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1.
Subcell Biochem ; 96: 563-577, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33252744

RESUMO

Anthrax toxin is a major virulence factor of Bacillus anthracis, a Gram-positive bacterium which can form highly stable spores that are the causative agents of the disease, anthrax. While chiefly a disease of livestock, spores can be "weaponized" as a bio-terrorist agent, and can be deadly if not recognized and treated early with antibiotics. The intracellular pathways affected by the enzymes are broadly understood and are not discussed here. This chapter focuses on what is known about the assembly of secreted toxins on the host cell surface and how the toxin is delivered into the cytosol. The central component is the "Protective Antigen", which self-oligomerizes and forms complexes with its pay-load, either Lethal Factor or Edema Factor. It binds a host receptor, CMG2, or a close relative, triggering receptor-mediated endocytosis, and forms a remarkably elegant yet powerful machine that delivers toxic enzymes into the cytosol, powered only by the pH gradient across the membrane. We now have atomic structures of most of the starting, intermediate and final assemblies in the infectious process. Together with a major body of biophysical, mutational and biochemical work, these studies reveal a remarkable story of both how toxin assembly is choreographed in time and space.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Antraz/microbiologia , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Bacillus anthracis/química , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Humanos , Transporte Proteico
2.
Proc Natl Acad Sci U S A ; 117(38): 23774-23781, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32878997

RESUMO

Intracellular pathogens are responsible for an enormous amount of worldwide morbidity and mortality, and each has evolved specialized strategies to establish and maintain their replicative niche. Listeria monocytogenes is a facultative intracellular pathogen that secretes a pore-forming cytolysin called listeriolysin O (LLO), which disrupts the phagosomal membrane and, thereby, allows the bacteria access to their replicative niche in the cytosol. Nonsynonymous and synonymous mutations in a PEST-like domain near the LLO N terminus cause enhanced LLO translation during intracellular growth, leading to host cell death and loss of virulence. Here, we explore the mechanism of translational control and show that there is extensive codon restriction within the PEST-encoding region of the LLO messenger RNA (mRNA) (hly). This region has considerable complementarity with the 5' UTR and is predicted to form an extensive secondary structure that overlaps the ribosome binding site. Analysis of both 5' UTR and synonymous mutations in the PEST-like domain that are predicted to disrupt the secondary structure resulted in up to a 10,000-fold drop in virulence during mouse infection, while compensatory double mutants restored virulence to WT levels. We showed by dynamic protein radiolabeling that LLO synthesis was growth phase-dependent. These data provide a mechanism to explain how the bacteria regulate translation of LLO to promote translation during starvation in a phagosome while repressing it during growth in the cytosol. These studies also provide a molecular explanation for codon bias at the 5' end of this essential determinant of pathogenesis.


Assuntos
Toxinas Bacterianas , Proteínas de Choque Térmico , Proteínas Hemolisinas , Listeria monocytogenes , RNA Bacteriano/química , RNA Mensageiro/química , Regiões 5' não Traduzidas/genética , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Replicação do DNA/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeriose , Camundongos , Conformação de Ácido Nucleico , RNA Bacteriano/genética , RNA Mensageiro/genética
3.
Nucleic Acids Res ; 48(18): 10527-10541, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32845304

RESUMO

YoeB-YefM, the widespread type II toxin-antitoxin (TA) module, binds to its own promoter to autoregulate its transcription: repress or induce transcription under normal or stress conditions, respectively. It remains unclear how YoeB-YefM regulates its transcription depending on the YoeB to YefM TA ratio. We find that YoeB-YefM complex from S.aureus exists as two distinct oligomeric assemblies: heterotetramer (YoeB-YefM2-YoeB) and heterohexamer (YoeB-YefM2-YefM2-YoeB) with low and high DNA-binding affinities, respectively. Structures of the heterotetramer alone and heterohexamer bound to promoter DNA reveals that YefM C-terminal domain undergoes disorder to order transition upon YoeB binding, which allosterically affects the conformation of N-terminal DNA-binding domain. At TA ratio of 1:2, unsaturated binding of YoeB to the C-terminal regions of YefM dimer forms an optimal heterohexamer for DNA binding, and two YefM dimers with N-terminal domains dock into the adjacent major grooves of DNA to specifically recognize the 5'-TTGTACAN6AGTACAA-3' palindromic sequence, resulting in transcriptional repression. In contrast, at TA ratio of 1:1, binding of two additional YoeB molecules onto the heterohexamer induces the completely ordered conformation of YefM and disassembles the heterohexamer into two heterotetramers, which are unable to bind the promoter DNA optimally due to steric clashes, hence derepresses TA operon transcription.


Assuntos
Proteínas de Bactérias/ultraestrutura , Endorribonucleases/ultraestrutura , Proteínas de Escherichia coli/genética , Staphylococcus aureus/ultraestrutura , Sistemas Toxina-Antitoxina/genética , Antitoxinas/genética , Antitoxinas/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Ligação a DNA/genética , Endorribonucleases/química , Endorribonucleases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Óperon/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , Multimerização Proteica/genética , Staphylococcus aureus/química , Staphylococcus aureus/genética
4.
Proc Natl Acad Sci U S A ; 117(36): 22090-22100, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839344

RESUMO

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.


Assuntos
Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/química , Citoplasma/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Inteínas , Neoplasias/tratamento farmacológico , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular Tumoral , Citoplasma/genética , Toxina Diftérica/administração & dosagem , Toxina Diftérica/química , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Feminino , Xenoenxertos , Humanos , Imunotoxinas/administração & dosagem , Imunotoxinas/química , Imunotoxinas/genética , Imunotoxinas/metabolismo , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/metabolismo , Domínios Proteicos , Transporte Proteico , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
5.
Arch Biochem Biophys ; 692: 108547, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32828796

RESUMO

Anthrax lethal factor (LF) is a critical component of the anthrax toxin, and functions intracellularly as a zinc-dependent endopeptidase targeting proteins involved in maintaining critical host signaling pathways. To reach the cytoplasm, LF requires to be unfolded and guided through the narrow protective antigen pore in a pH-dependent process. The current study sought to address the question as to whether LF is capable of retaining its metal ion when exposed to a low-pH environment (similar to that found in late endosomes) and an unfolding stress (induced by urea). Using a combination of tryptophan fluorescence spectroscopy and chelation studies, we show that a decrease in the pH value (from 7.0 to 5.0) leads to a pronounced shift in the onset of structural alterations in LF to lower urea concentrations. More importantly, the enzyme was found to retain its Zn2+ ion beyond the unfolding transitions monitored by Trp fluorescence, a finding indicative of tight metal binding to LF in a non-native state. In addition, an analysis of red-edge excitation shift (REES) spectra suggests the protein to maintain residual structure (a feature necessary for metal binding) even at very high denaturant concentrations. Furthermore, studies using the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) revealed LF's Zn2+ ion to become accessible to complexation at urea concentrations in between those required to cause structural changes and metal dissociation. This phenomenon likely originates from the conversion of a PAR-inaccessible (closed) to a PAR-accessible (open) state of LF at intermediate denaturant concentrations.


Assuntos
Antígenos de Bactérias/química , Bacillus anthracis/química , Toxinas Bacterianas/química , Quelantes/química , Zinco/química , Concentração de Íons de Hidrogênio , Desnaturação Proteica
6.
PLoS Pathog ; 16(8): e1008530, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810181

RESUMO

Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first.


Assuntos
Antraz/microbiologia , Antígenos de Bactérias/química , Bacillus anthracis/fisiologia , Toxinas Bacterianas/química , Microscopia Crioeletrônica/métodos , Animais , Humanos , Modelos Moleculares , Conformação Proteica
7.
Nature ; 583(7817): 631-637, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32641830

RESUMO

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.


Assuntos
Toxinas Bacterianas/metabolismo , Citidina Desaminase/metabolismo , DNA Mitocondrial/genética , Edição de Genes/métodos , Genes Mitocondriais/genética , Mitocôndrias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sequência de Bases , Burkholderia cenocepacia/enzimologia , Burkholderia cenocepacia/genética , Respiração Celular/genética , Citidina/metabolismo , Citidina Desaminase/química , Citidina Desaminase/genética , Genoma Mitocondrial/genética , Células HEK293 , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/terapia , Mutação , Fosforilação Oxidativa , Engenharia de Proteínas , RNA Guia/genética , Especificidade por Substrato , Sistemas de Secreção Tipo VI/metabolismo
8.
Nucleic Acids Res ; 48(13): 7532-7544, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32501503

RESUMO

Escherichia coli ItaT toxin reportedly acetylates the α-amino group of the aminoacyl-moiety of Ile-tRNAIle specifically, using acetyl-CoA as an acetyl donor, thereby inhibiting protein synthesis. The mechanism of the substrate specificity of ItaT had remained elusive. Here, we present functional and structural analyses of E. coli ItaT, which revealed the mechanism of ItaT recognition of specific aminoacyl-tRNAs for acetylation. In addition to Ile-tRNAIle, aminoacyl-tRNAs charged with hydrophobic residues, such as Val-tRNAVal and Met-tRNAMet, were acetylated by ItaT in vivo. Ile-tRNAIle, Val-tRNAVal and Met-tRNAMet were acetylated by ItaT in vitro, while aminoacyl-tRNAs charged with other hydrophobic residues, such as Ala-tRNAAla, Leu-tRNALeu and Phe-tRNAPhe, were less efficiently acetylated. A comparison of the structures of E. coli ItaT and the protein N-terminal acetyltransferase identified the hydrophobic residues in ItaT that possibly interact with the aminoacyl moiety of aminoacyl-tRNAs. Mutations of the hydrophobic residues of ItaT reduced the acetylation activity of ItaT toward Ile-tRNAIlein vitro, as well as the ItaT toxicity in vivo. Altogether, the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of the specific aminoacyl-moieties of aminoacyl-tRNAs, and ItaT has broader specificity toward aminoacyl-tRNAs charged with certain hydrophobic amino acids.


Assuntos
Acetiltransferases/química , Toxinas Bacterianas/química , Proteínas de Escherichia coli/química , Aminoacilação de RNA de Transferência , Acetiltransferases/genética , Acetiltransferases/metabolismo , Motivos de Aminoácidos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Especificidade por Substrato
9.
Nat Commun ; 11(1): 2694, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483155

RESUMO

Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.


Assuntos
Toxinas Bacterianas/toxicidade , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Polissacarídeos/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacocinética , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Heparina/química , Heparina/metabolismo , Humanos , Insetos/microbiologia , Antígenos CD15/química , Antígenos CD15/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Morganella morganii/patogenicidade , Photorhabdus/patogenicidade , Polissacarídeos/química , Xenorhabdus/patogenicidade
10.
Biochim Biophys Acta Biomembr ; 1862(9): 183364, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32450142

RESUMO

Clostridium perfringens epsilon toxin (ETX) is a heptameric pore-forming toxin of the aerolysin toxin family. ETX is the most potent toxin of this toxin family and the third most potent bacterial toxin with high cytotoxic and lethal activities in animals. In addition, ETX shows a demyelinating activity in nervous tissue leading to devastating multifocal central nervous system white matter disease in ruminant animals. Pore formation in target cell membrane is most likely the initial critical step in ETX biological activity. Eight single to quadruple ETX mutants were generated by replacement of polar residues (serine, threonine, glutamine) in middle positions of the ß-strands forming the ß-barrel and facing the channel lumen with charged glutamic residues. Channel activity and ion selectivity were monitored in artificial lipid monolayer membranes and cytotoxicity was investigated in MDCK cells by the viability MTT test and propidium iodide entry. All the mutants formed channels with similar conductance in artificial lipid membranes and increasing cation selectivity for increasing number of mutations. Here, we show that residues in the central position of each ß-strand of the amphipathic ß-hairpin loop that forms the transmembrane pore, control the size and ion selectivity of the channel. While the highest cationic ETX mutants were not cytotoxic, no strict correlation was observed between ion selectivity and cytotoxicity.


Assuntos
Toxinas Bacterianas/química , Membrana Celular/química , Clostridium perfringens/química , Animais , Toxinas Bacterianas/farmacologia , Membrana Celular/metabolismo , Cães , Células Madin Darby de Rim Canino , Estrutura Secundária de Proteína
11.
Proc Natl Acad Sci U S A ; 117(12): 6792-6800, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152097

RESUMO

Intestinal bile acids are known to modulate the germination and growth of Clostridioides difficile Here we describe a role for intestinal bile acids in directly binding and neutralizing TcdB toxin, the primary determinant of C. difficile disease. We show that individual primary and secondary bile acids reversibly bind and inhibit TcdB to varying degrees through a mechanism that requires the combined oligopeptide repeats region to which no function has previously been ascribed. We find that bile acids induce TcdB into a compact "balled up" conformation that is no longer able to bind cell surface receptors. Lastly, through a high-throughput screen designed to identify bile acid mimetics we uncovered nonsteroidal small molecule scaffolds that bind and inhibit TcdB through a bile acid-like mechanism. In addition to suggesting a role for bile acids in C. difficile pathogenesis, these findings provide a framework for development of a mechanistic class of C. difficile antitoxins.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Ácidos e Sais Biliares/metabolismo , Intestinos/fisiologia , Receptores de Superfície Celular/metabolismo , Células CACO-2 , Infecções por Clostridium/microbiologia , Células HCT116 , Humanos
12.
Nat Commun ; 11(1): 1502, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198389

RESUMO

Although certain microbial lipids are toxins, the structural features important for cytotoxicity remain unknown. Increased functional understanding is essential for developing therapeutics against toxic microbial lipids. Group B Streptococci (GBS) are bacteria associated with preterm births, stillbirths, and severe infections in neonates and adults. GBS produce a pigmented, cytotoxic lipid, known as granadaene. Despite its importance to all manifestations of GBS disease, studies towards understanding granadaene's toxic activity are hindered by its instability and insolubility in purified form. Here, we report the synthesis and screening of lipid derivatives inspired by granadaene, which reveal features central to toxin function, namely the polyene chain length. Furthermore, we show that vaccination with a non-toxic synthetic analog confers the production of antibodies that inhibit granadaene-mediated hemolysis ex vivo and diminish GBS infection in vivo. This work provides unique structural and functional insight into granadaene and a strategy to mitigate GBS infection, which will be relevant to other toxic lipids encoded by human pathogens.


Assuntos
Hemólise , Lipídeos/química , Polienos/química , Nascimento Prematuro/microbiologia , Infecções Estreptocócicas/metabolismo , Adulto , Animais , Linfócitos B , Toxinas Bacterianas/química , Vacinas Bacterianas , Linfócitos T CD4-Positivos , Modelos Animais de Doenças , Feminino , Humanos , Recém-Nascido , Lipídeos/imunologia , Lipídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polienos/imunologia , Gravidez , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/prevenção & controle , Streptococcus agalactiae , Vacinação
13.
Sci Adv ; 6(11): eaaz2094, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32195351

RESUMO

We identified a glucosyltransferase (YGT) and an ADP-ribosyltransferase (YART) in Yersinia mollaretii, highly related to glucosylating toxins from Clostridium difficile, the cause of antibiotics-associated enterocolitis. Both Yersinia toxins consist of an amino-terminal enzyme domain, an autoprotease domain activated by inositol hexakisphosphate, and a carboxyl-terminal translocation domain. YGT N-acetylglucosaminylates Rab5 and Rab31 at Thr52 and Thr36, respectively, thereby inactivating the Rab proteins. YART ADP-ribosylates Rab5 and Rab31 at Gln79 and Gln64, respectively. This activates Rab proteins by inhibiting GTP hydrolysis. We determined the crystal structure of the glycosyltransferase domain of YGT (YGTG) in the presence and absence of UDP at 1.9- and 3.4-Å resolution, respectively. Thereby, we identified a previously unknown potassium ion-binding site, which explains potassium ion-dependent enhanced glycosyltransferase activity in clostridial and related toxins. Our findings exhibit a novel type of inverse regulation of Rab proteins by toxins and provide new insights into the structure-function relationship of glycosyltransferase toxins.


Assuntos
ADP Ribose Transferases , Proteínas de Bactérias , Toxinas Bacterianas , Glicosiltransferases , Yersinia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Glicosilação , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Células HeLa , Humanos , Domínios Proteicos , Difosfato de Uridina/química , Difosfato de Uridina/metabolismo , Yersinia/química , Yersinia/enzimologia
14.
Nat Struct Mol Biol ; 27(3): 288-296, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32123390

RESUMO

The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and translocation of Ia across the cell membrane. Here we report cryo-EM structures of the translocation channel Ib-pore and its complex with Ia. The high-resolution Ib-pore structure demonstrates a similar structural framework to that of the catalytic ϕ-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure shows a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia amino-terminal domain forms multiple weak interactions with two additional Ib-pore constriction sites. Furthermore, Ib-binding induces tilting and partial unfolding of the Ia N-terminal α-helix, permitting its extension to the ϕ-clamp gate. This new mechanism of N-terminal unfolding is crucial for protein translocation.


Assuntos
ADP Ribose Transferases/química , Antígenos de Bactérias/química , Toxinas Bacterianas/química , Clostridium perfringens/química , Subunidades Proteicas/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Clonagem Molecular , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
15.
Nat Commun ; 11(1): 840, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32047164

RESUMO

Following assembly, the anthrax protective antigen (PA) forms an oligomeric translocon that unfolds and translocates either its lethal factor (LF) or edema factor (EF) into the host cell. Here, we report the cryo-EM structures of heptameric PA channels with partially unfolded LF and EF at 4.6 and 3.1-Å resolution, respectively. The first α helix and ß strand of LF and EF unfold and dock into a deep amphipathic cleft, called the α clamp, which resides at the interface of two PA monomers. The α-clamp-helix interactions exhibit structural plasticity when comparing the structures of lethal and edema toxins. EF undergoes a largescale conformational rearrangement when forming the complex with the channel. A critical loop in the PA binding interface is displaced for about 4 Å, leading to the weakening of the binding interface prior to translocation. These structures provide key insights into the molecular mechanisms of translocation-coupled protein unfolding and translocation.


Assuntos
Antígenos de Bactérias/química , Toxinas Bacterianas/química , Desdobramento de Proteína , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Toxinas Bacterianas/genética , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas
16.
Sci Rep ; 10(1): 2781, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066776

RESUMO

Harmful algal blooms formed by colony-forming cyanobacteria deteriorate water resources by producing cyanotoxins, which frequently occur at high intracellular concentrations. We aimed to localize toxic microcystins (MCs) and bioactive anabaenopeptins (APs) at the subcellular level under noninvasive conditions. Since both metabolites are synthesized nonribosomally, the relaxed specificity of key enzymes catalyzing substrate activation allowed chemical labeling through a standard copper-catalyzed click chemistry reaction. The genera Planktothrix and Microcystis specifically incorporated unnatural amino acids such as N-propargyloxy-carbonyl-L-lysine or O-propargyl-L-tyrosine, resulting in modified AP or MC peptides carrying the incorporated alkyne moiety. The labeled cells were quantitatively differentiated from the unlabeled control cells. MCs and APs occurred intracellularly as distinct entities showing a cell-wide distribution but a lowered spatial overlap with natural autofluorescence. Using the immunofluorescence technique, colocalization with markers of individual organelles was utilized to relate the distribution of labeled MCs to cellular compartments, e.g., using RbcL and FtsZ (cytosol) and PsbA (thylakoids). The colocalization correlation coefficients calculated pairwise between organelles and autofluorescence were highly positive as opposed to the relatively low positive indices derived from labeled MCs. The lower correlation coefficients imply that only a portion of the labeled MC molecules were related spatially to the organelles in the cell.


Assuntos
Toxinas Bacterianas/isolamento & purificação , Cianobactérias/química , Microcistinas/isolamento & purificação , Peptídeos Cíclicos/isolamento & purificação , Aminoácidos/química , Aminoácidos/metabolismo , Toxinas Bacterianas/química , Química Click , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/metabolismo , Água Doce/química , Proliferação Nociva de Algas , Microcistinas/química , Peptídeos Cíclicos/química
17.
Sci Rep ; 10(1): 3059, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080292

RESUMO

The repeating unit of the C. difficile Toxin A (rARU, also known as CROPS [combined repetitive oligopeptides]) C-terminal region, was shown to elicit protective immunity against C. difficile and is under consideration as a possible vaccine against this pathogen. However, expression of recombinant rARU in E. coli using the standard vaccine production process was very low. Transcriptome and proteome analyses showed that at restricted dissolved oxygen (DO) the numbers of differentially expressed genes (DEGs) was 2.5-times lower than those expressed at unrestricted oxygen. Additionally, a 7.4-times smaller number of ribosome formation genes (needed for translation) were down-regulated as compared with unrestricted DO. Higher rARU expression at restricted DO was associated with up-regulation of 24 heat shock chaperones involved in protein folding and with the up-regulation of the global regulator RNA chaperone hfq. Cellular stress response leading to down-regulation of transcription, translation, and energy generating pathways at unrestricted DO were associated with lower rARU expression. Investigation of the C. difficile DNA sequence revealed the presence of cell wall binding profiles, which based on structural similarity prediction by BLASTp, can possibly interact with cellular proteins of E. coli such as the transcriptional repressor ulaR, and the ankyrins repeat proteins. At restricted DO, rARU mRNA was 5-fold higher and the protein expression 27-fold higher compared with unrestricted DO. The report shows a strategy for improved production of C. difficile vaccine candidate in E. coli by using restricted DO growth. This strategy could improve the expression of recombinant proteins from anaerobic origin or those with cell wall binding profiles.


Assuntos
Toxinas Bacterianas/genética , Enterotoxinas/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Oxigênio/metabolismo , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Toxinas Bacterianas/química , Enterotoxinas/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Cinética , Modelos Biológicos , Subunidades Proteicas/química , Proteômica , Solubilidade
18.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 1): 31-39, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31929184

RESUMO

The Escherichia coli rnlAB operon encodes a toxin-antitoxin module that is involved in protection against infection by bacteriophage T4. The full-length RnlA-RnlB toxin-antitoxin complex as well as the toxin RnlA were purified to homogeneity and crystallized. When the affinity tag is placed on RnlA, RnlB is largely lost during purification and the resulting crystals exclusively comprise RnlA. A homogeneous preparation of RnlA-RnlB containing stoichiometric amounts of both proteins could only be obtained using a His tag placed C-terminal to RnlB. Native mass spectrometry and SAXS indicate a 1:1 stoichiometry for this RnlA-RnlB complex. Crystals of the RnlA-RnlB complex belonged to space group C2, with unit-cell parameters a = 243.32, b = 133.58, c = 55.64 Å, ß = 95.11°, and diffracted to 2.6 Šresolution. The presence of both proteins in the crystals was confirmed and the asymmetric unit is likely to contain a heterotetramer with RnlA2:RnlB2 stoichiometry.


Assuntos
Proteínas de Escherichia coli/química , Antitoxinas/química , Toxinas Bacterianas/química , Bacteriófago T4/metabolismo , Cromatografia Líquida , Cristalização , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Expressão Gênica/genética , Óperon/genética , Espalhamento a Baixo Ângulo , Espectrometria de Massas em Tandem , Difração de Raios X
19.
PLoS Pathog ; 16(1): e1008184, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31951643

RESUMO

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.


Assuntos
Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , Infecções por Yersinia pseudotuberculosis/microbiologia , Yersinia pseudotuberculosis/metabolismo , Regiões 5' não Traduzidas , Animais , Toxinas Bacterianas/química , Feminino , Humanos , Sequências Repetidas Invertidas , Camundongos , Camundongos Endogâmicos BALB C , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , Temperatura , Virulência , Yersinia pseudotuberculosis/química , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidade
20.
Nat Commun ; 11(1): 432, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974369

RESUMO

Large Clostridial Toxins (LCTs) are a family of six homologous protein toxins that are implicated in severe disease. LCTs infiltrate host cells using a translocation domain (LCT-T) that contains both cell-surface receptor binding sites and a membrane translocation apparatus. Despite much effort, LCT translocation remains poorly understood. Here we report the identification of 1104 LCT-T homologs, with 769 proteins from bacteria outside of clostridia. Sequences are widely distributed in pathogenic and host-associated species, in a variety of contexts and architectures. Consistent with these homologs being functional toxins, we show that a distant LCT-T homolog from Serratia marcescens acts as a pH-dependent translocase to deliver its effector into host cells. Based on evolutionary footprinting of LCT-T homologs, we further define an evolutionarily conserved translocase region that we show is an autonomous translocase capable of delivering heterologous cargo into host cells. Our work uncovers a broad class of translocating toxins and provides insights into LCT translocation.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Evolução Biológica , /metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Chlorocebus aethiops , Dicroísmo Circular , Sequência Conservada , Evolução Molecular , Células HCT116 , Interações Hospedeiro-Patógeno , Humanos , Concentração de Íons de Hidrogênio , Domínios Proteicos , Transporte Proteico , Homologia de Sequência de Aminoácidos , Serratia marcescens/metabolismo , Serratia marcescens/patogenicidade , Células Vero
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