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1.
Cell ; 185(7): 1157-1171.e22, 2022 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-35259335

RESUMEN

Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.


Asunto(s)
Toxinas Bacterianas/metabolismo , Enterococcus , Leucocitos Mononucleares , Factores de Virulencia/metabolismo , Animales , Bovinos , Enterococcus/metabolismo , Enterococcus/patogenicidad , Caballos , Ratones , Pruebas de Sensibilidad Microbiana , Porcinos
2.
Nature ; 610(7931): 349-355, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36171290

RESUMEN

Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.


Asunto(s)
Toxinas Bacterianas , Sistemas CRISPR-Cas , Proteínas de Drosophila , Drosophila melanogaster , Edición Génica , Factores de Virulencia , Animales , Toxinas Bacterianas/metabolismo , Agentes de Control Biológico , Culicidae , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/microbiología , Cuerpo Adiposo/citología , Técnicas de Silenciamiento del Gen , Hemocitos , Humanos , Mariposas Nocturnas , Mucinas , Control Biológico de Vectores , Fagocitosis , Photorhabdus/metabolismo , Secuencias Repetitivas de Aminoácido , Transgenes , Factores de Virulencia/metabolismo
3.
PLoS Biol ; 21(11): e3002353, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37943878

RESUMEN

Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of C. difficile toxin B (TcdBFBD), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdBFBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7-positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.


Asunto(s)
Toxinas Bacterianas , Neoplasias de la Mama , Clostridioides difficile , Neoplasias Mamarias Animales , Humanos , Animales , Ratones , Femenino , Vía de Señalización Wnt , Neoplasias de la Mama/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Cisplatino
4.
FASEB J ; 35(7): e21647, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34165206

RESUMEN

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance, and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. Especially the C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to a hypothetical protein receptor may support the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface.


Asunto(s)
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Linfocitos/metabolismo , Proteínas Recombinantes/metabolismo , Yersinia pseudotuberculosis/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Conformación Proteica , Proteínas Recombinantes/genética
5.
Genet Med ; 23(6): 1158-1162, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33531666

RESUMEN

PURPOSE: The endoplasmic reticulum membrane complex (EMC) is a highly conserved, multifunctional 10-protein complex related to membrane protein biology. In seven families, we identified 13 individuals with highly overlapping phenotypes who harbor a single identical homozygous frameshift variant in EMC10. METHODS: Using exome, genome, and Sanger sequencing, a recurrent frameshift EMC10 variant was identified in affected individuals in an international cohort of consanguineous families. Multiple families were independently identified and connected via Matchmaker Exchange and internal databases. We assessed the effect of the frameshift variant on EMC10 RNA and protein expression and evaluated EMC10 expression in normal human brain tissue using immunohistochemistry. RESULTS: A homozygous variant EMC10 c.287delG (Refseq NM_206538.3, p.Gly96Alafs*9) segregated with affected individuals in each family, who exhibited a phenotypic spectrum of intellectual disability (ID) and global developmental delay (GDD), variable seizures and variable dysmorphic features (elongated face, curly hair, cubitus valgus, and arachnodactyly). The variant arose on two founder haplotypes and results in significantly reduced EMC10 RNA expression and an unstable truncated EMC10 protein. CONCLUSION: We propose that a homozygous loss-of-function variant in EMC10 causes a novel syndromic neurodevelopmental phenotype. Remarkably, the recurrent variant is likely the result of a hypermutable site and arose on distinct founder haplotypes.


Asunto(s)
Discapacidades del Desarrollo , Discapacidad Intelectual , Niño , Discapacidades del Desarrollo/genética , Mutación del Sistema de Lectura , Homocigoto , Humanos , Discapacidad Intelectual/genética , Proteínas de la Membrana/genética , Linaje , Fenotipo , Convulsiones/genética
6.
PLoS Biol ; 16(11): e2006951, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30481169

RESUMEN

Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific "activator" for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.


Asunto(s)
Ricina/genética , Toxinas Shiga/genética , Toxinas Bacterianas/metabolismo , Sistemas CRISPR-Cas , Endosomas/metabolismo , Estudio de Asociación del Genoma Completo/métodos , Glucolípidos/metabolismo , Glicoesfingolípidos , Glicosilación , Aparato de Golgi/metabolismo , Aparato de Golgi/fisiología , Células HEK293 , Células HeLa , Humanos , Mutación con Pérdida de Función/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/fisiología , Proteínas Oncogénicas/metabolismo , Transporte de Proteínas , Ricina/metabolismo , Toxinas Shiga/metabolismo , Trihexosilceramidas/metabolismo , Trihexosilceramidas/fisiología
7.
Proc Natl Acad Sci U S A ; 107(11): 5154-9, 2010 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-20133735

RESUMEN

Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 of Mycobacterium tuberculosis is a unique nucleoid-associated protein that binds AT-rich regions of the genome, including genomic islands acquired by horizontal gene transfer and regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM and PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain-swapping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA-binding domain. Nuclear magnetic resonance analysis of the DNA-binding domain of Lsr2 and its interaction with DNA reveals a unique structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve a function similar to the Enterobacteriaceae H-NS, likely coordinating global gene regulation and virulence in this group of medically important bacteria.


Asunto(s)
Secuencia Rica en At/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Bacterianos/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Proteínas de Unión al ADN/química , Modelos Moleculares , Mycobacterium tuberculosis/inmunología , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Soluciones , Virulencia/genética
8.
Bioengineering (Basel) ; 10(8)2023 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-37627769

RESUMEN

Genetic screen technology has been applied to study the mechanism of action of bacterial toxins-a special class of virulence factors that contribute to the pathogenesis caused by bacterial infections. These screens aim to identify host factors that directly or indirectly facilitate toxin intoxication. Additionally, specific properties of certain toxins, such as membrane interaction, retrograde trafficking, and carbohydrate binding, provide robust probes to comprehensively investigate the lipid biosynthesis, membrane vesicle transport, and glycosylation pathways, respectively. This review specifically focuses on recent representative toxin-based genetic screens that have identified new players involved in and provided new insights into fundamental biological pathways, such as glycosphingolipid biosynthesis, protein glycosylation, and membrane vesicle trafficking pathways. Functionally characterizing these newly identified factors not only expands our current understanding of toxin biology but also enables a deeper comprehension of fundamental biological questions. Consequently, it stimulates the development of new therapeutic approaches targeting both bacterial infectious diseases and genetic disorders with defects in these factors and pathways.

9.
Cell Rep ; 38(10): 110476, 2022 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-35263584

RESUMEN

Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful. Delivery of Cas13a and CasRx, together with guide RNA expression, reduces mRNAs encoding GFP, SARS-CoV-2 fragments, and endogenous proteins PPIB, KRAS, and CXCR4 in multiple cell lines. Delivery of Cre recombinase modifies the reporter loci in cells. Delivery of Cas9, together with guide RNA expression, generates mutations at the targeted genomic sites in cell lines and induced pluripotent stem cell (iPSC)-derived human neurons. These findings establish modular delivery systems based on single-chain bacterial toxins for delivery of membrane-impermeable therapeutics into targeted cells.


Asunto(s)
Toxinas Bacterianas , COVID-19 , Toxinas Bacterianas/genética , Sistemas CRISPR-Cas , Edición Génica , Humanos , ARN Guía de Kinetoplastida/metabolismo , SARS-CoV-2
10.
Life Sci Alliance ; 5(6)2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35292538

RESUMEN

Clostridioides difficile toxin A and B (TcdA and TcdB) are two major virulence factors responsible for diseases associated with C. difficile infection (CDI). Here, we report the 3.18-Å resolution crystal structure of a TcdA fragment (residues L843-T2481), which advances our understanding of the complete structure of TcdA holotoxin. Our structural analysis, together with complementary single molecule FRET and limited proteolysis studies, reveal that TcdA adopts a dynamic structure and its CROPs domain can sample a spectrum of open and closed conformations in a pH-dependent manner. Furthermore, a small globular subdomain (SGS) and the CROPs protect the pore-forming region of TcdA in the closed state at neutral pH, which could contribute to modulating the pH-dependent pore formation of TcdA. A rationally designed TcdA mutation that trapped the CROPs in the closed conformation showed drastically reduced cytotoxicity. Taken together, these studies shed new lights into the conformational dynamics of TcdA and its roles in TcdA intoxication.


Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Conformación Molecular
11.
Nat Commun ; 13(1): 6786, 2022 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-36351897

RESUMEN

Toxin B (TcdB) is a major exotoxin responsible for diseases associated with Clostridioides difficile infection. Its sequence variations among clinical isolates may contribute to the difficulty in developing effective therapeutics. Here, we investigate receptor-binding specificity of major TcdB subtypes (TcdB1 to TcdB12). We find that representative members of subtypes 2, 4, 7, 10, 11, and 12 do not recognize the established host receptor, frizzled proteins (FZDs). Using a genome-wide CRISPR-Cas9-mediated screen, we identify tissue factor pathway inhibitor (TFPI) as a host receptor for TcdB4. TFPI is recognized by a region in TcdB4 that is homologous to the FZD-binding site in TcdB1. Analysis of 206 TcdB variant sequences reveals a set of six residues within this receptor-binding site that defines a TFPI binding-associated haplotype (designated B4/B7) that is present in all TcdB4 members, a subset of TcdB7, and one member of TcdB2. Intragenic micro-recombination (IR) events have occurred around this receptor-binding region in TcdB7 and TcdB2 members, resulting in either TFPI- or FZD-binding capabilities. Introduction of B4/B7-haplotype residues into TcdB1 enables dual recognition of TFPI and FZDs. Finally, TcdB10 also recognizes TFPI, although it does not belong to the B4/B7 haplotype, and shows species selectivity: it recognizes TFPI of chicken and to a lesser degree mouse, but not human, dog, or cattle versions. These findings identify TFPI as a TcdB receptor and reveal IR-driven changes on receptor-specificity among TcdB variants.


Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Animales , Bovinos , Perros , Ratones , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Clostridioides difficile/genética , Recombinación Genética , Humanos
12.
Toxins (Basel) ; 13(3)2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33803852

RESUMEN

Shiga toxins (Stxs) are classic bacterial toxins and major virulence factors of toxigenic Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC). These toxins recognize a glycosphingolipid globotriaosylceramide (Gb3/CD77) as their receptor and inhibit protein synthesis in cells by cleaving 28S ribosomal RNA. They are the major cause of life-threatening complications such as hemolytic uremic syndrome (HUS), associated with severe cases of EHEC infection, which is the leading cause of acute kidney injury in children. The threat of Stxs is exacerbated by the lack of toxin inhibitors and effective treatment for HUS. Here, we briefly summarize the Stx structure, subtypes, in vitro and in vivo models, Gb3 expression and HUS, and then introduce recent studies using CRISPR-Cas9-mediated genome-wide screens to identify the host cell factors required for Stx action. We also summarize the latest progress in utilizing and engineering Stx components for biomedical applications.


Asunto(s)
Infecciones por Escherichia coli/metabolismo , Síndrome Hemolítico-Urémico/metabolismo , Toxinas Shiga/metabolismo , Escherichia coli Shiga-Toxigénica/metabolismo , Trihexosilceramidas/metabolismo , Animales , Sistemas CRISPR-Cas , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Síndrome Hemolítico-Urémico/genética , Síndrome Hemolítico-Urémico/microbiología , Interacciones Huésped-Patógeno , Humanos , Inmunotoxinas/uso terapéutico , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Conformación Proteica , Toxinas Shiga/química , Toxinas Shiga/genética , Toxinas Shiga/uso terapéutico , Escherichia coli Shiga-Toxigénica/genética , Relación Estructura-Actividad
13.
Sci Adv ; 7(43): eabi4582, 2021 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-34678063

RESUMEN

Toxin B (TcdB) is a primary cause of Clostridioides difficile infection (CDI). This toxin acts by glucosylating small GTPases in the Rho/Ras families, but the structural basis for TcdB recognition and selectivity of specific GTPase substrates remain unsolved. Here, we report the cocrystal structures of the glucosyltransferase domain (GTD) of two distinct TcdB variants in complex with human Cdc42 and R-Ras, respectively. These structures reveal a common structural mechanism by which TcdB recognizes Rho and R-Ras. Furthermore, we find selective clustering of adaptive residue changes in GTDs that determine their substrate preferences, which helps partition all known TcdB variants into two groups that display distinct specificities toward Rho or R-Ras. Mutations that selectively disrupt GTPases binding reduce the glucosyltransferase activity of the GTD and the toxicity of TcdB holotoxin. These findings establish the structural basis for TcdB recognition of small GTPases and reveal strategies for therapeutic interventions for CDI.

14.
Nat Commun ; 12(1): 3748, 2021 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-34145250

RESUMEN

C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB-CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB.


Asunto(s)
Antígenos/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/patología , Proteoglicanos/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Antígenos/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Sitios de Unión/fisiología , Anticuerpos ampliamente neutralizantes/farmacología , Microscopía por Crioelectrón , Enterocolitis Seudomembranosa/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multiproteicos/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica , Proteoglicanos/genética
15.
Cell Host Microbe ; 27(5): 782-792.e7, 2020 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-32302524

RESUMEN

The exotoxin TcsL is a major virulence factor in Paeniclostridium (Clostridium) sordellii and responsible for the high lethality rate associated with P. sordellii infection. Here, we present a genome-wide CRISPR-Cas9-mediated screen using a human lung carcinoma cell line and identify semaphorin (SEMA) 6A and 6B as receptors for TcsL. Disrupting SEMA6A/6B expression in several distinct human cell lines and primary human endothelial cells results in reduced TcsL sensitivity, while SEMA6A/6B over-expression increases their sensitivity. TcsL recognizes the extracellular domain (ECD) of SEMA6A/6B via a region homologous to the receptor-binding site in Clostridioides difficile toxin B (TcdB), which binds the human receptor Frizzled. Exchanging the receptor-binding interfaces between TcsL and TcdB switches their receptor-binding specificity. Finally, administration of SEMA6A-ECD proteins protects human cells from TcsL toxicity and reduces TcsL-induced damage to lung tissues and the lethality rate in mice. These findings establish SEMA6A and 6B as pathophysiologically relevant receptors for TcsL.


Asunto(s)
Toxinas Bacterianas/metabolismo , Clostridium sordellii/genética , Clostridium sordellii/metabolismo , Semaforinas/genética , Semaforinas/aislamiento & purificación , Células A549 , Animales , Proteínas Bacterianas , Sitios de Unión , Sistemas CRISPR-Cas , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Células Endoteliales/metabolismo , Femenino , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Neoplasias Pulmonares , Masculino , Ratones , Unión Proteica , Semaforinas/metabolismo , Factores de Virulencia/metabolismo
16.
Life Sci Alliance ; 3(5)2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32234750

RESUMEN

Osteosarcoma (OS) is a primary malignant bone neoplasm with high frequencies of tumor metastasis and recurrence. Although the Akt/PKB signaling pathway is known to play key roles in tumorigenesis, the roles of cyclin-dependent kinase-like 3 (CDKL3) in OS progression remain largely elusive. We have demonstrated the high expression levels of CDKL3 in OS human specimens and comprehensively investigated the role of CDKL3 in promoting OS progression both in vitro and in vivo. We found that CDKL3 regulates Akt activation and its downstream effects, including cell growth and autophagy. The up-regulation of CDKL3 in OS specimens appeared to be associated with Akt activation and shorter overall patient survival (P = 0.003). Our findings identify CDKL3 as a critical regulator that stimulates OS progression by enhancing Akt activation. CDKL3 represents both a biomarker for OS prognosis, and a potential therapeutic target in precision medicine by targeting CDKL3 to treat Akt hyper-activated OS.


Asunto(s)
Osteosarcoma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Apoptosis/genética , Autofagia/genética , Neoplasias Óseas/genética , Carcinogénesis/genética , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , China , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Recurrencia Local de Neoplasia/genética , Osteosarcoma/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genética
17.
Metallomics ; 11(11): 1925-1936, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31631207

RESUMEN

Auranofin (AuRF) has been reported to display anticancer activity and has entered several clinical trials; however, its mechanism of action remains largely unknown. In this work, the anticancer mechanism of auranofin was investigated using a proteomics strategy entailing subcellular fractionation prior to mass spectrometric analysis. Bioinformatics analysis of the nuclear sub-proteomes revealed that tumor suppressor p14ARF is a key regulator of transcription. Through independent analysis, we validated that up-regulation of p14ARF is associated with E2F-dependent transcription and increased p53 expression. Our analyses further reveal that 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), which is the rate-determining enzyme of the mevalonate pathway, is a novel target of auranofin with half maximal inhibitory concentration at micromolar levels. The auranofin-induced cancer cell death could be partially reverted by the addition of downstream products of the mevalonate pathway (mevalonolactone or geranyleranyl pyrophosphate (GGPP)), implying that auranofin may target the mevalonate pathway to exert its anticancer effect.


Asunto(s)
Antineoplásicos/farmacología , Auranofina/farmacología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Terapia Molecular Dirigida , Línea Celular Tumoral , Factores de Transcripción E2F/metabolismo , Oro/farmacología , Humanos , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacos
18.
Cell Rep ; 28(10): 2517-2526.e5, 2019 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-31484065

RESUMEN

The endoplasmic reticulum (ER) membrane protein complex (EMC) is a key contributor to biogenesis and membrane integration of transmembrane proteins, but our understanding of its mechanisms and the range of EMC-dependent proteins remains incomplete. Here, we carried out an unbiased mass spectrometry (MS)-based quantitative proteomic analysis comparing membrane proteins in EMC-deficient cells to wild-type (WT) cells and identified 36 EMC-dependent membrane proteins and 171 EMC-independent membrane proteins. Of these, six EMC-dependent and six EMC-independent proteins were further independently validated. We found that a common feature among EMC-dependent proteins is that they contain transmembrane domains (TMDs) with polar and/or charged residues. Mutagenesis studies demonstrate that EMC dependency can be converted in cells by removing or introducing polar and/or charged residues within TMDs. Our studies expand the list of validated EMC-dependent and EMC-independent proteins and suggest that the EMC is involved in handling TMDs with residues challenging for membrane integration.


Asunto(s)
Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Proteómica , Línea Celular , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/química , Mutagénesis/genética , Dominios Proteicos , Reproducibilidad de los Resultados , Respuesta de Proteína Desplegada
19.
Nat Microbiol ; 4(10): 1760-1769, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31160825

RESUMEN

Clostridium difficile toxin A (TcdA) is a major exotoxin contributing to disruption of the colonic epithelium during C. difficile infection. TcdA contains a carbohydrate-binding combined repetitive oligopeptides (CROPs) domain that mediates its attachment to cell surfaces, but recent data suggest the existence of CROPs-independent receptors. Here, we carried out genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated screens using a truncated TcdA lacking the CROPs, and identified sulfated glycosaminoglycans (sGAGs) and low-density lipoprotein receptor (LDLR) as host factors contributing to binding and entry of TcdA. TcdA recognizes the sulfation group in sGAGs. Blocking sulfation and glycosaminoglycan synthesis reduces TcdA binding and entry into cells. Binding of TcdA to the colonic epithelium can be reduced by surfen, a small molecule that masks sGAGs, by GM-1111, a sulfated heparan sulfate analogue, and by sulfated cyclodextrin, a sulfated small molecule. Cells lacking LDLR also show reduced sensitivity to TcdA, although binding between LDLR and TcdA are not detected, suggesting that LDLR may facilitate endocytosis of TcdA. Finally, GM-1111 reduces TcdA-induced fluid accumulation and tissue damage in the colon in a mouse model in which TcdA is injected into the caecum. These data demonstrate in vivo and pathological relevance of TcdA-sGAGs interactions, and reveal a potential therapeutic approach of protecting colonic tissues by blocking these interactions.


Asunto(s)
Toxinas Bacterianas/metabolismo , Clostridioides difficile/química , Enterotoxinas/metabolismo , Glicosaminoglicanos/metabolismo , Receptores de LDL/metabolismo , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Membrana Celular/metabolismo , Colon/efectos de los fármacos , Colon/metabolismo , Endocitosis , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/toxicidad , Glicosaminoglicanos/deficiencia , Células HeLa , Heparitina Sulfato/análogos & derivados , Heparitina Sulfato/farmacología , Humanos , Mucosa Intestinal/metabolismo , Ratones , Mutación , Oligopéptidos/genética , Unión Proteica , Receptores de LDL/deficiencia
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