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1.
Front Cell Infect Microbiol ; 11: 641920, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33816347

RESUMO

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen which causes chronic infections in immunocompromised patients and leads to high mortality rate. It is identified as a common coinfecting pathogen in COVID-19 patients causing exacerbation of illness. In our hospital, P. aeruginosa is one of the top coinfecting bacteria identified among COVID-19 patients. We collected a strong biofilm-forming P. aeruginosa strain displaying small colony variant morphology from a severe COVID-19 patient. Genomic and transcriptomic sequencing analyses were performed with phenotypic validation to investigate its adaptation in SARS-CoV-2 infected environment. Genomic characterization predicted specific genomic islands highly associated with virulence, transcriptional regulation, and DNA restriction-modification systems. Epigenetic analysis revealed a specific N6-methyl adenine (m6A) methylating pattern including methylation of alginate, flagellar and quorum sensing associated genes. Differential gene expression analysis indicated that this isolate formed excessive biofilm by reducing flagellar formation (7.4 to 1,624.1 folds) and overproducing extracellular matrix components including CdrA (4.4 folds), alginate (5.2 to 29.1 folds) and Pel (4.8-5.5 folds). In summary, we demonstrated that P. aeuginosa clinical isolates with novel epigenetic markers could form excessive biofilm, which might enhance its antibiotic resistance and in vivo colonization in COVID-19 patients.


Assuntos
Adaptação Fisiológica/fisiologia , Coinfecção/complicações , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Alginatos , Bactérias , Biofilmes/crescimento & desenvolvimento , Metilação de DNA , Epigenômica , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Humanos , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Percepção de Quorum/genética , Transcriptoma , Virulência
2.
Anal Chem ; 93(9): 4149-4153, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33635624

RESUMO

Loop-mediated isothermal amplification (LAMP) holds great potential for point-of-care (POC) diagnostics due to its speed and sensitivity. However, differentiation between spurious amplification and amplification of the target sequence is a challenge. Herein, we develop the use of molecular beacon (MB) probes for the sequence-specific detection of LAMP on commercially available lateral flow immunoassay (LFIA) strips. The detection of three unique DNA sequences, including ORF1a from SARS-CoV-2, is demonstrated. In addition, the method is capable of detecting clinically relevant single-nucleotide polymorphisms (BRAF V600E). For all sequences tested, the LFIA method offers similar sensitivity to fluorescence detection using a qPCR instrument. We also demonstrate the coupling of the method with solid-phase microextraction to enable isolation and detection of the target sequences from human plasma, pond water, and artificial saliva. Lastly, a 3D printed device is designed and implemented to prevent contamination caused by opening the reaction containers after LAMP.


Assuntos
Adesinas Bacterianas/genética , Imunoensaio , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Virais/genética , Humanos , Poliproteínas/genética , Fitas Reagentes/química , Sensibilidade e Especificidade , Análise de Sequência de DNA , Vibrio cholerae/genética
3.
Int J Food Microbiol ; 343: 109091, 2021 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-33639477

RESUMO

This study investigated the antimicrobial resistance determinants, virulence factors and identified serovars in 37 Salmonella enterica strains isolated from human stool and contaminated foods linked to outbreaks that occurred in Brazil over 7 years using whole genome sequencing (WGS). Phylogenetic analysis of selected serovars (S. Typhimurium, S. Infantis, S. London, and S. Johannesburg) was performed. Ten distinct serovars were identified and, 51% of the tested strains (n = 19) showed disagreement with the previous conventional serotyping. The antimicrobial resistance (AMR) determinants or plasmids varied among the strains. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac (6')-laa, aph (3″)-lb, aph (6)-ld, aadA1 and aadA2], sulfonamides (sul1), trimethoprin (dfrA8), fosfomycin (fosA7) and tetracyclines (tetA, tetB, tetC), as well as point mutations in parC (T57S) and gyrA (S83F). Plasmidome showed the presence of IncHI2, IncHI2A, IncFIB (S), IncFII (S), IncI1 and p0111 plasmids. Eight Salmonella pathogenicity islands and up to 102 stress and/or virulence genes were identified in the evaluated genomes. Virulence genes of K88 fimbrial adhesin were first reported in S. enterica (S. Pomona, S. Bredeney and S. Mbandaka strains). pilW gene was first identified in S. Pomona. Phylogenetic analysis showed that some serovars circulated in Brazil for decades, primarily within the poultry production chain. Findings highlighted the virulence and AMR determinants in strains that may lead to recurring food outbreaks.


Assuntos
Farmacorresistência Bacteriana/genética , Doenças Transmitidas por Alimentos/microbiologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética , Fatores de Virulência/genética , Adesinas Bacterianas/genética , Animais , Antibacterianos/farmacologia , Brasil , Fezes/microbiologia , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Genoma Bacteriano/genética , Ilhas Genômicas/genética , Humanos , Filogenia , Plasmídeos/genética , Aves Domésticas/microbiologia , Doenças das Aves Domésticas/microbiologia , Salmonella enterica/isolamento & purificação , Salmonella enterica/patogenicidade , Sorotipagem , Virulência/genética , Sequenciamento Completo do Genoma
4.
J Biosci Bioeng ; 131(4): 381-389, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33495047

RESUMO

Initial work to generate physically robust biofilms for biocatalytic applications revealed that Escherichia coli K-12 can form a floating biofilm at the air-liquid interface, commonly referred to as a pellicle. Unlike other species where pellicle formation is well-characterised, such as Bacillus subtilis, there are few reports of E. coli K-12 pellicles in the literature. In order to study pellicle formation, a growth model was developed and pellicle formation was monitored over time. Mechanical forces, both motility and shaking, were shown to have effects on pellicle formation and development. The role and regulation of curli, an amyloid protein adhesin critical in E. coli K-12 biofilm formation, was studied by using promoter-green fluorescent protein reporters; flow cytometry and confocal laser scanning microscopy were used to monitor curli expression over time and in different locations. Curli were found to be not only crucial for pellicle formation, but also heterogeneously expressed within the pellicle. The components of the extracellular polymeric substances (EPS) in pellicles were analysed by confocal microscopy using lectins, revealing distinct pellicle morphology on the air-facing and medium-facing sides, and spatially- and temporally-regulated generation of the EPS components poly-N-acetyl glucosamine and colanic acid. We discuss the difference between pellicles formed by E. coli K-12, pathogenic E. coli strains and other species, and the relationship between E. coli K-12 pellicles and solid surface-attached biofilms.


Assuntos
Adesinas Bacterianas/metabolismo , Escherichia coli K12/metabolismo , Adesinas Bacterianas/genética , Biofilmes , Escherichia coli K12/genética , Polissacarídeos/metabolismo
5.
Appl Environ Microbiol ; 87(7)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33483309

RESUMO

Prodiginines are a family of red-pigmented secondary metabolites with multiple biological activities. The biosynthesis of prodiginines is affected by various physiological and environmental factors. Thus, prodiginine biosynthesis regulation is highly complex and multifaceted. Although the regulatory mechanism for prodiginine biosynthesis has been extensively studied in Serratia and Streptomyces species, little is known about that in the marine betaproteobacterium Pseudoalteromonas In this study, we report that stringent starvation protein A (SspA), an RNA polymerase-associated regulatory protein, is required for the biosynthesis of prodiginine in Pseudoalteromonas sp. strain R3. The strain lacking sspA (ΔsspA) fails to produce prodiginine, which resulted from the downregulation of the prodiginine biosynthetic gene (pig) cluster. The effect of SspA on prodiginine biosynthesis is independent of histone-like nucleoid structuring protein (H-NS) and RpoS (σS). Further analysis demonstrates that the ΔsspA strain has a significant decrease in the transcription of the siderophore biosynthesis gene (pvd) cluster, leading to the inhibition of siderophore production and iron uptake. The ΔsspA strain regains the ability to synthesize prodiginine by cocultivation with siderophore producers or the addition of iron. Therefore, we conclude that SspA-regulated prodiginine biosynthesis is due to decreased siderophore levels and iron deficiency. We further show that the iron homeostasis master regulator Fur is also essential for pig transcription and prodiginine biosynthesis. Overall, our results suggest that SspA indirectly regulates the biosynthesis of prodiginine, which is mediated by the siderophore-dependent iron uptake pathway.IMPORTANCE The red-pigmented prodiginines are attracting increasing interest due to their broad biological activities. As with many secondary metabolites, the biosynthesis of prodiginines is regulated by both environmental and physiological factors. At present, studies on the regulation of prodiginine biosynthesis are mainly restricted to Serratia and Streptomyces species. This work focused on the regulatory mechanism of prodiginine biosynthesis in Pseudoalteromonas sp. R3. We found that stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis via affecting the siderophore-dependent iron uptake pathway. The connections among SspA, iron homeostasis, and prodiginine biosynthesis were investigated. These findings uncover a novel regulatory mechanism for prodigiosin biosynthesis.


Assuntos
Adesinas Bacterianas/genética , Prodigiosina/análogos & derivados , Pseudoalteromonas/genética , Sideróforos/metabolismo , Adesinas Bacterianas/metabolismo , Ferro/metabolismo , Prodigiosina/biossíntese , Pseudoalteromonas/metabolismo
6.
PLoS Pathog ; 17(1): e1009222, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465168

RESUMO

Bacterial binding to platelets is a key step in the development of infective endocarditis (IE). Sialic acid, a common terminal carbohydrate on host glycans, is the major receptor for streptococci on platelets. So far, all defined interactions between streptococci and sialic acid on platelets are mediated by serine-rich repeat proteins (SRRPs). However, we identified Streptococcus oralis subsp. oralis IE-isolates that bind sialic acid but lack SRRPs. In addition to binding sialic acid, some SRRP- isolates also bind the cryptic receptor ß-1,4-linked galactose through a yet unknown mechanism. Using comparative genomics, we identified a novel sialic acid-binding adhesin, here named AsaA (associated with sialic acid adhesion A), present in IE-isolates lacking SRRPs. We demonstrated that S. oralis subsp. oralis AsaA is required for binding to platelets in a sialic acid-dependent manner. AsaA comprises a non-repeat region (NRR), consisting of a FIVAR/CBM and two Siglec-like and Unique domains, followed by 31 DUF1542 domains. When recombinantly expressed, Siglec-like and Unique domains competitively inhibited binding of S. oralis subsp. oralis and directly interacted with sialic acid on platelets. We further demonstrated that AsaA impacts the pathogenesis of S. oralis subsp. oralis in a rabbit model of IE. Additionally, we found AsaA orthologues in other IE-causing species and demonstrated that the NRR of AsaA from Gemella haemolysans blocked binding of S. oralis subsp. oralis, suggesting that AsaA contributes to the pathogenesis of multiple IE-causing species. Finally, our findings provide evidence that sialic acid is a key factor for bacterial-platelets interactions in a broader range of species than previously appreciated, highlighting its potential as a therapeutic target.


Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Endocardite Bacteriana/patologia , Ácido N-Acetilneuramínico/metabolismo , Streptococcus/metabolismo , Adesinas Bacterianas/genética , Animais , Proteínas de Bactérias/genética , Endocardite Bacteriana/metabolismo , Endocardite Bacteriana/microbiologia , Masculino , Coelhos , Streptococcus/classificação , Streptococcus/genética , Streptococcus/isolamento & purificação
7.
Methods Mol Biol ; 2210: 113-121, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32815132

RESUMO

The type IX secretion system (T9SS) is the most recently discovered secretion system in the gram-negative bacteria and is specific to the Bacteroidetes phylum. It is comprised of at least 19 proteins, which together allows for the secretion and cell surface attachment of a specific group of proteins (T9SS substrates), that harbor a signal sequence at the C-terminus. Here we describe the structural characterization of the PorK, PorN and PorG components of the Porphyromonas gingivalis T9SS using electron microscopy and cross-linking mass spectrometry.


Assuntos
Sistemas de Secreção Bacterianos/metabolismo , Porphyromonas gingivalis/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Espectrometria de Massas/métodos , Microscopia Eletrônica/métodos , Porphyromonas gingivalis/genética , Sinais Direcionadores de Proteínas/genética
8.
Int J Food Microbiol ; 337: 108929, 2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33157488

RESUMO

Campylobacter jejuni is the leading cause of bacterial food poisoning worldwide. Chickens are considered to be one of the major reservoirs of Campylobacter infection in humans due to colonization of their intestinal tract. When the chickens are slaughtered and processed, the entire skin of the carcass becomes contaminated with campylobacters. We observed that the number of C. jejuni attached to chicken skin was reduced significantly after treatment of the skin with sodium hydroxide followed by washing with PBS, implying that adhesion factors involved in binding to C. jejuni may exist on skin. Such potential binding-related proteins present in alkaline extracts of the skin surface were detected by a two-dimensional overlay assay and identified by liquid chromatography mass spectrometry (LC-MS). Chicken serum albumin (CSA) was identified as a major protein in these alkaline extracts and confirmed by ELISA to bind specifically to C. jejuni. Moreover, using the same approach, flagellar hook protein E (FlgE) and major outer membrane protein (MOMP) in C. jejuni were identified as bacterial adhesins that bound to the CSA. The ability to bind CSA was also confirmed using recombinant FlgE and MOMP of C. jejuni expressed in Escherichia coli. The present findings suggest that adhesins expressed on C. jejuni cells may bind specifically via proteins present on the skin, as well as by physical attachment.


Assuntos
Campylobacter jejuni/fisiologia , Galinhas/microbiologia , Pele/microbiologia , Adesinas Bacterianas/análise , Adesinas Bacterianas/genética , Animais , Infecções por Campylobacter/microbiologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética
9.
Nat Chem Biol ; 17(3): 351-359, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33349707

RESUMO

Living organisms have evolved sophisticated cell-mediated biomineralization mechanisms to build structurally ordered, environmentally adaptive composite materials. Despite advances in biomimetic mineralization research, it remains difficult to produce mineralized composites that integrate the structural features and 'living' attributes of their natural counterparts. Here, inspired by natural graded materials, we developed living patterned and gradient composites by coupling light-inducible bacterial biofilm formation with biomimetic hydroxyapatite (HA) mineralization. We showed that both the location and the degree of mineralization could be regulated by tailoring functional biofilm growth with spatial and biomass density control. The cells in the composites remained viable and could sense and respond to environmental signals. Additionally, the composites exhibited a maximum 15-fold increase in Young's modulus after mineralization and could be applied to repair damage in a spatially controlled manner. Beyond insights into the mechanism of formation of natural graded composites, our study provides a viable means of fabricating living composites with dynamic responsiveness and environmental adaptability.


Assuntos
Adesinas Bacterianas/genética , Biofilmes/efeitos da radiação , Durapatita/química , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos da radiação , Proteínas/genética , Adesinas Bacterianas/metabolismo , Adesinas Bacterianas/efeitos da radiação , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/efeitos da radiação , Biofilmes/crescimento & desenvolvimento , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Materiais Biomiméticos/efeitos da radiação , Biomineralização/efeitos da radiação , Engenharia Celular/métodos , Relação Dose-Resposta à Radiação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/efeitos da radiação , Expressão Gênica , Luz , Mytilus , Proteínas/metabolismo , Proteínas/efeitos da radiação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação
10.
PLoS One ; 15(12): e0244713, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382795

RESUMO

The prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) was determined by evaluating its presence in faecal samples from 155 heifers, and 254 dairy cows in 21 farms at North of Portugal sampled between December 2017 and June 2019. The prevalence of STEC in heifers (45%) was significantly higher than in lactating cows (16%) (p<0.05, Fisher exact test statistic value is <0.00001). A total of 133 STEC were isolated, 24 (13.8%) carried Shiga-toxin 1 (stx1) genes, 69 (39.7%) carried Shiga-toxin 2 (stx2) genes, and 40 (23%) carried both stx1 and stx2. Intimin (eae) virulence gene was detected in 29 (21.8%) of the isolates. STEC isolates belonged to 72 different O:H serotypes, comprising 40 O serogroups and 23 H types. The most frequent serotypes were O29:H12 (15%) and O113:H21 (5.2%), found in a large number of farms. Two isolates belonged to the highly virulent serotypes associated with human disease O157:H7 and O26:H11. Many other bovine STEC serotypes founded in this work belonged to serotypes previously described as pathogenic to humans. Thus, this study highlights the need for control strategies that can reduce STEC prevalence at the farm level and, thus, prevent food and environmental contamination.


Assuntos
Adesinas Bacterianas/genética , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Bovinos , Infecções por Escherichia coli/microbiologia , Feminino , Portugal , Sorogrupo , Sorotipagem , Escherichia coli Shiga Toxigênica/genética , Virulência
11.
PLoS One ; 15(10): e0240101, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33007036

RESUMO

Bacterial phytopathogen Xylella fastidiosa specifically colonizes the plant vascular tissue through a complex process of cell adhesion, biofilm formation, and dispersive movement. Adaptation to the chemical environment of the xylem is essential for bacterial growth and progression of infection. Grapevine xylem sap contains a range of plant secondary metabolites such as phenolics, which fluctuate in response to pathogen infection and plant physiological state. Phenolic compounds are often involved in host-pathogen interactions and influence infection dynamics through signaling activity, antimicrobial properties, and alteration of bacterial phenotypes. The effect of biologically relevant concentrations of phenolic compounds coumaric acid, gallic acid, epicatechin, and resveratrol on growth of X. fastidiosa was assessed in vitro. None of these compounds inhibited bacterial growth, but epicatechin and gallic acid reduced cell-surface adhesion. Cell-cell aggregation decreased with resveratrol treatment, but the other phenolic compounds tested had minimal effect on aggregation. Expression of attachment (xadA) and aggregation (fimA) related genes were altered by presence of the phenolic compounds, consistent with observed phenotypes. All four of the phenolic compounds bound to purified X. fastidiosa lipopolysaccharide (LPS), a major cell-surface component. Information regarding the impact of chemical environment on pathogen colonization in plants is important for understanding the infection process and factors associated with host susceptibility.


Assuntos
Aderência Bacteriana/efeitos dos fármacos , Membrana Celular/metabolismo , Lipopolissacarídeos/metabolismo , Fenóis/farmacologia , Vitis/química , Xylella/citologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Aderência Bacteriana/genética , Catequina/farmacologia , Membrana Celular/efeitos dos fármacos , Meios de Cultura/química , Fímbrias Bacterianas/efeitos dos fármacos , Fímbrias Bacterianas/genética , Ácido Gálico/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Resveratrol/farmacologia , Xylella/efeitos dos fármacos , Xylella/genética , Xylella/crescimento & desenvolvimento
12.
Nat Commun ; 11(1): 5431, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33110079

RESUMO

Physical forces have profound effects on cellular behavior, physiology, and disease. Perhaps the most intruiguing and fascinating example is the formation of catch-bonds that strengthen cellular adhesion under shear stresses. Today mannose-binding by the Escherichia coli FimH adhesin remains one of the rare microbial catch-bond thoroughly characterized at the molecular level. Here we provide a quantitative demonstration of a catch-bond in living Gram-positive pathogens using force-clamp spectroscopy. We show that the dock, lock, and latch interaction between staphylococcal surface protein SpsD and fibrinogen is strong, and exhibits an unusual catch-slip transition. The bond lifetime first grows with force, but ultimately decreases to behave as a slip bond beyond a critical force (~1 nN) that is orders of magnitude higher than for previously investigated complexes. This catch-bond, never reported for a staphylococcal adhesin, provides the pathogen with a mechanism to tightly control its adhesive function during colonization and infection.


Assuntos
Adesinas Bacterianas/química , Infecções Estafilocócicas/microbiologia , Staphylococcus/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Fibrinogênio/química , Fibrinogênio/metabolismo , Humanos , Ligação Proteica , Análise Espectral , Infecções Estafilocócicas/metabolismo , Staphylococcus/química , Staphylococcus/genética , Staphylococcus/crescimento & desenvolvimento
13.
PLoS Negl Trop Dis ; 14(9): e0008613, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898134

RESUMO

Although enteroaggregative E. coli (EAEC) has been implicated as a common cause of diarrhea in multiple settings, neither its essential genomic nature nor its role as an enteric pathogen are fully understood. The current definition of this pathotype requires demonstration of cellular adherence; a working molecular definition encompasses E. coli which do not harbor the heat-stable or heat-labile toxins of enterotoxigenic E. coli (ETEC) and harbor the genes aaiC, aggR, and/or aatA. In an effort to improve the definition of this pathotype, we report the most definitive characterization of the pan-genome of EAEC to date, applying comparative genomics and functional characterization on a collection of 97 EAEC strains isolated in the course of a multicenter case-control diarrhea study (Global Enteric Multi-Center Study, GEMS). Genomic analysis revealed that the EAEC strains mapped to all phylogenomic groups of E. coli. Circa 70% of strains harbored one of the five described AAF variants; there were no additional AAF variants identified, and strains that lacked an identifiable AAF generally did not have an otherwise complete AggR regulon. An exception was strains that harbored an ETEC colonization factor (CF) CS22, like AAF a member of the chaperone-usher family of adhesins, but not phylogenetically related to the AAF family. Of all genes scored, sepA yielded the strongest association with diarrhea (P = 0.002) followed by the increased serum survival gene, iss (p = 0.026), and the outer membrane protease gene ompT (p = 0.046). Notably, the EAEC genomes harbored several genes characteristically associated with other E. coli pathotypes. Our data suggest that a molecular definition of EAEC could comprise E. coli strains harboring AggR and a complete AAF(I-V) or CS22 gene cluster. Further, it is possible that strains meeting this definition could be both enteric bacteria and urinary/systemic pathogens.


Assuntos
Aderência Bacteriana/genética , Infecções por Escherichia coli/epidemiologia , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/patogenicidade , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Adesinas Bacterianas/genética , Aderência Bacteriana/fisiologia , Estudos de Casos e Controles , Linhagem Celular , Pré-Escolar , Diarreia/microbiologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Genoma Bacteriano/genética , Genômica , Humanos , Lactente , Recém-Nascido , Transativadores/genética , Virulência/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma
14.
PLoS Pathog ; 16(8): e1008707, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32780778

RESUMO

Proteus mirabilis, a Gram-negative uropathogen, is a major causative agent in catheter-associated urinary tract infections (CAUTI). Mannose-resistant Proteus-like fimbriae (MR/P) are crucially important for P. mirabilis infectivity and are required for biofilm formation and auto-aggregation, as well as for bladder and kidney colonization. Here, the X-ray crystal structure of the MR/P tip adhesin, MrpH, is reported. The structure has a fold not previously described and contains a transition metal center with Zn2+ coordinated by three conserved histidine residues and a ligand. Using biofilm assays, chelation, metal complementation, and site-directed mutagenesis of the three histidines, we show that an intact metal binding site occupied by zinc is essential for MR/P fimbria-mediated biofilm formation, and furthermore, that P. mirabilis biofilm formation is reversible in a zinc-dependent manner. Zinc is also required for MR/P-dependent agglutination of erythrocytes, and mutation of the metal binding site renders P. mirabilis unfit in a mouse model of UTI. The studies presented here provide important clues as to the mechanism of MR/P-mediated biofilm formation and serve as a starting point for identifying the physiological MR/P fimbrial receptor.


Assuntos
Adesinas Bacterianas/metabolismo , Biofilmes , Proteínas de Fímbrias/metabolismo , Proteus mirabilis/metabolismo , Infecções Urinárias/microbiologia , Zinco/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Humanos , Infecções por Proteus/metabolismo , Infecções por Proteus/microbiologia , Proteus mirabilis/química , Proteus mirabilis/genética , Alinhamento de Sequência , Infecções Urinárias/metabolismo , Zinco/química
15.
Arch Microbiol ; 202(9): 2453-2459, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32607723

RESUMO

Determinant genes controlling biofilm formation in a plant commensal bacterium, Pseudomonas protegens Pf-5, were identified by transposon mutagenesis. Comprehensive screening of 7500 transposon-inserted mutants led to the isolation of four mutants exhibiting decreased and five mutants exhibiting increased biofilm formation. Mutations in the genes encoding MFS drug resistance transporter, LapA adhesive protein, RetS sensor histidine kinase/response regulator, and HecA adhesin/hemagglutinin led to decreased biofilm formation, indicating that these genes are necessary for biofilm formation in Pf-5. The mutants exhibiting increased biofilm formation had transposon insertions in the genes coding for an outer membrane protein, a GGDEF domain-containing protein, AraC transcriptional regulator, non-ribosomal peptide synthetase OfaB, and the intergenic region of a DNA-binding protein and the Aer aerotaxis receptor, suggesting that these genes are negative regulators of biofilm formation. Some of these mutants also showed altered swimming and swarming motilities, and a negative correlation between biofilm formation and swarming motility was observed. Thus, sessile-motile lifestyle is regulated by divergent regulatory genes in Pf-5.


Assuntos
Proteínas de Bactérias/genética , Biofilmes , Pseudomonas/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Genes Reguladores , Histidina Quinase/genética , Mutação
16.
PLoS One ; 15(6): e0235115, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32569268

RESUMO

BACKGROUND: Microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) facilitate Staphylococcus aureus adherence to host tissue. We hypothesized that S. aureus isolates from implant-associated infections (IAIs) would differ in MSCRAMM profile and biofilm formation in vitro compared to skin and soft tissue infection (SSTI) isolates. METHODS: Pediatric patients and their isolates were identified retrospectively. IAI and SSTI isolates were matched (1:4). Pulsed field gel electrophoresis was performed to group isolates as USA300 vs. non-USA300. Whole genome sequencing was performed and raw sequence data were interrogated for presence of MSCRAMMs (clfA, clfB, cna, ebh, efb, fnbpA, fnbpB, isdA, isdB, sdrC, sdrD, sdrE), biofilm-associated (icaA,D,B,C), and Panton-Valentine leukocidin (lukSF-PV) genes, accessory gene regulator group, and multilocus sequence types. In vitro biofilm formation was assessed for 47 IAI and 47 SSTI isolates using a microtiter plate assay. Conditional logistic regression was performed for analysis of matched data (STATA11, College Station, TX). RESULTS: Forty-seven IAI and 188 SSTI isolates were studied. IAI isolates were more often methicillin susceptible S. aureus and non-USA300 vs. SSTI isolates [34 (72%) vs. 79 (42%), p = 0.001 and 38 (81%) vs. 57 (30%) p <0.001, respectively]. Greater than 98% of isolates carried clfA, clfB, efb, isdA, isdB, and icaA,D,B,C while cna was more frequently found among IAI vs. SSTI isolates (p = 0.003). Most isolates were strong biofilm producers. CONCLUSIONS: S. aureus IAI isolates were significantly more likely to be MSSA and non-USA300 than SSTI isolates. Carriage of MSCRAMMs and biofilm formation did not differ significantly between isolates. Evaluation of genetic polymorphisms and gene expression profiles are needed to further delineate the role of adhesins in the pathogenesis of IAIs.


Assuntos
Adesinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Genes Bacterianos , Infecções Relacionadas à Prótese/genética , Infecções Relacionadas à Prótese/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Criança , Humanos , Pele/patologia , Infecções dos Tecidos Moles/genética , Infecções dos Tecidos Moles/microbiologia
17.
PLoS Pathog ; 16(5): e1008500, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32401811

RESUMO

Pertussis toxin is the preeminent virulence factor and major protective antigen produced by Bordetella pertussis, the human respiratory pathogen and etiologic agent of whooping cough. Genes for its synthesis and export are encoded by the 12 kb ptx-ptl operon, which is under the control of the pertussis promoter, Pptx. Expression of this operon, like that of all other known protein virulence factors, is regulated by the BvgAS two-component global regulatory system. Although Pptx has been studied for years, characterization of its promoter architecture vis-à-vis BvgA-binding has lagged behind that of other promoters, mainly due to its lower affinity for BvgA~P. Here we take advantage of a mutant BvgA protein (Δ127-129), which enhances ptx transcription in B. pertussis and also demonstrates enhanced binding affinity to Pptx. By using this mutant protein labeled with FeBABE, binding of six head-to-head dimers of BvgA~P was observed, with a spacing of 22 bp, revealing a binding geometry similar to that of other BvgA-activated promoters carrying at least one strong binding site. All of these six BvgA-binding sites lack sequence features associated with strong binding. A genetic analysis indicated the degree to which each contributes to Pptx activity. Thus the weak/medium binding affinity of Pptx revealed in this study explains its lower responsiveness to phosphorylated BvgA, relative to other promoters containing a high affinity binding site, such as that of the fha operon.


Assuntos
Proteínas de Bactérias , Bordetella pertussis , DNA Bacteriano , Toxina Pertussis , Regiões Promotoras Genéticas , Fatores de Transcrição , Transcrição Genética , Adesinas Bacterianas/biossíntese , Adesinas Bacterianas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Toxina Pertussis/biossíntese , Toxina Pertussis/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência de Bordetella/biossíntese , Fatores de Virulência de Bordetella/genética
18.
mBio ; 11(2)2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317318

RESUMO

The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C-DNase X complex mediates the ROS blockade was unknown. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia-induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia-mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation.IMPORTANCE Ehrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies.


Assuntos
Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Basigina/metabolismo , Ehrlichia chaffeensis/fisiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas c-vav/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Animais , Basigina/genética , Humanos , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
19.
Lett Appl Microbiol ; 71(2): 179-186, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32333799

RESUMO

Shiga toxin-producing Escherichia coli is carried in the intestine of ruminant animals, and outbreaks have occurred after contact with ruminant animals or their environment. The presence of STEC virulence genes in the environment was investigated along recreational walking paths in the North West and East Anglia regions of England. In all, 720 boot sock samples from walkers' shoes were collected between April 2013 and July 2014. Multiplex PCR was used to detect E. coli based on the amplification of the uidA gene and investigate STEC-associated virulence genes eaeA, stx1 and stx2. The eaeA virulence gene was detected in 45·5% of the samples, where stx1 and/or stx2 was detected in 12·4% of samples. There was a difference between the two regions sampled, with the North West exhibiting a higher proportion of positive boot socks for stx compared to East Anglia. In univariate analysis, ground conditions, river flow and temperature were associated with positive boot socks. The detection of stx genes in the soil samples suggests that STEC is present in the English countryside and individuals may be at risk for infection after outdoor activities even if there is no direct contact with animals. SIGNIFICANCE AND IMPACT OF THE STUDY: Several outbreaks within the UK have highlighted the danger of contracting Shiga toxin-producing Escherichia coli from contact with areas recently vacated by livestock. This is more likely to occur for STEC infections compared to other zoonotic bacteria given the low infectious dose required. While studies have determined the prevalence of STEC within farms and petting zoos, determining the risk to individuals enjoying recreational outdoor activities that occur near where livestock may be present is less researched. This study describes the prevalence with which stx genes, indicative of STEC bacteria, were found in the environment in the English countryside.


Assuntos
Adesinas Bacterianas/genética , Proteínas de Escherichia coli/genética , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/patogenicidade , Animais , Inglaterra , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Geografia , Humanos , Gado/microbiologia , Reação em Cadeia da Polimerase Multiplex , Escherichia coli Shiga Toxigênica/isolamento & purificação , Sapatos , Virulência/genética , Fatores de Virulência/genética
20.
Int J Mol Sci ; 21(8)2020 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-32290560

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis and hemolytic uremic syndrome. EHEC infection begins with bacterial adherence to the host intestine via lectin-like adhesins that bind to the intestinal wall. However, EHEC-related lectin-glycan interactions (LGIs) remain unknown. Here, we conducted a genome-wide investigation of putative adhesins to construct an LGI network. We performed microarray-based transcriptomic and proteomic analyses with E. coli EDL933. Using PSORTb-based analysis, potential outer-membrane-embedded adhesins were predicted from the annotated genes of 318 strains. Predicted proteins were classified using TMHMM v2.0, SignalP v5.0, and LipoP v1.0. Functional and protein-protein interaction analyses were performed using InterProScan and String databases, respectively. Structural information of lectin candidate proteins was predicted using Iterative Threading ASSEmbly Refinement (I-TASSER) and Spatial Epitope Prediction of Protein Antigens (SEPPA) tools based on 3D structure and B-cell epitopes. Pathway analysis returned 42,227 Gene Ontology terms; we then selected 2585 lectin candidate proteins by multi-omics analysis and performed homology modeling and B-cell epitope analysis. We predicted a total of 24,400 outer-membrane-embedded proteins from the genome of 318 strains and integrated multi-omics information into the genomic information of the proteins. Our integrated multi-omics data will provide a useful resource for the construction of LGI networks of E. coli.


Assuntos
Escherichia coli Êntero-Hemorrágica/genética , Lectinas/genética , Polissacarídeos/genética , Proteoma/genética , Transcriptoma/genética , Adesinas Bacterianas/genética , Aderência Bacteriana/genética , Epitopos de Linfócito B/genética , Proteínas de Escherichia coli/genética , Proteômica/métodos
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