Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.115
Filtrar
1.
PLoS Pathog ; 16(9): e1008852, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32960931

RESUMO

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridium difficile , Enterocolite Pseudomembranosa , Glucosiltransferases , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clostridium difficile/enzimologia , Clostridium difficile/genética , Clostridium difficile/patogenicidade , Cricetinae , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/enzimologia , Enterocolite Pseudomembranosa/genética , Enterocolite Pseudomembranosa/patologia , Feminino , Deleção de Genes , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Masculino , Camundongos
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.
PLoS Pathog ; 16(8): e1008708, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32785266

RESUMO

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the "flagellar switch" upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5' leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Infecções por Clostridium/microbiologia , Clostridium difficile/metabolismo , Flagelos/metabolismo , Fator Rho/metabolismo , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridium difficile/genética , Clostridium difficile/patogenicidade , Feminino , Flagelos/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óperon , Fator Rho/genética , Virulência
6.
PLoS One ; 15(8): e0236713, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750089

RESUMO

Coagulase-negative staphylococci (CoNS) are the most common isolates from blood culture in neonates resulting in high mortality and morbidity. This study investigated CoNS obtained from blood cultures of neonates for antibiotic resistance and virulence factors, and possible association with inflammatory response (C-reactive protein). A total of 93 CoNS isolates were collected from 76 blood cultures of neonates at the Maternity hospital in Kuwait in a six-month period and investigated for susceptibility to antibiotics, carriage of staphylococcal cassette chromosome mec (SCCmec), and virulence-associated genes. The 93 CoNS isolates consisted of S. epidermidis (76; 81.7%), S. capitis (12; 12.9%), S. hominis (2; 2.1%), S. warneri (2; 2.1%) and S. haemolyticus (1; 1.0%). Eighty-six (92.4%) of the isolates were resistant to cefoxitin (MR-CoNS) while 49 (52.7%) expressed multi-antibiotic resistance. The methicillin-resistant isolates (MR-CoNS) carried SCCmec III, SCCmec IVa and four combinations of SCCmec types including SCCmec types I+IVa (one S. warneri and 25 S. epidermidis isolates), types I+III (one S. epidermidis isolate), types III+IVa (six S. epidermidis isolates) and types I+III+IVa (one S. epidermidis isolate). The most common virulence-related genes were icaC, seb, arc detected in 69.7%, 60.5%, 40.8% of the isolates respectively. Two isolates were positive for tst1. No association between C-reactive protein and antibiotic resistance or virulence factors was established. This study revealed that S. epidermidis carrying different SCCmec genetic elements, was the dominant CoNS species isolated from neonatal blood cultures with 90.3% and 36.6% of the isolates positive for genes for biofilm and ACME production respectively.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Recém-Nascido Prematuro , Infecções Estafilocócicas/microbiologia , Staphylococcus/efeitos dos fármacos , Toxinas Bacterianas/genética , Biofilmes , Coagulase/metabolismo , Feminino , Genes Bacterianos , Humanos , Recém-Nascido , Kuweit , Masculino , Testes de Sensibilidade Microbiana , Estudos Retrospectivos , Staphylococcus/enzimologia , Staphylococcus/isolamento & purificação , Staphylococcus/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
7.
Zhonghua Er Ke Za Zhi ; 58(7): 564-569, 2020 Jul 02.
Artigo em Chinês | MEDLINE | ID: mdl-32605340

RESUMO

Objective: To explore the infection rate and clinical characteristics of toxigenic Clostridium difficile in children with inflammatory bowel disease (IBD). Methods: From July 2015 to October 2016, the fecal samples and clinical data of 30 IBD children admitted to Department of Gastroenterology, Beijing Children's Hospital, Capital Medical University, as well as the specimens and data of 30 healthy children were collected in the meantime. The toxin gene of Clostridium difficile was detected and clinical characteristics of children with positive toxin gene were analyzed retrospectively. χ(2) test was used to compare the variables between groups. Results: Among the 30 IBD patients, 15 were in ulcerative colitis (UC) group and 15 in Crohn's disease (CD) group. In the IBD group, 6 (3 in UC and 3 in CD group) had positive result of toxigenic Clostridium difficile (20%), among whom 5 were toxin Clostridium difficile A (tcdA) +toxin Clostridium difficile B (tcdB) -, and 1 was tcdA+tcdB+. In the healthy group, only one had positive result of toxigenic Clostridium difficile (3%), which was tcdA+tcdB-. Binary toxin gene was negative in both groups. The infection rate of toxigenic Clostridium difficile in IBD group was significantly higher than that in healthy control group (χ(2)=4.043, P=0.044). In UC group, no Clostridium difficile toxin gene was detected during the remission period (0/1), one case was positive for toxin gene (1/11) during mild active period, and 2 cases were (2/3) during moderately active period. There were significant differences in the infection rate of toxigenic Clostridium difficile between patients in different active period (χ(2)=4.000, P=0.046). The main manifestations of the 6 cases were diarrhea, abdominal pain and bloody stool, and the relapsed case was characterized by sudden aggravation. TcdA was detected in all toxin gene positive samples, and 1 case combined with tcdB had more serious bloody mucopurulent stool. Five cases had colonoscopy, but there was no obvious characteristics of toxigenic Clostridium difficile colitis such as yellow white plaques or pseudomembranous spot. Three cases had antibiotic exposure history. All 6 cases were sensitive to metronidazole treatment, and stable without relapse during the 3-month follow-up. Conclusions: The infection rate of toxigenic Clostridium difficile in children with IBD is higher than that in healthy children. The patients with both tcdA and tcdB could have more serious clinical symptoms, although there may not be specific pathological changes of toxigenic Clostridium difficile colitis. The recognition of toxigenic Clostridium difficile infection in IBD children should be strengthened in clinical work.


Assuntos
Toxinas Bacterianas , Infecções por Clostridium , Clostridium difficile , Doenças Inflamatórias Intestinais , Proteínas de Bactérias , Toxinas Bacterianas/genética , Criança , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/epidemiologia , Clostridium difficile/genética , Clostridium difficile/patogenicidade , Enterotoxinas/genética , Fezes , Humanos , Doenças Inflamatórias Intestinais/microbiologia , Estudos Retrospectivos
8.
PLoS One ; 15(7): e0236551, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726339

RESUMO

PhoH2 proteins are highly conserved across bacteria and archaea yet their biological function is poorly characterised. We examined the growth profiles of Mycobacterium smegmatis strains mc2155 and mc2155 ΔphoH2 and observed the same growth profile and growth rate in a variety of conditions. In light of the comparable growth, we used RNAseq to provide a snapshot of the differences between the transcriptomes of M. smegmatis mc2155 and M. smegmatis mc2155 ΔphoH2 during normal growth. At 48 hours, elevated expression of the sigF regulon was observed in ΔphoH2 relative to wild type. In biochemical assays, PhoH2 showed activity toward sigF mRNA insinuating a role of PhoH2 in modulating the pool of sigF mRNA in the cell during normal growth, adding further complexity to the repertoire of reported mechanisms of post-translational regulation. Multiple copies of the preferred target site of PhoH2 were identified in loops of the sigF mRNA structure, leading us to propose a mechanism for the activity of PhoH2 that is initiated after assembly on specific single-stranded loops of RNA. We hypothesise that PhoH2 is a toxin-antitoxin that contributes to the regulation of SigF at a post-transcriptional level through targeted activity on sigF mRNA. This work presents the first evidence for post-transcriptional regulation of SigF along with the biological function of PhoH2 from M. smegmatis. This has implications for the highly conserved PhoH2 toxin-antitoxin module across the mycobacteria including the important human pathogen M. tuberculosis.


Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Mycobacterium smegmatis/metabolismo , Fator sigma/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium smegmatis/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Fator sigma/genética
9.
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
10.
PLoS One ; 15(7): e0235633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628709

RESUMO

The antibacterial efficacy of the tetracycline antibiotics has been greatly reduced by the development of resistance, hence a decline in their clinical use. The hok/sok locus is a type I toxin/antitoxin plasmid stability element, often associated with multi-drug resistance plasmids, especially ESBL-encoding plasmids. It enhances host cell survivability and pathogenicity in stressful growth conditions, and increases bacterial tolerance to ß-lactam antibiotics. The hok/sok locus forms dsRNA by RNA:RNA interactions between the toxin encoding mRNA and antitoxin non-coding RNA, and doxycycline has been reported to bind dsRNA structures and inhibit their cleavage/processing by the dsRNase, RNase III. This study investigated the antibacterial activities of doxycycline in hok/sok host bacteria cells, the effects on hok/sok-induced changes in growth and the mechanism(s) involved. Diverse strains of E. coli were transformed with hok/sok plasmids and assessed for doxycycline susceptibility and growth changes. The results show that the hok/sok locus increases bacterial susceptibility to doxycycline, which is more apparent in strains with more pronounced hok/sok-induced growth effects. The increased doxycycline susceptibility occurs despite ß-lactam resistance imparted by hok/sok. Doxycycline was found to induce bacterial death in a manner phenotypically characteristic of Hok toxin expression, suggesting that it inhibits the toxin/antitoxin dsRNA degradation, leading to Hok toxin expression and cell death. In this way, doxycycline could counteract the multi-drug resistance plasmid maintenance/propagation, persistence and pathogenicity mechanisms associated with the hok/sok locus, which could potentially help in efforts to mitigate the rise of antimicrobial resistance.


Assuntos
Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Doxiciclina/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , RNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Bacteriano/metabolismo , RNA de Cadeia Dupla/metabolismo
11.
PLoS One ; 15(6): e0234119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492051

RESUMO

Recently, a new rapid assay for the detection of tcdB gene of Clostridioides difficile was developed using the GENECUBE. The assay can directly detect the tcdB gene from stool samples without a purification in approximately 35 minutes with a few minutes of preparation process. We performed a prospective comparative study of the performance of the assay at eight institutions in Japan. Fresh residual stool samples (Bristol stool scale ≥5) were used and comparisons were performed with the BD MAX Cdiff assay and toxigenic cultures. For the evaluation of 383 stool samples compared with the BD MAX Cdiff assay, the sensitivity, and specificity of the two assays was 99.0% (379/383), 98.1% (52/53), 99.1% (327/330), respectively. In the comparison with toxigenic culture, the total, sensitivity, and specificity were 96.6% (370/383), 85.0% (51/60), and 98.8% (319/323), respectively. The current investigation indicated the GENECUBE Clostridioides difficile assay has equivalent performance with the BD MAX Cdiff assay for the detection of tcdB gene of C. difficile.


Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridium difficile/genética , Fezes/microbiologia , Infecções por Clostridium/diagnóstico , Clostridium difficile/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Enterotoxinas/genética , Humanos , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico , Proteínas Repressoras/genética , Sensibilidade e Especificidade
12.
Nucleic Acids Res ; 48(15): 8617-8625, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32597957

RESUMO

Type II toxin-antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin-antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin-antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 activity induces ribosome stalling at all four glycyl codons but does not evoke a stringent response. In vitro, AtaT2 acetylates the aminoacyl moiety of isoaccepting glycyl tRNAs, thus precluding their participation in translation. Our study broadens the known target specificity of GNAT toxins beyond the earlier described isoleucine and formyl methionine tRNAs, and suggest that various GNAT toxins may have evolved to specificaly target other if not all individual aminoacyl tRNAs.


Assuntos
Acetiltransferases/genética , Escherichia coli O157/genética , Glicina-tRNA Ligase/genética , Biossíntese de Proteínas/genética , Antitoxinas/genética , Toxinas Bacterianas/genética , Escherichia coli O157/patogenicidade , Sistemas Toxina-Antitoxina/genética
13.
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
14.
Appl Environ Microbiol ; 86(13)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32358012

RESUMO

Pseudomonas putida S12 is highly tolerant of organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies revealed that P. putida S12 contains the single-copy 583-kbp megaplasmid pTTS12. pTTS12 carries several important operons and gene clusters facilitating P. putida S12 survival and growth in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid from P. putida S12 and conclusively confirmed that the SrpABC efflux pump is the major determinant of solvent tolerance on the megaplasmid pTTS12. In addition, we identified a novel toxin-antitoxin module (proposed gene names slvT and slvA, respectively) encoded on pTTS12 which contributes to the solvent tolerance phenotype and is important for conferring stability to the megaplasmid. Chromosomal introduction of the srp operon in combination with the slvAT gene pair created a solvent tolerance phenotype in non-solvent-tolerant strains, such as P. putida KT2440, Escherichia coli TG1, and E. coli BL21(DE3).IMPORTANCE Sustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in bacterial biocatalysis. However, during the bioproduction of such chemicals and biopolymers, aromatic compounds that function as products, substrates, or intermediates in the production process may exert toxicity to microbial host cells and limit the production yield. Therefore, solvent tolerance is a highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerant Pseudomonas putida S12 for molecular adaptation to an organic solvent. In addition to the solvent extrusion pump (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to short-term tolerance in moderate solvent concentrations, as well as to the stability of pTTS12. These two gene clusters were successfully expressed in non-solvent-tolerant strains of P. putida and Escherichia coli strains to confer and enhance solvent tolerance.


Assuntos
Toxinas Bacterianas/genética , Plasmídeos/efeitos dos fármacos , Pseudomonas putida/efeitos dos fármacos , Solventes/metabolismo , Toxinas Bacterianas/metabolismo , Pseudomonas putida/genética
15.
Epidemiol Infect ; 148: e153, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32321605

RESUMO

This study presents enhanced surveillance data from 2004 to 2018 for all community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) specimens collected in Western Australia (WA), and describes the changing epidemiology over this period. A total of 57 557 cases were reviewed. Annual incidence rates increased from 86.2 cases per 100 000 population to 245.6 per 100 000 population (IRR = 2.9, CI95 2.7-3.0). The proportion of isolates carrying Panton-Valentine leucocidin (PVL)-associated genes increased from 3.4% to 59.8% (χ2 test for trend 7021.9, P < 0.001). The emergence of PVL-positive, 'Queensland CA-MRSA' (ST93-IV) and 'WA 121' (ST5-IV) accounted for the majority of increases in CA-MRSA across the study period. It is unclear why some clones are more prolific in certain regions. In WA, CA-MRSA rates increase as indices of temperature and humidity increase after controlling for socioeconomic disadvantage. We suggest climatic conditions may contribute to transmission, along with other socio-behavioural factors. A better understanding of the ability for certain clones to form ecological niches and cause outbreaks is required.


Assuntos
Infecções Comunitárias Adquiridas/microbiologia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Criança , Pré-Escolar , Infecções Comunitárias Adquiridas/epidemiologia , Exotoxinas/genética , Exotoxinas/metabolismo , Feminino , Genótipo , Humanos , Incidência , Lactente , Leucocidinas/genética , Leucocidinas/metabolismo , Masculino , Staphylococcus aureus Resistente à Meticilina/classificação , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Austrália Ocidental/epidemiologia , Adulto Jovem
16.
Harmful Algae ; 93: 101767, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32307065

RESUMO

Reports of anatoxins poisoning of wildlife and domestic animals by toxigenic cyanobacteria in streams and rivers are increasing globally. Little is known about the taxonomy, morphology and genomics of anatoxins producing species, limiting our knowledge about their environmental preferences. We isolated three benthic non-heterocystous filamentous cyanobacterial strains from the Russian River in Northern California (USA), which produce anatoxin-a and dihydroanatoxin-a. Both 16S rRNA and protein sequence phylogenetic analyses showed that the strains represent a distinct new member of the cyanobacterial genus Microcoleus (Oscillatoriales). A novel species, Microcoleus anatoxicus is described and accompanied with light microscope photomicrographs, toxin profiles and the complete anatoxin-a gene cassette with the first description of the anaK gene in Microcoleus.


Assuntos
Toxinas Bacterianas , Cianobactérias , Animais , Toxinas Bacterianas/genética , California , Cianobactérias/genética , Filogenia , Prolina/análogos & derivados , RNA Ribossômico 16S/genética , Rios , Federação Russa
17.
Harmful Algae ; 93: 101792, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32307073

RESUMO

Cylindrospermopsis and Raphidiopsis (C/R group) are closely related species responsible for cyanobacterial blooms worldwide. Paralytic shellfish toxins (PSTs) and cylindrospermopsins (CYNs) have been identified in different C/R group strains. However, the evolutionary relationship between PST- and CYN-producing strains has not been systematically evaluated. In this study, C/R group strains and their toxin biosynthesis genes were evaluated by phylogenetic analysis and sequence comparison. None of the tested strains are able to produce PSTs and CYNs simultaneously. The C/R group strains were clustered into five clades, including two non-toxic, two CYN-producing and one PST-producing clades. A high degree of similarity was observed for rpoC1 (> 96%) and ITS-L (> 97%) sequences within each clade with the exception of the ITS-L (87% to 100%) region in CYN-producing R. curvata, which has been shown to contain variable sequence insertions. Genomic analysis revealed that sxtY and sxtZ could be found in both toxic and non-toxic strains. The transposase gene IS4 was only observed in strains from the PST-producing clade. The sxt and cyr gene clusters share five gene families with similar functions. The amino acid sequences of the adenylyl-sulfate kinase genes, sxtO and cyrN, are more similar (45% to 81%) than other pairs of genes (8.0% to 40%). SxtO and CyrN proteins from C/R group strains forms an independent clade on the phylogenetic tree with a high degree of sequence similarity (78% to 100%). In conclusion, PST- and CYN- producing C/R group species can be classified into different clades based on their phylogenetic profile. The sxtO and cyrN genes have probably diverged from a single ancestral adenylyl-sulfate kinase gene, and may be specifically used for toxin biosynthesis in C/R group species.


Assuntos
Toxinas Bacterianas , Cylindrospermopsis , Toxinas Bacterianas/genética , Filogenia , Frutos do Mar , Uracila/análogos & derivados
18.
Mol Biotechnol ; 62(6-7): 335-343, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32236842

RESUMO

Emergence of multidrug resistant strains and extremely drug resistant strains of Mycobacterium tuberculosis is due to its ability to form persister cells. The formation of persister cells is assumed to be triggered due to the presence of large number of toxin-antitoxin (TA) systems in its genome. Mtb genome encodes 47 VapBC TA systems. In this work, we aim to biochemically characterize VapC46 toxin of the VapBC46 TA operon from Mycobacterium tuberculosis. Heterologous expression of VapC46 in E. coli is shown to exhibit bacteriostasis and toxicity alters the surface morphology of the E. coli cells. VapC46 is shown to possess ribonuclease activity in a magnesium-dependent manner. Using FRET and pull down assay, VapC46 is shown to interact with VapB46 antitoxin. A model of VapC46 is shown to resemble PIN domain family of proteins and reveals the putative active site required for its ribonuclease activity.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Genoma Bacteriano/genética , Mycobacterium tuberculosis/genética , Ribonucleases/genética , Ribonucleases/metabolismo , Sistemas Toxina-Antitoxina/genética , Sistemas Toxina-Antitoxina/fisiologia
19.
RNA ; 26(7): 803-813, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32284351

RESUMO

The ribonuclease A family of proteins is well studied from the biochemical and biophysical points of view, but its evolutionary origins are obscure, as no sequences homologous to this family have been reported outside of vertebrates. Recently, the spatial structure of the ribonuclease domain from a bacterial polymorphic toxin was shown to be closely similar to the structure of vertebrate ribonuclease A. The absence of sequence similarity between the two structures prompted a speculation of convergent evolution of bacterial and vertebrate ribonuclease A-like enzymes. We show that bacterial and homologous archaeal polymorphic toxin ribonucleases with a known or predicted ribonuclease A-like fold are distant homologs of the ribonucleases from the EndoU family, found in all domains of cellular life and in viruses. We also detected a homolog of vertebrate ribonucleases A in the transcriptome assembly of the sea urchin Mesocentrotus franciscanus These observations argue for the common ancestry of prokaryotic ribonuclease A-like and ubiquitous EndoU-like ribonucleases, and suggest a better-grounded scenario for the origin of animal ribonucleases A, which could have emerged in the deuterostome lineage, either by an extensive modification of a copy of an EndoU gene, or, more likely, by a horizontal acquisition of a prokaryotic immunity-mediating ribonuclease gene.


Assuntos
Toxinas Bacterianas/genética , Ribonuclease Pancreático/genética , Sequência de Aminoácidos , Animais , Archaea/genética , Bactérias/genética , Evolução Molecular , Filogenia , Alinhamento de Sequência , Vertebrados/genética
20.
Microbiol Res ; 235: 126448, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32114363

RESUMO

Vibrio parahaemolyticus is a common foodborne pathogen in seafood and represents a major threat to human health worldwide. In this study, we identified that PhoR, a histidine kinase, is involved in the regulation of swarming and flagella assembly. RNA sequencing analysis showed that 1122 genes were differentially expressed in PhoR mutant, including 394 upregulated and 728 downregulated genes. KEGG enrichment and heatmap analysis demonstrated that the bacterial secretion system, flagella assembly and chemotaxis pathways were significantly downregulated in PhoR mutant, while the microbial metabolism in diverse environments and carbon metabolism pathways were upregulated in PhoR mutant. qRT-PCR further confirmed that genes responsible for the type III secretion system (T3SS), swarming and the thermostable direct hemolysin were positively regulated by PhoR. Phosphorylation assays suggested that PhoR was highly activated in BHI medium compared to LB medium. Taken together, these data suggested that activated PhoR contributes to the expression of swarming motility and secretion system genes in Vibrio parahaemolyticus.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Transcriptoma , Sistemas de Secreção Tipo III/genética , Vibrio parahaemolyticus/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Regulação para Baixo , Perfilação da Expressão Gênica , Proteínas Hemolisinas/genética , Movimento , Regulação para Cima , Vibrio parahaemolyticus/enzimologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA