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1.
GM Crops Food ; 12(1): 382-395, 2021 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-34193022

RESUMO

The idea of enhanced methanol production from cell wall by pectin methyl esterase enzymes (PME) combined with expression of cry genes from Bacillus thuringiensis as a strategy to improve insect pest control in cotton is presented. We constructed a cassette containing two cry genes (cry1Fa and Cry32Aa) and two pme genes, one from Arabidopsis thaliana (AtPME), and other from Aspergillus. niger (AnPME) in pCAMBIA1301 plant expression vector using CAMV-35S promoter. This construction was transformed in Eagle-2 cotton variety by using shoot apex-cut Agrobacterium-mediated transformation. Expression of cry genes and pme genes was confirmed by qPCR. Methanol production was measured in control and in the cry and pme transformed plants showing methanol production only in transformed plants, in contrast to the non-transgenic cotton plants. Finally, insect bioassays performed with transgenic plants expressing cry and pme genes showed 100% mortality for Helicoverpa armigera (cotton bollworm) larvae, 70% mortality for Pectinophora gossypiella (pink bollworm) larvae and 95% mortality of Earias fabia, (spotted bollworm) larvae, that was higher than the transgenic plants expressing only cry genes that showed 84%, 49% and 79% mortality, respectively. These results demonstrate that Bt. cry-genes coupled with pme genes are an effective strategy to improve the control of different insect pests.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Gossypium/genética , Proteínas Hemolisinas/genética , Resistência a Inseticidas , Larva , Metanol , Plantas Geneticamente Modificadas
2.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205064

RESUMO

Cortactin is a well-known regulatory protein of the host actin cytoskeleton and represents an attractive target of microbial pathogens like Helicobacter pylori. H. pylori manipulates cortactin's phosphorylation status by type-IV secretion-dependent injection of its virulence protein CagA. Multiple host tyrosine kinases, like FAK, Src, and Abl, are activated during infection, but the pathway(s) involved is (are) not yet fully established. Among them, Src and Abl target CagA and stimulate tyrosine phosphorylation of the latter at its EPIYA-motifs. To investigate the role of cortactin in more detail, we generated a CRISPR/Cas9 knockout of cortactin in AGS gastric epithelial cells. Surprisingly, we found that FAK, Src, and Abl kinase activities were dramatically downregulated associated with widely diminished CagA phosphorylation in cortactin knockout cells compared to the parental control. Together, we report here a yet unrecognized cortactin-dependent signaling pathway involving FAK, Src, and Abl activation, and controlling efficient phosphorylation of injected CagA during infection. Thus, the cortactin status could serve as a potential new biomarker of gastric cancer development.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Quinase 1 de Adesão Focal/genética , Infecções por Helicobacter/genética , Helicobacter pylori/genética , Proteínas Oncogênicas v-abl/genética , Regulação Bacteriana da Expressão Gênica/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Fosforilação/genética , Quinases da Família src/genética
3.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205867

RESUMO

The increase in the use of antimicrobials such as colistin for the treatment of infectious diseases has led to the appearance of Aeromonas strains resistant to this drug. However, resistance to colistin not only occurs in the clinical area but has also been determined in Aeromonas isolates from the environment or animals, which has been determined by the detection of mcr genes that confer a resistance mechanism to colistin. The variants mcr-1, mcr-3, and mcr-5 have been detected in the genus Aeromonas in animal, environmental, and human fluids samples. In this article, an overview of the resistance to colistin in Aeromonas is shown, as well as the generalities of this molecule and the recommended methods to determine colistin resistance to be used in some of the genus Aeromonas.


Assuntos
Aeromonas/genética , Antibacterianos/química , Colistina/química , Farmacorresistência Bacteriana/genética , Aeromonas/efeitos dos fármacos , Aeromonas/patogenicidade , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Colistina/uso terapêutico , Humanos , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética
4.
BMC Genomics ; 22(1): 507, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34225670

RESUMO

BACKGROUND: Salmonella is a major bacterial pathogen associated with a large number of outbreaks of foodborne diseases. Many highly virulent serovars that cause human illness belong to Salmonella serogroup C1, and Salmonella ser. Choleraesuis is a prominent cause of invasive infections in Asia. Comparative genomic analysis in our previous study showed that two homologous genes, SC0368 and SC0595 in Salmonella ser. Choleraesuis were unique to serogroup C1. In this study, two single-deletion mutants (Δ0368 and Δ0595) and one double-deletion mutant (Δ0368Δ0595) were constructed based on the genome. All these mutants and the wild-type strain were subjected to RNA-Seq analysis to reveal functional relationships of the two serogroup C1-specific genes. RESULTS: Data from RNA-Seq indicated that deletion of SC0368 resulted in defects in motility through repression of σ28 in flagellar regulation Class 3. Consistent with RNA-Seq data, results from transmission electron microcopy (TEM) showed that flagella were not present in △0368 and △0368△0595 mutants resulting in both swimming and swarming defects. Interestingly, the growth rates of two non-motile mutants △0368 and △0368△0595 were significantly greater than the wild-type, which may be associated with up-regulation of genes encoding cytochromes, enhancing bacterial proliferation. Moreover, the △0595 mutant was significantly more invasive in Caco-2 cells as shown by bacterial enumeration assays, and the expression of lipopolysaccharide (LPS) core synthesis-related genes (rfaB, rfaI, rfaQ, rfaY, rfaK, rfaZ) was down-regulated only in the △0368△0595 mutant. In addition, this study also speculated that these two genes might be contributing to serotype conversion for Salmonella C1 serogroup based on their apparent roles in biosynthesis of LPS and the flagella. CONCLUSION: A combination of biological and transcriptomic (RNA-Seq) analyses has shown that the SC0368 and SC0595 genes are involved in biosynthesis of flagella and complete LPS, as well as in bacterial growth and virulence. Such information will aid to revealing the role of these specific genes in bacterial physiology and evolution within the serogroup C1.


Assuntos
Flagelos , Salmonella , Ásia , Proteínas de Bactérias/genética , Células CACO-2 , Flagelos/genética , Humanos , Sorogrupo
5.
Molecules ; 26(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206459

RESUMO

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson's disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).


Assuntos
Proteínas de Bactérias , Enzimas Imobilizadas , Halomonas , Levodopa/biossíntese , Microrganismos Geneticamente Modificados , Monofenol Mono-Oxigenase , Nanopartículas , Poli-Hidroxialcanoatos , Verrucomicrobia/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Enzimas Imobilizadas/biossíntese , Enzimas Imobilizadas/genética , Halomonas/enzimologia , Halomonas/genética , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética , Monofenol Mono-Oxigenase/biossíntese , Monofenol Mono-Oxigenase/genética , Poli-Hidroxialcanoatos/biossíntese , Poli-Hidroxialcanoatos/genética , Verrucomicrobia/enzimologia
6.
Front Cell Infect Microbiol ; 11: 666030, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235092

RESUMO

Background: Increasing use of colistin has led to the world-wide emergence of mobile colistin resistant gene (mcr). The present study aimed to identify and characterise mcr and other drug-resistant genes in colistin resistant Klebsiella pneumoniae clinical isolates. Methods: Twenty-two colistin resistant K. pneumoniae were analysed for mcr and other drug-resistant genes, efflux pumps, and virulence genes, and for their biofilm forming ability. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed for all mcr-1 positive isolates. S1-PFGE and Southern hybridisation were performed for localisation of mcr-1 and bla NDM. Results: Nineteen colistin resistant K. pneumoniae harboured mcr-1 and 3 had mgrB disruption. All isolates harboured bla OXA-48-type and ESBL genes; eight strains (five with mcr-1 and three with mgrB disruption) co-harboured bla NDM. Efflux pumps genes AcrAB and mdtK were detected in all 22 and tol-C in 21 isolates. Virulence-related genes entB and irp-1 were detected in all 22, mrkD in 20, and fimH-1 in 18 isolates; 11 isolates were strong biofilm producers. PFGE clustered mcr-1 positive isolates into eight groups based on ≥90% similarity; MLST revealed diverse sequence types, predominant being ST-15 (n = 4) and ST-16 (n = 4). Both mcr-1 and bla NDM were localised on plasmid and chromosome; mcr-1 was present on IncFII type and bla NDM on IncFIB and IncA/C type plasmids. Conclusions: Colistin resistance in K. pneumoniae was predominantly mediated by mcr-1. Co-existence of colistin, carbapenem, and other drug-resistant genes along with efflux pumps indicates towards enormous genomic plasticity in K. pneumoniae with ability to emerge as super-spreader of drug-resistance.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Humanos , Índia , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Plasmídeos , beta-Lactamases/genética
7.
Front Cell Infect Microbiol ; 11: 679982, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235094

RESUMO

Sulfate Transport Anti-Sigma antagonist domains (Pfam01740) are found in all branches of life, from eubacteria to mammals, as a conserved fold encoded by highly divergent amino acid sequences. These domains are present as part of larger SLC26/SulP anion transporters, where the STAS domain is associated with transmembrane anchoring of the larger multidomain protein. Here, we focus on STAS Domain only Proteins (SDoPs) in eubacteria, initially described as part of the Bacillus subtilis Regulation of Sigma B (RSB) regulatory system. Since their description in B. subtilis, SDoPs have been described to be involved in the regulation of sigma factors, through partner-switching mechanisms in various bacteria such as: Mycobacterium. tuberculosis, Listeria. monocytogenes, Vibrio. fischeri, Bordetella bronchiseptica, among others. In addition to playing a canonical role in partner-switching with an anti-sigma factor to affect the availability of a sigma factor, several eubacterial SDoPs show additional regulatory roles compared to the original RSB system of B. subtilis. This is of great interest as these proteins are highly conserved, and often involved in altering gene expression in response to changes in environmental conditions. For many of the bacteria we will examine in this review, the ability to sense environmental changes and alter gene expression accordingly is critical for survival and colonization of susceptible hosts.


Assuntos
Proteínas de Transporte de Ânions , Genes Bacterianos , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Imidazóis , Estrutura Terciária de Proteína , Fator sigma/genética
8.
BMC Genomics ; 22(1): 530, 2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34247587

RESUMO

BACKGROUND: Acinetobacter baumannii is a common nosocomial pathogen that poses a huge threat to global health. Owing to the severity of A. baumannii infections, it became necessary to investigate the epidemiological characteristics of A. baumannii in Chinese hospitals and find the reasons for the high antibiotic resistance rate and mortality. This study aimed to investigate the epidemiologic and genetic characteristics of A. baumannii isolated from patients with hospital acquired pneumonia (HAP), bloodstream infection (BSI) and urinary tract infection (UTI) in China and uncover potential mechanisms for multi-drug resistance and virulence characteristics of A. baumannii isolates. RESULTS: All isolates were classified into two primary clades in core gene-based phylogenetic relationship. Clonal complex 208 (CC208) mainly consisted of ST195 (32 %) and ST208 (24.6 %). CC208 and non-CC208 isolates had carbapenem resistance rates of 96.2 and 9.1 %, respectively. Core genes were enriched in 'Amino acid transport and metabolism', 'Translation', 'Energy production and conversion', 'Transcription', 'Inorganic ion transport and metabolism' and 'Cell wall/membrane/envelope synthesis'. Most isolates possessed virulence factors related to polysaccharide biosynthesis, capsular polysaccharide synthesis and motility. Eleven isolates belong to ST369 or ST191 (oxford scheme) all had the virulence factor cap8E and it had a higher positive rate in UTI (35.3 %) than in BSI (18.9 %) and HAP (12.9 %). ABGRI1 antibiotic resistance islands were responsible for streptomycin, tetracycline and sulfonate resistance. The blaOXA-23 gene was the most probable cause for carbapenem resistance, although the blaOXA-66 gene with nonsynonymous SNPs (F82L, I129L) was not. CONCLUSIONS: A. baumannii is a genomically variable pathogen that has the potential to cause a range of infectious diseases. There is high proportion of carbapenem resistance in isolates from all three infection sites (HAP, BSI and UTI), which can be attributed to the blaOXA-23 gene. CC208 is the predominant clone in blaOXA-23-carrying A. baumannii that should be monitored. Virulence factors involving bacteria motility and polysaccharide biosynthesis which are widespread in clinical A. baumannii strains deserve our attention.


Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Doenças Transmissíveis , Infecções por Acinetobacter/epidemiologia , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , China/epidemiologia , Farmacorresistência Bacteriana Múltipla , Genômica , Humanos , Testes de Sensibilidade Microbiana , Filogenia , beta-Lactamases/genética
9.
J Med Microbiol ; 70(7)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34232118

RESUMO

Introduction. Carbapenem resistant Enterobacterales (CRE) are one of the leading causes of systemic and nosocomial infections and are multidrug-resistant organisms producing different carbapenemases. There are many genotypic and phenotypic methods for detecting the carbapenemases; however, there is a limitation for each. Modified carbapenem inactivation method (mCIM) assay is a recent phenotypic method which has been published by the Clinical and Laboratory Standards Institute.Hypothesis / Gap Statement. mCIM assay could provide a reliable method for the detection of carbapenemases in CRE.Aim. Evaluation of the mCIM assay performance for the detection of carbapenemases in Enterobacterales and the identification of the common carbapenemase genes at Eastern Province of Saudi Arabia and Kingdom of Bahrain.Methodology. A collection of 197 non-duplicate carbapenem resistant Enterobacterales clinical isolates, were evaluated with the mCIM test comparing its performance to multiplex PCR. The minimum inhibitory concentration susceptibility testing was done by the Etest method for imipenem, meropenem, and ertapenem.Results. The sensitivity of the mCIM assay was 94 % (95 % CI, (89.3-97.1)). In Saudi Arabia and Bahrain, OXA-48 was the most prevalent carbapenemase gene followed by NDM. Coexistence of multiple carbapenemase genes is reported in eleven cases.Conclusion. These findings indicate that the mCIM test is a reliable and simple assay for detecting the activity of carbapenemase in Enterobacterales, especially in resource-limited laboratories.


Assuntos
Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Carbapenêmicos/farmacologia , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/microbiologia , Testes de Sensibilidade Microbiana/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Barein , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Carbapenêmicos/uso terapêutico , Infecções por Enterobacteriaceae/tratamento farmacológico , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana/normas , Pessoa de Meia-Idade , Oriente Médio , Arábia Saudita , Sensibilidade e Especificidade , Adulto Jovem , beta-Lactamases/genética
10.
J Med Microbiol ; 70(7)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34236301

RESUMO

Introduction. Tigecycline is currently acknowledged to be one of the most effective antibiotics against infections caused by Mycobacteroides abscessus.Gap statement. The genetic determinants of tigecycline resistance in M. abscessus are not well understood.Aim. In this study, we characterized a tigecycline-resistant M. abscessus mutant, designated CL7, to identify the potential resistance mechanism.Methodology. CL7 was characterized using antimicrobial susceptibility testing, whole-genome sequencing, PCR and RT-qPCR. For biological verification, gene overexpression assays were carried out.Results. Whole-genome sequencing and the subsequent gene overexpression assays showed that CL7 harboured a stop-gain mutation in MAB_3543 c, which may be responsible for the tigecycline resistance phenotype. This gene encodes an orthologue of SigH, which is involved in the positive regulation of physiological stress response and is negatively regulated by the RshA anti-sigma factor in Mycobacterium tuberculosis. We hypothesized that the MAB_3543 c mutation may disrupt the interaction between SigH and RshA (MAB_3542 c). RT-qPCR analyses revealed the upregulation of MAB_3543 c and other key stress response genes, which has previously been shown to be a hallmark of SigH-RshA bond disruption and tigecycline resistance.Conclusion. The MAB_3543c mutation may represent a novel determinant of tigecycline resistance in M. abscessus. The findings of this study will hopefully contribute to our knowledge of potential tigecycline resistance mechanisms in M. abscessus, which may lead to better diagnostics and treatment modalities in the future.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/genética , Fator sigma/genética , Tigeciclina/farmacologia , Genoma Bacteriano , Mutação , Sequenciamento Completo do Genoma
11.
Nat Commun ; 12(1): 4223, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244518

RESUMO

The bacterial flagellar MS ring is a transmembrane complex acting as the core of the flagellar motor and template for flagellar assembly. The C ring attached to the MS ring is involved in torque generation and rotation switch, and a large symmetry mismatch between these two rings has been a long puzzle, especially with respect to their role in motor function. Here, using cryoEM structural analysis of the flagellar basal body and the MS ring formed by full-length FliF from Salmonella enterica, we show that the native MS ring is formed by 34 FliF subunits with no symmetry variation. Symmetry analysis of the C ring shows a variation with a peak at 34-fold, suggesting flexibility in C ring assembly. Finally, our data also indicate that FliF subunits assume two different conformations, contributing differentially to the inner and middle parts of the M ring and thus resulting in 23- and 11-fold subsymmetries in the inner and middle M ring, respectively. The internal core of the M ring, formed by 23 subunits, forms a hole of the right size to accommodate the protein export gate.


Assuntos
Proteínas de Bactérias/ultraestrutura , Flagelos/ultraestrutura , Proteínas de Membrana/ultraestrutura , Sistemas de Secreção Tipo III/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Fracionamento Celular , Microscopia Crioeletrônica , Flagelos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/ultraestrutura , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
12.
J Med Microbiol ; 70(7)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34251298

RESUMO

Carbapenem-resistant Gram-negative bacteria (CR-GNB) are a major source of nosocomial infections worldwide. In this study, the ability of a loop-mediated isothermal amplification (LAMP)-based method (Isoplex CRE-ART) to rapidly detect carbapenemase-encoding genes bla OXA-48-like, bla OXA-23-like, bla OXA-24-like, bla KPC, bla VIM, bla NDM and bla IMP in 231 carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii isolates was investigated. The accuracy of the LAMP test was compared to results of molecular isolate characterization using a Laboratory Developed Test multiplex carbapenemase PCR assay. The LAMP test correctly identified the presence of on-panel carbapenemases with a sensitivity of 99.16 % [95 % confidence interval (CI): 95.39-99.96 %] and a specificity of 98.21 % (95 % CI: 93.72-99.68 %) in 60 min. Our findings suggest that the Isoplex CRE-ART assay is able to rapidly identify carbapenemase genes in CR-GNB and improves options for pathogen characterization in the context of clinical microbiological and infection control diagnostics.


Assuntos
Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Bactérias Gram-Negativas/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Genes Bacterianos
13.
BMC Genomics ; 22(1): 550, 2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34275445

RESUMO

BACKGROUND: Fibrillar adhesins are long multidomain proteins that form filamentous structures at the cell surface of bacteria. They are an important yet understudied class of proteins composed of adhesive and stalk domains that mediate interactions of bacteria with their environment. This study aims to characterize fibrillar adhesins in a wide range of bacterial phyla and to identify new fibrillar adhesin-like proteins to improve our understanding of host-bacteria interactions. RESULTS: Through careful literature and computational searches, we identified 82 stalk and 27 adhesive domain families in fibrillar adhesins. Based on the presence of these domains in the UniProt Reference Proteomes database, we identified and analysed 3,542 fibrillar adhesin-like proteins across species of the most common bacterial phyla. We further enumerate the adhesive and stalk domain combinations found in nature and demonstrate that fibrillar adhesins have complex and variable domain architectures, which differ across species. By analysing the domain architecture of fibrillar adhesins, we show that in Gram positive bacteria, adhesive domains are mostly positioned at the N-terminus and cell surface anchors at the C-terminus of the protein, while their positions are more variable in Gram negative bacteria. We provide an open repository of fibrillar adhesin-like proteins and domains to enable further studies of this class of bacterial surface proteins. CONCLUSION: This study provides a domain-based characterization of fibrillar adhesins and demonstrates that they are widely found in species across the main bacterial phyla. We have discovered numerous novel fibrillar adhesins and improved our understanding of pathogenic adhesion and invasion mechanisms.


Assuntos
Adesinas Bacterianas , Proteínas de Bactérias , Adesinas Bacterianas/genética , Bactérias/genética , Aderência Bacteriana , Proteínas de Bactérias/genética , Bactérias Gram-Positivas , Proteínas de Membrana
14.
Nat Commun ; 12(1): 3388, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099676

RESUMO

Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.


Assuntos
Proteínas de Bactérias/efeitos da radiação , Diabetes Mellitus Tipo 2/terapia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Optogenética/métodos , Transativadores/efeitos da radiação , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia Celular , Diabetes Mellitus Tipo 2/genética , Feminino , Engenharia Genética , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células HEK293 , Humanos , Luz , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Obesos , Optogenética/instrumentação , Fotopletismografia/instrumentação , Domínios Proteicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Thermus thermophilus/genética , Transativadores/genética , Transativadores/metabolismo , Transgenes , Dispositivos Eletrônicos Vestíveis
15.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070642

RESUMO

Urate oxidase initiates the uric acid degradation pathways and is extensively used for protein drug development for gout therapy and serum uric acid diagnosis. We first present the biochemical and structural elucidation of a urate oxidase from the extremophile microorganism Deinococcus radiodurans (DrUox). From enzyme characterization, DrUox showed optimal catalytic ability at 30 °C and pH 9.0 with high stability under physiological conditions. Only the Mg2+ ion moderately elevated its activity, which indicates the characteristic of the cofactor-free urate oxidase family. Of note, DrUox is thermostable in mesophilic conditions. It retains almost 100% activity when incubated at 25 °C and 37 °C for 24 h. In this study, we characterized a thermostable urate oxidase, DrUox with high catalytic efficiency and thermal stability, which strengthens its potential for medical applications.


Assuntos
Proteínas de Bactérias , Deinococcus , Gota/tratamento farmacológico , Hiperuricemia/tratamento farmacológico , Urato Oxidase , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/uso terapêutico , Deinococcus/enzimologia , Deinococcus/genética , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Urato Oxidase/química , Urato Oxidase/genética , Urato Oxidase/uso terapêutico
16.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073255

RESUMO

Whole genome sequences of two Acinetobacter baumannii clinical isolates, 48-1789 and MAR24, revealed that they carry the KL106 and KL112 capsular polysaccharide (CPS) biosynthesis gene clusters, respectively, at the chromosomal K locus. The KL106 and KL112 gene clusters are related to the previously described KL11 and KL83 gene clusters, sharing genes for the synthesis of l-rhamnose (l-Rhap) and 6-deoxy-l-talose (l-6dTalp). CPS material isolated from 48-1789 and MAR24 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The structures of K106 and K112 oligosaccharide repeats (K units) l-6dTalp-(1→3)-D-GlcpNAc tetrasaccharide fragment share the responsible genes in the respective gene clusters. The K106 and K83 CPSs also have the same linkage between K units. The KL112 cluster includes an additional glycosyltransferase gene, Gtr183, and the K112 unit includes α l-Rhap side chain that is not found in the K106 structure. K112 further differs in the linkage between K units formed by the Wzy polymerase, and a different wzy gene is found in KL112. However, though both KL106 and KL112 share the atr8 acetyltransferase gene with KL83, only K83 is acetylated.


Assuntos
Acinetobacter baumannii , Desoxiaçúcares , Hexoses , Polissacarídeos Bacterianos , Acinetobacter baumannii/química , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desoxiaçúcares/química , Desoxiaçúcares/genética , Desoxiaçúcares/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Hexoses/química , Hexoses/genética , Hexoses/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Especificidade da Espécie
17.
Biosens Bioelectron ; 189: 113382, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34087725

RESUMO

The sensitive and accurate detection of rare mutations has profound clinical implications; however, current methods require expensive instrumentation and are laborious and time-consuming. Thus, there is a need for a probe-based alternative that can effectively discriminate single-base mutations. Recently, several groups have shown the potential of the CRISPR/Cas12a system for sensitive and selective DNA detection but its application on single nucleotide variants (SNVs) detection is limited by the requirement of a protospacer adjacent motif (PAM) directly upstream to the SNV site and the amplification of non-specific signals due to the rapid and indiscriminate trans cleavage activity. Here, we report an ultra-selective Cas12a-based system that eliminates the need for the PAM sequence in the target with lower noise from the wild-type sequence by using its non-canonical double-stranded trans-cleavage activity. We show that our strategy can allow the detection of an EGFR gene mutation in sub-femtomolar concentrations up to 0.1% variant allele frequency using either fluorescence or electrochemical readouts.


Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , Alelos , Proteínas de Bactérias/genética , DNA/genética
18.
Nat Commun ; 12(1): 3748, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145250

RESUMO

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.


Assuntos
Antígenos/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/patogenicidade , Enterocolite Pseudomembranosa/patologia , Proteoglicanas/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sítios de Ligação/fisiologia , Anticorpos Amplamente Neutralizantes/farmacologia , Microscopia Crioeletrônica , Enterocolite Pseudomembranosa/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Proteoglicanas/genética
19.
Nat Commun ; 12(1): 3690, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140468

RESUMO

CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.


Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroidetes/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/genética , Bacteroidetes/enzimologia , Biologia Computacional , DNA Primase/genética , Primers do DNA/biossíntese , DNA Polimerase Dirigida por DNA/genética , Dimerização , Escherichia coli/metabolismo , Expressão Gênica , Mutação , Filogenia , Células Procarióticas/metabolismo , Proteínas Recombinantes , Ribonucleotídeos/metabolismo
20.
Res Vet Sci ; 138: 1-10, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34087563

RESUMO

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.


Assuntos
Proteínas de Bactérias/genética , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/metabolismo , Bovinos , Feminino , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Virulência/genética , Fatores de Virulência/metabolismo
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