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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.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672911

RESUMO

The Gram-negative bacterium Flavobacterium johnsoniae employs gliding motility to move rapidly over solid surfaces. Gliding involves the movement of the adhesin SprB along the cell surface. F. johnsoniae spreads on nutrient-poor 1% agar-PY2, forming a thin film-like colony. We used electron microscopy and time-lapse fluorescence microscopy to investigate the structure of colonies formed by wild-type (WT) F. johnsoniae and by the sprB mutant (ΔsprB). In both cases, the bacteria were buried in the extracellular polymeric matrix (EPM) covering the top of the colony. In the spreading WT colonies, the EPM included a thick fiber framework and vesicles, revealing the formation of a biofilm, which is probably required for the spreading movement. Specific paths that were followed by bacterial clusters were observed at the leading edge of colonies, and abundant vesicle secretion and subsequent matrix formation were suggested. EPM-free channels were formed in upward biofilm protrusions, probably for cell migration. In the nonspreading ΔsprB colonies, cells were tightly packed in layers and the intercellular space was occupied by less matrix, indicating immature biofilm. This result suggests that SprB is not necessary for biofilm formation. We conclude that F. johnsoniae cells use gliding motility to spread and maturate biofilms.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Flavobacterium/fisiologia , Locomoção/fisiologia , Proteínas de Bactérias/genética , Flavobacterium/genética , Flavobacterium/ultraestrutura , Locomoção/genética , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Mutação , Imagem com Lapso de Tempo/métodos
3.
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
4.
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
5.
J Med Microbiol ; 70(3)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33492206

RESUMO

Introduction. Staphylococcus epidermidis is predominant in implant-associated infections due to its capability to form biofilms. It can deploy several strategies for biofilm development using either polysaccharide intercellular adhesin (PIA), extracellular DNA (eDNA) and/or proteins, such as the extracellular matrix-binding protein (Embp).Hypothesis/Gap Statement. We hypothesize that the dichotomic regulation of S. epidermidis adhesins is linked to whether it is inside a host or not, and that in vitro biofilm investigations in laboratory media may not reflect actual biofilms in vivo.Aim. We address the importance of PIA and Embp in biofilm grown in 'humanized' media to understand if these components play different roles in biofilm formation under conditions where bacteria can incorporate host proteins in the biofilm matrix.Methodology. S. epidermidis 1585 WT (deficient in icaADBC), and derivative strains that either lack embp, express embp from an inducible promotor, or express icaADBC from a plasmid, were cultivated in standard laboratory media, or in media with human plasma or serum. The amount, structure, elasticity and antimicrobial penetration of biofilms was quantified to describe structural differences caused by the different matrix components and growth conditions. Finally, we quantified the initiation of biofilms as suspended aggregates in response to host factors to determine how quickly the cells aggregate in response to the host environment and reach a size that protects them from phagocytosis.Results. S. epidermidis 1585 required polysaccharides to form biofilm in laboratory media. However, these observations were not representative of the biofilm phenotype in the presence of human plasma. If human plasma were present, polysaccharides and Embp were redundant for biofilm formation. Biofilms formed in human plasma were loosely attached and existed mostly as suspended aggregates. Aggregation occurred after 2 h of exposing cells to plasma or serum. Despite stark differences in the amount and composition of biofilms formed by polysaccharide-producing and Embp-producing strains in different media, there were no differences in vancomycin penetration or susceptibility.Conclusion. We suggest that the assumed importance of polysaccharides for biofilm formation is an artefact from studying biofilms in laboratory media void of human matrix components. The cell-cell aggregation of S. epidermidis can be activated by host factors without relying on either of the major adhesins, PIA and Embp, indicating a need to revisit the basic question of how S. epidermidis deploys self-produced and host-derived matrix components to form antibiotic-tolerant biofilms in vivo.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Polissacarídeos Bacterianos/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/fisiologia , Aderência Bacteriana , Regulação Bacteriana da Expressão Gênica , Humanos
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.
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
8.
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
9.
Methods Mol Biol ; 2210: 123-133, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32815133

RESUMO

The type IX secretion system (T9SS) is a protein secretion system for gingipain proteases and is found on the cell surface of Porphyromonas gingivalis. Proteins secreted by T9SS contain a signal peptide, functional domains, an immunoglobulin (Ig)-like domain, and a C-terminal domain (CTD). Thirty genes on the P. gingivalis chromosome encode proteins that possess the CTD, which is important for T9SS-mediated translocation to the cell surface across the outer membrane. In T9SS mutant strains, proteins accumulate as precursors in the cell and therefore exhibit a phenotype similar to that of secreted protein-deficient mutants. Black pigment productivity and hemagglutination are phenotypic features of P. gingivalis associated with the activity of gingipains. In P. gingivalis T9SS mutants, unprocessed gingipains with high molecular weights accumulate in the cell, and colony pigmentation and hemagglutination are not observed in the same phenotype as a gingipain null mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Porphyromonas gingivalis/metabolismo , Adesinas Bacterianas/metabolismo , Animais , Cisteína Endopeptidases Gingipaínas/metabolismo , Hemaglutinação/fisiologia , Coelhos , Fatores de Virulência/metabolismo
10.
PLoS Biol ; 18(12): e3000986, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33378358

RESUMO

Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs.


Assuntos
Cálcio/metabolismo , Proteínas de Escherichia coli/metabolismo , Piroptose/fisiologia , Receptores de Superfície Celular/metabolismo , Actinas/metabolismo , Adesinas Bacterianas/metabolismo , Adesinas Bacterianas/fisiologia , Análise por Conglomerados , Escherichia coli Enteropatogênica/metabolismo , Escherichia coli Enteropatogênica/patogenicidade , Células Epiteliais/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Células HeLa , Humanos , Mucosa Intestinal/metabolismo , Intestinos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Transporte Proteico , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/fisiologia , Sistemas de Secreção Tipo III/metabolismo
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 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
14.
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
15.
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
16.
Nat Microbiol ; 5(7): 917-928, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32251370

RESUMO

Bacteria have evolved diverse mechanisms to fend off predation by bacteriophages. We previously identified the Dnd system, which uses DndABCDE to insert sulfur into the DNA backbone as a double-stranded phosphorothioate (PT) modification, and DndFGH, a restriction component. Here, we describe an unusual SspABCD-SspE PT system in Vibrio cyclitrophicus, Escherichia coli and Streptomyces yokosukanensis, which has distinct genetic organization, biochemical functions and phenotypic behaviour. SspABCD confers single-stranded and high-frequency PTs with SspB acting as a nickase and possibly introducing nicks to facilitate sulfur incorporation. Strikingly, SspABCD coupled with SspE provides protection against phages in unusual ways: (1) SspE senses sequence-specific PTs by virtue of its PT-stimulated NTPase activity to exert its anti-phage activity, and (2) SspE inhibits phage propagation by introducing nicking damage to impair phage DNA replication. These results not only expand our knowledge about the diversity and functions of DNA PT modification but also enhance our understanding of the known arsenal of defence systems.


Assuntos
Bacteriófagos/fisiologia , Interações Hospedeiro-Patógeno , Fosfatos/metabolismo , Streptomyces/fisiologia , Streptomyces/virologia , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sítios de Ligação , Ativação Enzimática , Genoma Bacteriano , Interações Hospedeiro-Patógeno/genética , Modelos Moleculares , Fases de Leitura Aberta , Fosfatos/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica
17.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32122940

RESUMO

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative ß-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted ß-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as ß-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly ß-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal ß-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.


Assuntos
Formação de Anticorpos/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Dermatite Digital/imunologia , Dermatite Digital/microbiologia , Treponema/imunologia , Treponema/patogenicidade , Adesinas Bacterianas/metabolismo , Animais , Aderência Bacteriana/fisiologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , DNA Bacteriano/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/microbiologia , Imunoglobulina G/metabolismo , Proteínas de Membrana/metabolismo , Filogenia , Conformação Proteica em Folha beta , Serina Endopeptidases/metabolismo , Virulência/fisiologia
18.
Sci Adv ; 6(10): eaay6616, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32181348

RESUMO

The gliding bacterium Flavobacterium johnsoniae is known to have an adhesin, SprB, that moves along the cell surface on a spiral track. Following viscous shear, cells can be tethered by the addition of an anti-SprB antibody, causing spinning at 3 Hz. Labeling the type 9 secretion system (T9SS) with a YFP fusion of GldL showed a yellow fluorescent spot near the rotation axis, indicating that the motor driving the motion is associated with the T9SS. The distance between the rotation axis and the track (90 nm) was determined after adding a Cy3 label for SprB. A rotary motor spinning a pinion of radius 90 nm at 3 Hz would cause a spot on its periphery to move at 1.5 µm/s, the gliding speed. We suggest the pinion drives a flexible tread that carries SprB along a track fixed to the cell surface. Cells glide when this adhesin adheres to the solid substratum.


Assuntos
Adesinas Bacterianas/genética , Sistemas de Secreção Bacterianos/genética , Flavobacterium/genética , Adesinas Bacterianas/metabolismo , Anticorpos Antibacterianos/química , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Fenômenos Biomecânicos , Carbocianinas/química , Flavobacterium/metabolismo , Corantes Fluorescentes/química , Expressão Gênica , Genes Reporter , Locomoção/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rotação
19.
PLoS One ; 15(3): e0230138, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176708

RESUMO

Surface-expressed colonization factors and their subunits are promising candidates for inclusion into a multivalent vaccine targeting enterotoxigenic Escherichia coli (ETEC), a leading cause of acute bacterial diarrhea in developing regions. However, soluble antigens are often poorly immunogenic in the absence of an adjuvant. We show here that the serum immune response to CfaE, the adhesin of the ETEC colonization factor CFA/I, can be enhanced in BALB/c mice by immunization with a chimeric antigen containing CfaE and pentameric cholera toxin B subunit (CTB) of cholera toxin from Vibrio cholerae. We constructed this antigen by replacing the coding sequence for the A1 domain of the cholera toxin A subunit (CTA) with the sequence of donor strand complemented CfaE (dscCfaE) within the cholera toxin operon, resulting in a dscCfaE-CTA2 fusion. After expression, via non-covalent interactions between CTA2 and CTB, the fusion and CTB polypeptides assemble into a complex containing a single dscCfaE-CTA2 protein bound to pentameric CTB (dscCfaE-CTA2/CTB). This holotoxin-like chimera retained the GM1 ganglioside binding activity of CTB, as well as the ability of CfaE to mediate the agglutination of bovine red blood cells when adsorbed to polystyrene beads. When administered intranasally to mice, the presence of CTB in the chimera significantly increased the serum immune response to CfaE compared to dscCfaE alone, stimulating a response similar to that obtained with a matched admixture of dscCfaE and CTB. However, by the orogastric route, immunization with the chimera elicited a superior functional immune response compared to an equivalent admixture of dscCfaE and CTB, supporting further investigation of the chimera as an ETEC vaccine candidate.


Assuntos
Toxina da Cólera , Escherichia coli Enterotoxigênica/imunologia , Vacinas contra Escherichia coli/imunologia , Proteínas de Fímbrias , Proteínas Recombinantes de Fusão , Adesinas Bacterianas/imunologia , Adesinas Bacterianas/metabolismo , Adjuvantes Imunológicos/administração & dosagem , Animais , Quimera/genética , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Toxina da Cólera/metabolismo , Feminino , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/imunologia , Proteínas de Fímbrias/metabolismo , Imunização , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo
20.
Mol Microbiol ; 113(3): 650-658, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32185835

RESUMO

The Gram-positive bacterium Streptococcus pneumoniae, the pneumococcus, is an important commensal resident of the human nasopharynx. Carriage is usually asymptomatic, however, S. pneumoniae can become invasive and spread from the upper respiratory tract to the lungs causing pneumonia, and to other organs to cause severe diseases such as bacteremia and meningitis. Several pneumococcal proteins important for its disease-causing capability have been described and many are expressed on the bacterial surface. The surface located pneumococcal type-1 pilus has been associated with virulence and the inflammatory response, and it is present in 20%-30% of clinical isolates. Its tip protein RrgA has been shown to be a major adhesin to human cells and to promote invasion through the blood-brain barrier. In this review we discuss recent findings of the impact of RrgA on bacterial colonization of the upper respiratory tract and on pneumococcal virulence, and use epidemiological data and genome-mining to suggest trade-off mechanisms potentially explaining the rather low prevalence of pilus-1 expressing pneumococci in humans.


Assuntos
Proteínas de Fímbrias/metabolismo , Streptococcus pneumoniae/metabolismo , Fatores de Virulência/metabolismo , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/fisiologia , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/fisiologia , Ligação Proteica , Streptococcus pneumoniae/patogenicidade , Virulência/genética , Fatores de Virulência/fisiologia
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