN
Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.
Asunto(s)
Codón/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Biosíntesis de Proteínas/genética , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biosíntesis , Genes Sintéticos/genética , Semivida , Cinética , Modelos Logísticos , Modelos Genéticos , Datos de Secuencia Molecular , Oportunidad Relativa , Extensión de la Cadena Peptídica de Translación , Pliegue del ARN , Estabilidad del ARN , ARN Bacteriano/genética , ARN Mensajero/genética , Transcripción Genética/genética , Proteínas Virales/metabolismoRESUMEN
ABC-F proteins have evaded functional characterization even though they compose one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that YjjK, the most prevalent eubacterial ABC-F protein, gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. Accordingly, we rename this protein energy-dependent translational throttle A (EttA). We determined the crystal structure of Escherichia coli EttA and used it to design mutants for biochemical studies including enzymological assays of the initial steps of protein synthesis. These studies suggest that EttA may regulate protein synthesis in energy-depleted cells, which have a low ATP/ADP ratio. Consistently with this inference, EttA-deleted cells exhibit a severe fitness defect in long-term stationary phase. These studies demonstrate that an ABC-F protein regulates protein synthesis via a new mechanism sensitive to cellular energy status.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Proteínas de Escherichia coli/fisiología , Extensión de la Cadena Peptídica de Translación , Ribosomas/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Biológicos , Modelos Moleculares , Filogenia , Estructura Terciaria de ProteínaRESUMEN
Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. Here, we elucidate the mechanism of a newly characterized regulatory translation factor, energy-dependent translational throttle A (EttA), which is an Escherichia coli representative of the ATP-binding cassette F (ABC-F) protein family. Using cryo-EM, we demonstrate that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, where it forms bridging interactions between the ribosomal L1 stalk and the tRNA bound in the peptidyl-tRNA-binding site. Using single-molecule fluorescence resonance energy transfer, we show that the ATP-bound form of EttA restricts ribosome and tRNA dynamics required for protein synthesis. This work represents the first example, to our knowledge, in which the detailed molecular mechanism of any ABC-F family protein has been determined and establishes a framework for elucidating the mechanisms of other regulatory translation factors.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Proteínas de Escherichia coli/fisiología , Extensión de la Cadena Peptídica de Translación , Ribosomas/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Microscopía por Crioelectrón , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Biológicos , Modelos Moleculares , Filogenia , Estructura Terciaria de Proteína , ARN de Transferencia/químicaRESUMEN
Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially contributing to the difficulties encountered in IMP crystallization.
Asunto(s)
Proteínas de Escherichia coli/biosíntesis , Escherichia coli/crecimiento & desarrollo , Proteínas de la Membrana/biosíntesis , Análisis por Matrices de Proteínas/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Pliegue de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial virulence. Eukaryotic-type Ser/Thr kinases reported in prokaryotes, including that in group A Streptococcus (GAS) (Streptococcus pyogenes Ser/Thr kinase (SP-STK)), regulate cell division, growth, and virulence. The mechanism of this regulation is, however, unknown. In this study, we demonstrated that SP-STK-controlled cell division is mediated under the positive regulation of secretory protein that possesses a cysteine and histidine-dependent aminohydrolases/peptidases (CHAP) domain with functionally active cell wall hydrolase activity (henceforth named as CdhA (CHAP-domain-containing and chain-forming cell wall hydrolase). Deletion of the CdhA-encoding gene resulted in severe cell division and growth defects in GAS mutants. The mutant expressing the truncated CdhA (devoid of the CHAP domain), although displayed no such defects, it became attenuated for virulence in mice and highly susceptible to cell wall-acting antibiotics, as observed for the mutant lacking CdhA. When CdhA was overexpressed in the wild-type GAS as well as in heterologous strains, Escherichia coli and Staphylococcus aureus, we observed a distinct increase in bacterial chain length. Our data reveal that CdhA is a multifunctional protein with a major function of the N-terminal region as a cell division plane-recognizing domain and that of the C-terminal CHAP domain as a virulence-regulating domain. CdhA is thus an important therapeutic target.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pared Celular/enzimología , Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Infecciones Estreptocócicas/metabolismo , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/patogenicidad , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Pared Celular/metabolismo , Escherichia coli/genética , Eliminación de Gen , Humanos , Hidrolasas/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Serina-Treonina Quinasas/genética , Staphylococcus aureus/genética , Infecciones Estreptocócicas/genética , Infecciones Estreptocócicas/terapia , Streptococcus pyogenes/genética , Factores de Virulencia/genéticaRESUMEN
The entire genome of Lactobacillus casei BL23, a strain with probiotic properties, has been sequenced. The genomes of BL23 and the industrially used probiotic strain Shirota YIT 9029 (Yakult) seem to be very similar.
Asunto(s)
Genoma Bacteriano/genética , Lacticaseibacillus casei/genética , Datos de Secuencia MolecularRESUMEN
Genome sequencing of two different Lactobacillus casei strains (ATCC334 and BL23) is presently going on and preliminary data revealed that this lactic acid bacterium possesses numerous carbohydrate transport systems probably reflecting its capacity to proliferate under varying environmental conditions. Many carbohydrate transporters belong to the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but all different kinds of non-PTS transporters are present as well and their substrates are known in a few cases. In L. casei regulation of carbohydrate transport and carbon metabolism is mainly achieved by PTS proteins. Carbon catabolite repression (CCR) is mediated via several mechanisms, including the major P-Ser-HPr/catabolite control protein A (CcpA)-dependent mechanism. Catabolite response elements, the target sites for the P-Ser-HPr/CcpA complex, precede numerous genes and operons. PTS regulation domain-containing antiterminators and transcription activators are also present in both L. casei strains. Their activity is usually controlled by two PTS-mediated phosphorylation reactions exerting antagonistic effects on the transcription regulators: P~EIIB-dependent phosphorylation regulates induction of the corresponding genes and P~His-HPr-mediated phosphorylation plays a role in CCR. Carbohydrate transport of L. casei is also regulated via inducer exclusion and inducer expulsion. The presence of glucose, fructose, etc. leads to inhibition of the transport or metabolism of less favorable carbon sources (inducer exclusion) or to the export of accumulated non-metabolizable carbon sources (inducer expulsion). While P-Ser-HPr is essential for inducer exclusion of maltose, it is not necessary for the expulsion of accumulated thio-methyl-beta-D-galactopyranoside. Surprisingly, recent evidence suggests that the PTS of L. casei also plays a role in cold shock response.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Carbono/metabolismo , Frío , Lacticaseibacillus casei/metabolismo , Fosfotransferasas/metabolismo , Transporte Biológico , FosforilaciónRESUMEN
Surface dehydrogenase (SDH) is an anchorless, multifunctional protein displayed on the surfaces of group A Streptococcus (GAS) organisms. SDH is encoded by a single gene, sdh (gap or plr) that is essential for bacterial survival. Hence, the resulting nonfeasibility of creating a knockout mutant is a major limiting factor in studying its role in GAS pathogenesis. An insertion mutagenesis strategy was devised in which a nucleotide sequence encoding a hydrophobic tail of 12 amino acids ((337)IVLVGLVMLLLS(348)) was added at the 3' end of the sdh gene, successfully creating a viable mutant strain (M1-SDH(HBtail)). In this mutant strain, the SDH(HBtail) protein was not secreted in the medium but was retained in the cytoplasm and to some extent trapped within the cell wall. Hence, SDH(HBtail) was not displayed on the GAS surface. The mutant strain, M1-SDH(HBtail), grew at the same rate as the wild-type strain. The SDH(HBtail) protein displayed the same GAPDH activity as the wild-type SDH protein. Although the whole-cell extracts of the wild-type and mutant strains showed similar GAPDH activities, cell wall extracts of the mutant strain showed 5.5-fold less GAPDH activity than the wild-type strain. The mutant strain, M1-SDH(HBtail), bound significantly less human plasminogen, adhered poorly to human pharyngeal cells, and lost its innate antiphagocytic activity. These results indicate that the prevention of the cell surface export of SDH affects the virulence properties of GAS. The anchorless SDH protein, thus, is an important virulence factor.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Fagocitosis , Receptores de Péptidos/metabolismo , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/patogenicidad , Secuencia de Aminoácidos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Línea Celular , Membrana Celular/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/análisis , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Datos de Secuencia Molecular , Mutación , Fagocitos/microbiología , Plasminógeno/metabolismo , Transporte de Proteínas , Receptores de Péptidos/análisis , Receptores de Péptidos/genética , Streptococcus pyogenes/enzimologíaRESUMEN
Streptococcal surface dehydrogenase (SDH) is a multifunctional, anchorless protein present on the surface of group A Streptococcus (GAS). It plays a regulatory role in GAS-mediated intracellular signaling events in human pharyngeal cells. Using ligand-binding assays, we have identified an approximately 55 kDa protein as an SDH-specific receptor protein on the surface of Detroit human pharyngeal cells. LC-MS/MS analyses identified this SDH-binding pharyngeal cell-surface-exposed membrane-bound protein as uPAR (urokinase plasminogen activator receptor)/CD87. Ligand-binding assays also revealed that only the N-terminal domain (D1) of uPAR bound to SDH. uPAR-D1 more specifically bound to the C-terminal alpha-helix and two immediate flanking regions of the S-loop of the SDH molecule. Site-directed mutagenesis in GAS resulting in SDH with altered C-terminal ends, and the removal of uPAR from pharyngeal cells by phosphatidylinositol-phopsholipase C treatment decreased GAS ability to adhere to pharyngeal cells. When compared to uninfected Detroit pharyngeal cells, GAS-infected pharyngeal cells showed a transient but a significant increase in the expression of uPAR-specific mRNA, and a prolonged recycling process of uPAR on the cell surface. Together, these results indicate that the specific streptococcal surface protein-pharyngeal cell receptor interaction mediated by SDH and uPAR is modulated during GAS infection of human pharyngeal cells. This interaction significantly contributes to bacterial adherence and thus may play a significant role in GAS pathogenesis by regulating intracellular signaling events in pharyngeal cells.
Asunto(s)
Adhesión Bacteriana , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Oxidorreductasas/metabolismo , Faringe/citología , Faringe/microbiología , Receptores de Superficie Celular/metabolismo , Streptococcus pyogenes/enzimología , Secuencia de Aminoácidos , Línea Celular , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Oxidorreductasas/química , Oxidorreductasas/genética , Faringe/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores de Superficie Celular/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Streptococcus pyogenes/metabolismoRESUMEN
A proteome analysis of Lactobacillus casei mutants that are affected in carbon catabolite repression revealed that a 15-kDa protein was strongly overproduced in a ptsHI47T mutant. This protein was identified as EIIA of a mannose class phosphotransferase system (PTS). A 7.1-kb DNA fragment containing the EIIA-encoding open reading frame and five other genes was sequenced. The first gene encodes a protein resembling the RpoN (sigma54)-dependent Bacillus subtilis transcription activator LevR. The following pentacistronic operon is oriented in the opposite direction and encodes four proteins with strong similarity to the proteins of the B. subtilis Lev-PTS and one protein of unknown function. The genes present on the 7.1-kb DNA fragment were therefore called levR and levABCDX. The levABCDX operon was induced by fructose and mannose. No "-12, -24" promoter typical of RpoN-dependent genes precedes the L. casei lev operon, and its expression was therefore RpoN independent but required LevR. Phosphorylation of LevR by P approximately His-HPr stimulates its activity, while phosphorylation by P approximately EIIBLev inhibits it. Disruption of the EIIBLev-encoding levB gene therefore led to strong constitutive expression of the lev operon, which was weaker in a strain carrying a ptsI mutation preventing phosphorylation by both P approximately EIIBLev and P approximately His-HPr. Expression of the L. casei lev operon is also subject to P-Ser-HPr-mediated catabolite repression. The observed slow phosphoenolpyruvate- and ATP-dependent phosphorylation of HPrI47T as well as the slow phosphoryl group transfer from the mutant P approximately His-HPr to EIIALev are assumed to be responsible for the elevated expression of the lev operon in the ptsHI47T mutant.