RESUMEN
Cells genetically deficient in sphingomyelin synthase-1 (SGMS1) or blocked in their synthesis pharmacologically through exposure to a serine palmitoyltransferase inhibitor (myriocin) show strongly reduced surface display of influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA). The transport of HA to the cell surface was assessed by accessibility of HA on intact cells to exogenously added trypsin and to HA-specific antibodies. Rates of de novo synthesis of viral proteins in wild-type and SGMS1-deficient cells were equivalent, and HA negotiated the intracellular trafficking pathway through the Golgi normally. We engineered a strain of influenza virus to allow site-specific labeling of HA and NA using sortase. Accessibility of both HA and NA to sortase was blocked in SGMS1-deficient cells and in cells exposed to myriocin, with a corresponding inhibition of the release of virus particles from infected cells. Generation of influenza virus particles thus critically relies on a functional sphingomyelin biosynthetic pathway, required to drive influenza viral glycoproteins into lipid domains of a composition compatible with virus budding and release.
Asunto(s)
Vías Biosintéticas/fisiología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Esfingomielinas/biosíntesis , Animales , Transporte Biológico/fisiología , Perros , Ácidos Grasos Monoinsaturados/farmacología , Técnica del Anticuerpo Fluorescente , Glicoproteínas Hemaglutininas del Virus de la Influenza/fisiología , Interacciones Huésped-Patógeno , Células de Riñón Canino Madin Darby , Polietilenglicoles , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Transferasas (Grupos de Otros Fosfatos Sustitutos)/deficiencia , TripsinaRESUMEN
A monoclonal antibody against the C-type lectin DEC205 (αDEC205) is an effective vehicle for delivery of antigens to dendritic cells through creation of covalent αDEC205-antigen adducts. These adducts can induce antigen-specific T-cell immune responses or tolerance. We exploit the transpeptidase activity of sortase to install modified peptides and protein-sized antigens onto the heavy chain of αDEC205, including linkers that contain nonnatural amino acids. We demonstrate stoichiometric site-specific labeling on a scale not easily achievable by genetic fusions (49 distinct fusions in this report). We conjugated a biotinylated version of a class I MHC-restricted epitope to unlabeled αDEC205 and monitored epitope generation upon binding of the adduct to dendritic cells. Our results show transfer of αDEC205 heavy chain to the cytoplasm, followed by proteasomal degradation. Introduction of a labile dipeptide linker at the N terminus of a T-cell epitope improves proteasome-dependent class I MHC-restricted peptide cross-presentation when delivered by αDEC205 in vitro and in vivo. We also conjugated αDEC205 with a linker-optimized peptide library of known CD8 T-cell epitopes from the mouse γ-herpes virus 68. Animals immunized with such conjugates displayed a 10-fold reduction in viral load.
Asunto(s)
Aminoaciltransferasas/metabolismo , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos Virales/inmunología , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Epítopos de Linfocito T/inmunología , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Antígenos CD/química , Antígenos CD/genética , Antígenos Virales/química , Antígenos Virales/genética , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/prevención & control , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunización , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Inmunoconjugados/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Ingeniería de Proteínas , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Rhadinovirus/genética , Rhadinovirus/inmunología , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/prevención & control , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.
Asunto(s)
ADP Ribosa Transferasas/toxicidad , Toxinas Bacterianas/toxicidad , Exotoxinas/toxicidad , Furina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factores de Virulencia/toxicidad , Red trans-Golgi/metabolismo , ADP Ribosa Transferasas/genética , Toxinas Bacterianas/genética , Secuencia de Bases , Cartilla de ADN , Endocitosis , Exotoxinas/genética , Mutación , Reacción en Cadena de la Polimerasa , Transporte de Proteínas , Proteolisis , Receptores Acoplados a Proteínas G/fisiología , Factores de Virulencia/genética , Exotoxina A de Pseudomonas aeruginosaRESUMEN
The ability to induce humoral and cellular immunity via antigen delivery through the unbroken skin (epicutaneous immunization, EPI) has immediate relevance for vaccine development. However, it is unclear which adjuvants induce protective memory CD8 T-cell responses by this route, and the molecular and cellular requirements for priming through intact skin are not defined. We report that cholera toxin (CT) is superior to other adjuvants in its ability to prime memory CD8 T cells that control bacterial and viral challenges. Epicutaneous immunization with CT does not require engagement of classic toll-like receptor (TLR) and inflammasome pathways and, surprisingly, is independent of skin langerin-expressing cells (including Langerhans cells). However, CT adjuvanticity required type-I IFN sensitivity, participation of a Batf3-dependent dendritic cell (DC) population and engagement of CT with suitable gangliosides. Chemoenzymatic generation of CT-antigen fusion proteins led to efficient priming of the CD8 T-cell responses, paving the way for development of this immunization strategy as a therapeutic option.
Asunto(s)
Adyuvantes Inmunológicos/metabolismo , Linfocitos T CD8-positivos/inmunología , Toxina del Cólera/administración & dosificación , Toxina del Cólera/inmunología , Transducción de Señal/efectos de los fármacos , Vacunación , Administración Cutánea , Animales , Antígenos/inmunología , Antígenos de Superficie/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Células Cultivadas , Gangliósido G(M1)/inmunología , Memoria Inmunológica/efectos de los fármacos , Interferón Tipo I/inmunología , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/farmacología , Proteínas Represoras/metabolismo , Transducción de Señal/inmunología , Receptores Toll-Like/inmunologíaRESUMEN
We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Bacteriófago M13/metabolismo , Proteínas de la Cápside/metabolismo , Técnicas de Visualización de Superficie Celular/métodos , Cisteína Endopeptidasas/metabolismo , Animales , Bacteriófago M13/química , Bacteriófago M13/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Ratones , Ratones Endogámicos C57BL , Propiedades de SuperficieRESUMEN
Cyclic peptides are highly valued tools in biomedical research. In many cases, they show higher receptor affinity, enhanced biological activity, and improved serum stability. Technical difficulties in producing cyclic peptides, especially larger ones, in appreciable yields have precluded a prolific use in biomedical research. Here, we describe a novel and efficient cyclization method that uses the peptidyl-transferase activity of the Staphylococcus aureus enzyme sortase A to cyclize linear synthetic precursor peptides. As a model, we used histatin 1, a 38-mer salivary peptide with motogenic activity. Chemical cyclization of histatin 1 resulted in ≤ 3% yields, whereas sortase-mediated cyclization provided a yield of >90%. The sortase-cyclized peptide displayed a maximum wound closure activity at 10 nM, whereas the linear peptide displayed maximal activity at 10 µM. Circular dichroism and NMR spectroscopic analysis of the linear and cyclic peptide in solution showed no evidence for conformational changes, suggesting that structural differences due to cyclization only became manifest when these peptides were located in the binding domain of the receptor. The sortase-based cyclization technology provides a general method for easy and efficient manufacturing of large cyclic peptides.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Histatinas/biosíntesis , Péptidos Cíclicos/biosíntesis , Secuencia de Aminoácidos , Aminoaciltransferasas/genética , Proteínas Bacterianas/genética , Línea Celular , Dicroismo Circular , Cisteína Endopeptidasas/genética , Histatinas/química , Histatinas/genética , Histatinas/farmacología , Humanos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , Péptidos Cíclicos/farmacología , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The recent development of successful CAR (chimeric antigen receptor) T cell therapies has been accompanied by a need to better control potentially fatal toxicities that can arise from adverse immune reactions. Here we present a ligand-controlled CAR system, based on the IKZF3 ZF2 ß-hairpin IMiD-inducible degron, which allows for the reversible control of expression levels of type I membrane proteins, including CARs. Testing this system in an established mouse xenotransplantation model for acute lymphoblastic leukemia, we validate the ability of the CAR19-degron to target and kill CD19-positive cells displaying complete control/clearance of the tumor. We also demonstrate that the activity of CAR19-degron can be regulated in vivo when dosing a US Food and Drug Administration-approved drug, lenalidomide.
Asunto(s)
Factor de Transcripción Ikaros/inmunología , Factores Inmunológicos/farmacología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Adolescente , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Factor de Transcripción Ikaros/química , Factores Inmunológicos/química , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Receptores Quiméricos de Antígenos/genética , Adulto JovenRESUMEN
The unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtA(strep) is used to label the protein's C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtA(staph) and LPXT derivatives. The generality of N-terminal labeling with SrtA(staph) is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Colorantes Fluorescentes/química , Péptidos/química , Alanina , Secuencia de Aminoácidos , Sitios de Unión , Staphylococcus aureus/enzimología , Streptococcus pyogenes/enzimología , Especificidad por SustratoRESUMEN
Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Ratones , Unión Proteica , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
ALDP, ALDPR, PMP70 and PMP70R are half ATP-binding cassette (ABC) transporters of the mammalian peroxisomal membrane. By analogy with other members of this family, it is assumed that peroxisomal ABC transporters must dimerize to become functional units. However, not much is known regarding the type of dimers (i.e., homodimers and/or heterodimers) that are formed in vivo under normal expression conditions. In this work, we have characterized the quaternary structure of mouse liver PMP70 and ALDP. The PMP70 protein complex was purified to apparent homogeneity using a two-step purification protocol. The ALDP-containing protein complex was characterized by preparative immunoprecipitation experiments. In both cases, no evidence for the existence of heteromeric interactions or for the presence of accessory proteins in these ABC transporter protein complexes could be obtained. Our data indicate that the majority (if not all) of mouse liver PMP70 and ALDP are homomeric proteins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP , Animales , Digitonina/química , Electroforesis en Gel de Poliacrilamida , Masculino , Ratones , Ratones Endogámicos C57BL , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of sortase A on a solid support (Sepharose beads). Immobilization of sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca(2+)-independent variant of sortase A with increased catalytic activity. This heptamutant variant of sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized sortase A takes 1-2 d. Batch reactions take 3-12 h and flow reactions proceed at 0.5 ml h(-1), depending on the geometry of the reactor used.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Enzimas Inmovilizadas/metabolismo , Peptidil Transferasas/metabolismo , Ingeniería de Proteínas/métodos , Proteínas/metabolismo , Aminoaciltransferasas/genética , Proteínas Bacterianas/genética , Catálisis , Cisteína Endopeptidasas/genética , Mutación/genética , SefarosaRESUMEN
Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.
Asunto(s)
Aeromonas hydrophila/metabolismo , Toxinas Bacterianas/metabolismo , Antígenos CD59/metabolismo , Glipicanos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Aeromonas hydrophila/química , Secuencia de Aminoácidos , Animales , Toxinas Bacterianas/análisis , Biotina/química , Antígenos CD59/análisis , Línea Celular , Colorantes Fluorescentes/química , Glipicanos/análisis , Humanos , Datos de Secuencia Molecular , Imagen Óptica , Proteínas Citotóxicas Formadoras de Poros/análisis , Receptores del Activador de Plasminógeno Tipo Uroquinasa/análisisRESUMEN
M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multiphage devices requires precise control of interactions between the M13 capsid proteins. Toward this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi-material devices.
Asunto(s)
Bacteriófago M13/genética , Bacteriófago M13/metabolismo , Proteínas de la Cápside/química , ADN Viral/metabolismo , Secuencias de Aminoácidos , Bacteriófago M13/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Hibridación de Ácido Nucleico/métodos , Imagen Óptica , Ingeniería de ProteínasRESUMEN
Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes â¼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.
Asunto(s)
Aminoaciltransferasas/química , Proteínas Bacterianas/química , Cisteína Endopeptidasas/química , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Pared Celular/metabolismo , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Espectrometría de MasasRESUMEN
This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPXTG (for Streptococcus aureus sortase A) or LPXTA (for Staphylococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h.
Asunto(s)
Aminoaciltransferasas/química , Proteínas Bacterianas/química , Cisteína Endopeptidasas/química , Ingeniería de Proteínas/métodos , Cromatografía Líquida de Alta Presión , Cinética , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Especificidad por SustratoRESUMEN
Chimeric proteins, including bispecific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-to-N fused recombinant proteins, but not unnaturally linked N-to-N and C-to-C fusion proteins. This protocol describes a simple procedure for the production of such chimeric proteins, starting from correctly folded proteins and readily available peptides. By equipping the N terminus or C terminus of the proteins of interest with a set of click handles using sortase A, followed by a strain-promoted click reaction, unnatural N-to-N and C-to-C linked (hetero) fusion proteins are established. Examples of proteins that have been conjugated via this method include interleukin-2, interferon-α, ubiquitin, antibodies and several single-domain antibodies. If the peptides, sortase A and the proteins of interest are in hand, the unnaturally N-to-N and C-to-C fused proteins can be obtained in 3-4 d.
Asunto(s)
Aminoaciltransferasas/química , Proteínas Bacterianas/química , Química Clic/métodos , Cisteína Endopeptidasas/química , Ingeniería de Proteínas/métodos , Carbono/química , Cromatografía Líquida de Alta Presión , Nitrógeno/química , Fragmentos de Péptidos/biosíntesis , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Staphylococcus aureus/enzimologíaRESUMEN
We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ~12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.
Asunto(s)
Toxina del Cólera/toxicidad , Toxina Diftérica/química , Transporte de Proteínas/genética , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Células Cultivadas , Cólera/fisiopatología , Toxina del Cólera/química , Toxina del Cólera/genética , Clonación Molecular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Gangliósidos/metabolismo , Gangliósidos/fisiología , Ingeniería Genética , Haploidia , Humanos , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Transporte de Monosacáridos/fisiología , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/toxicidad , Análisis de Secuencia de Proteína , Sialiltransferasas/química , Sialiltransferasas/metabolismo , Sialiltransferasas/fisiologíaRESUMEN
Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.