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1.
Nat Commun ; 11(1): 4321, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859904

RESUMO

Bacterial colonization of the human intestine requires firm adhesion of bacteria to insoluble substrates under hydrodynamic flow. Here we report the molecular mechanism behind an ultrastable protein complex responsible for resisting shear forces and adhering bacteria to cellulose fibers in the human gut. Using single-molecule force spectroscopy (SMFS), single-molecule FRET (smFRET), and molecular dynamics (MD) simulations, we resolve two binding modes and three unbinding reaction pathways of a mechanically ultrastable R. champanellensis (Rc) Dockerin:Cohesin (Doc:Coh) complex. The complex assembles in two discrete binding modes with significantly different mechanical properties, with one breaking at ~500 pN and the other at ~200 pN at loading rates from 1-100 nN s-1. A neighboring X-module domain allosterically regulates the binding interaction and inhibits one of the low-force pathways at high loading rates, giving rise to a catch bonding mechanism that manifests under force ramp protocols. Multi-state Monte Carlo simulations show strong agreement with experimental results, validating the proposed kinetic scheme. These results explain mechanistically how gut microbes regulate cell adhesion strength at high shear stress through intricate molecular mechanisms including dual-binding modes, mechanical allostery and catch bonds.


Assuntos
Aderência Bacteriana/fisiologia , Microbioma Gastrointestinal/fisiologia , Fenômenos Mecânicos , Fenômenos Físicos , Bactérias , Aderência Bacteriana/genética , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Trato Gastrointestinal/microbiologia , Técnicas de Inativação de Genes , Humanos , Cinética , Simulação de Dinâmica Molecular , Método de Monte Carlo , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula , Estresse Mecânico
2.
Invest Ophthalmol Vis Sci ; 61(4): 17, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32298434

RESUMO

Purpose: Extended contact lens (CL) wear predisposes the wearer to Pseudomonas aeruginosa infection of the cornea, but the mechanism involved remains incompletely understood. The purpose of this study was to investigate the role of the stress hormone norepinephrine (NE) in the pathogenesis of CL-induced P. aeruginosa keratitis. Methods: A total 195 adult C57BL/6 mice were used in this study. Corneal NE content was measured after 48 hours of sterile CL wear in mice. The effect of NE on P. aeruginosa adhesion and biofilm formation on the CL surface was examined in vitro. Moreover, mouse eyes were covered with P. aeruginosa-contaminated CLs, and either 500-µM NE was topically applied or the eyes were subconjunctivally injected with 100 µg of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to deplete local NE. Clinical scores, neutrophil infiltration, proinflammatory cytokine levels, and bacterial load on the corneas and CLs were evaluated. Results: Corneal NE content was elevated with extended CL wear in mice. In vitro, NE promoted the adhesion and biofilm formation of P. aeruginosa on the CL surface. In mice, topical application of NE aggravated P. aeruginosa infection, accompanied with increased clinical scores, neutrophil infiltration, proinflammatory cytokine expression, and bacterial burden on the corneas and CLs. However, pre-depletion of local NE with DSP-4 significantly alleviated the severity of P. aeruginosa keratitis. Conclusions: Extended CL wear elevates corneal NE content, which promotes the pathogenesis of CL-induced P. aeruginosa keratitis in mice. Targeting NE may provide a potential strategy for the treatment of CL-related corneal infection caused by P. aeruginosa.


Assuntos
Lentes de Contato de Uso Prolongado/efeitos adversos , Úlcera da Córnea/metabolismo , Infecções Oculares Bacterianas/metabolismo , Norepinefrina/metabolismo , Infecções por Pseudomonas/metabolismo , Animais , Aderência Bacteriana/fisiologia , Carga Bacteriana , Técnicas de Cocultura , Úlcera da Córnea/etiologia , Úlcera da Córnea/microbiologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Infecções Oculares Bacterianas/etiologia , Infecções Oculares Bacterianas/microbiologia , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/etiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia
3.
Proc Natl Acad Sci U S A ; 117(17): 9546-9553, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32295877

RESUMO

Understanding how bacteria colonize surfaces and regulate cell-cycle progression in response to cellular adhesion is of fundamental importance. Here, we use transposon sequencing in conjunction with fluorescence resonance energy transfer (FRET) microscopy to uncover the molecular mechanism for how surface sensing drives cell-cycle initiation in Caulobacter crescentus We identify the type IV pilin protein PilA as the primary signaling input that couples surface contact to cell-cycle initiation via the second messenger cyclic di-GMP (c-di-GMP). Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17-amino acid transmembrane helix of PilA to activate the PleC-PleD two-component signaling system, increase cellular c-di-GMP levels, and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for "cell-cycle initiating pilin" peptide. Addition of the chemically synthesized CIP peptide initiates cell-cycle progression and simultaneously inhibits surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer strategies to inhibit surface sensing, prevent biofilm formation and control persistent infections.


Assuntos
Aderência Bacteriana/fisiologia , Caulobacter crescentus/fisiologia , Ciclo Celular/fisiologia , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Fímbrias/genética
4.
PLoS One ; 15(4): e0231791, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302361

RESUMO

Periprosthetic joint infections (PJIs) are a devastating complication that occurs in 2% of patients following joint replacement. These infections are costly and difficult to treat, often requiring multiple corrective surgeries and prolonged antimicrobial treatments. The Gram-positive bacterium Staphylococcus aureus is one of the most common causes of PJIs, and it is often resistant to a number of commonly used antimicrobials. This tolerance can be partially attributed to the ability of S. aureus to form biofilms. Biofilms associated with the surface of indwelling medical devices have been observed on components removed during chronic infection, however, the development and localization of biofilms during PJIs remains unclear. Prior studies have demonstrated that synovial fluid, in the joint cavity, promotes the development of bacterial aggregates with many biofilm-like properties, including antibiotic resistance. We anticipate these aggregates have an important role in biofilm formation and antibiotic tolerance during PJIs. Therefore, we sought to determine specifically how synovial fluid promotes aggregate formation and the impact of this process on surface attachment. Using flow cytometry and microscopy, we quantified the aggregation of various clinical S. aureus strains following exposure to purified synovial fluid components. We determined that fibrinogen and fibronectin promoted bacterial aggregation, while cell free DNA, serum albumin, and hyaluronic acid had minimal effect. To determine how synovial fluid mediated aggregation affects surface attachment, we utilized microscopy to measure bacterial attachment. Surprisingly, we found that synovial fluid significantly impeded bacterial surface attachment to a variety of materials. We conclude from this study that fibrinogen and fibronectin in synovial fluid have a crucial role in promoting bacterial aggregation and inhibiting surface adhesion during PJI. Collectively, we propose that synovial fluid may have conflicting protective roles for the host by preventing adhesion to surfaces, but by promoting bacterial aggregation is also contributing to the development of antibiotic tolerance.


Assuntos
Aderência Bacteriana/fisiologia , Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/fisiologia , Líquido Sinovial/microbiologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Fibrinogênio/farmacologia , Fibronectinas/farmacologia , Humanos , Staphylococcus aureus/efeitos dos fármacos , Líquido Sinovial/efeitos dos fármacos , Fatores de Tempo
5.
Arch Microbiol ; 202(6): 1349-1357, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32152646

RESUMO

This study aimed to assess adhesion and anti-adhesion, aggregation, and surface properties of four selected oral Lactobacillus strains, L. fermentum SD7, L. paracasei SD1, L. rhamnosus SD4, and L. rhamnosus SD11, together with Lactobacillus rhamnosus GG. Human cells, enterocytes Caco-2 and oral keratinocyte H357 were used, and various enteric and oral pathogens were included. Results showed that all Lactobacillus tested gave high adhesion and internalization in both Caco-2 and H357 cells similar to L. rhamnosus GG, and it suggests that such properties are strain dependent and specific to host cells. Anti-adhesion was different; it depended on the internalization ability of individual Lactobacillus and pathogenic strains to Caco-2 and H357. Coaggregation ability depended on autoaggregation of both the Lactobacillus and pathogenic strains. A positive correlation between surface charges and aggregation, and internalization and anti-adhesion of all Lactobacillus was found. In conclusion, results suggests that the selected Lactobacillus might be potential probiotics for usage in both the oral cavity and intestinal tract due to their abilities of aggregation, adherence and anti-internalization to both Caco-2 and H357 cells.


Assuntos
Aderência Bacteriana , Lactobacillus/metabolismo , Probióticos , Aderência Bacteriana/fisiologia , Células CACO-2 , Humanos , Intestinos/microbiologia , Boca/microbiologia , Propriedades de Superfície
6.
Arch Microbiol ; 202(6): 1517-1527, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32222779

RESUMO

Biofilm formation is a harmful phenomenon in many areas, such as in industry and clinically, but offers advantages in the field of biocatalysis for the generation of robust biocatalytic platforms. In this work, we optimised growth conditions for the production of Escherichia coli biofilms by three strains (PHL644, a K-12 derivative with enhanced expression of the adhesin curli; the commercially-used strain BL21; and the probiotic Nissle 1917) on a variety of surfaces (plastics, stainless steel and PTFE). E. coli PHL644 and PTFE were chosen as optimal strain and substratum, respectively, and conditions (including medium, temperature, and glucose concentration) for biofilm growth were determined. Finally, the impact of these growth conditions on expression of the curli genes was determined using flow cytometry for planktonic and sedimented cells. We reveal new insights into the formation of biofilms and expression of curli in E. coli K-12 in response to environmental conditions.


Assuntos
Adesinas Bacterianas/biossíntese , Proteínas de Bactérias/biossíntese , Biofilmes/crescimento & desenvolvimento , Exposição Ambiental , Escherichia coli/metabolismo , Adesinas Bacterianas/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Plásticos/química , Politetrafluoretileno/química , Aço Inoxidável/química , Propriedades de Superfície
7.
FASEB J ; 34(1): 1783-1801, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914584

RESUMO

The natural product icariin (ICA) and its phosphorylated derivatives (pICA) have been shown to have outstanding anti-inflammatory and antioxidant properties. This study was to explore the protective effects of ICA and pICA on the intestinal epithelium of enterotoxigenic Escherichia coli (ETEC)-induced piglet diarrhea and its underlying mechanisms in vivo and in vitro. ETEC K88 increased pro-inflammatory cytokine expression, activated oxidative stress and inhibited antioxidant enzyme activity, induced phosphorylated p38 MAPK gene and protein expression, disrupted intestinal barrier function, and led to diarrhea in piglets. Pretreatment with ICA and pICA effectively alleviated ETEC-induced intestinal barrier dysfunction in vivo and in vitro. Pretreatment with p38 MAPK inhibitor (SB203580) significantly rescued the IPEC-J2 cells barrier function damaged by ETEC challenge. However, pretreatment with p38 MAPK activator (anisomycin) did not alleviated the IPEC-J2 cells barrier function damaged by ETEC challenge. Our data demonstrated that ICA and pICA regulate the inflammatory response and oxidative stress of intestinal epithelial cells by inhibiting the expression of p38 MAPK, thereby alleviating ETEC K88-induced disruption of intestinal barrier function and intestinal permeability. These findings provide new insights into the prevention and treatment of intestinal barrier dysfunction induced by ETEC K88.


Assuntos
Escherichia coli Enterotoxigênica/patogenicidade , Células Epiteliais/metabolismo , Infecções por Escherichia coli/metabolismo , Flavonoides/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Aderência Bacteriana/fisiologia , Linhagem Celular , Diarreia/metabolismo , Diarreia/microbiologia , Células Epiteliais/microbiologia , Infecções por Escherichia coli/microbiologia , Enteropatias/metabolismo , Enteropatias/microbiologia , Mucosa Intestinal/microbiologia , Intestinos/microbiologia , Masculino , Permeabilidade , Suínos , Doenças dos Suínos/metabolismo , Doenças dos Suínos/microbiologia
8.
Nat Prod Res ; 34(5): 710-713, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30445822

RESUMO

The correlation between the allocation of trisoxazole macrolides in the capitums, appendages, and bases of the sponge Penares cf. nux and the surface-attached bacteria on the corresponding parts was examined. The kabiramide contents were highest in the capitums, followed by the appendages and bases. Conversely, direct counts of cultivable surface-attached bacteria showed that the bacteria aggregate more densely on the surfaces of the bases. This suggested the repelling effects of the kabiramides against the fouling bacteria, particularly on the capitums and appendages. Twenty-two bacterial strains were isolated and identified to 15 species; however, none has shown the potentials as a producer of any secondary metabolites in the sponge P. nux.


Assuntos
Antibacterianos/metabolismo , Macrolídeos/farmacologia , Poríferos/metabolismo , Animais , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Aderência Bacteriana/fisiologia , Macrolídeos/metabolismo , Oxazóis/metabolismo , Oxazóis/farmacologia , Poríferos/química
9.
PLoS Pathog ; 15(12): e1008136, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790511

RESUMO

Sexually transmitted infections are a critical public health issue. However, the mechanisms underlying sexually transmitted infections in women and the link between the infection mechanism and the wide range of clinical outcomes remain elusive due to a lack of research models mimicking human infection in vivo. We established a human cervical tissue explant model to mimic local Neisseria gonorrhoeae (GC) infections. We found that GC preferentially colonize the ectocervix by activating integrin-ß1, which inhibits epithelial shedding. GC selectively penetrate into the squamocolumnar junction (TZ) and endocervical epithelia by inducing ß-catenin phosphorylation, which leads to E-cadherin junction disassembly. Epithelial cells in various cervical regions differentially express carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), the host receptor for GC opacity-associated proteins (OpaCEA). Relatively high levels were detected on the luminal membrane of ecto/endocervical epithelial cells but very low levels intracellularly in TZ epithelial cells. CEACAM-OpaCEA interaction increased ecto/endocervical colonization and reduced endocervical penetration by increasing integrin-ß1 activation and inhibiting ß-catenin phosphorylation respectively, through CEACAM downstream signaling. Thus, the intrinsic properties of cervical epithelial cells and phase-variation of bacterial surface molecules both play a role in controlling GC infection mechanisms and infectivity, preferential colonization or penetration, potentially leading to asymptomatic or symptomatic infection.


Assuntos
Aderência Bacteriana/fisiologia , Colo do Útero/microbiologia , Gonorreia/microbiologia , Membrana Mucosa/microbiologia , Neisseria gonorrhoeae/patogenicidade , Colo do Útero/metabolismo , Feminino , Humanos , Membrana Mucosa/metabolismo , Técnicas de Cultura de Órgãos
10.
World J Microbiol Biotechnol ; 36(1): 10, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31863307

RESUMO

Aggregation and adhesion capability and survival efficacy of candidate probiotic strain Pediococcus acidilactici NCDC 252 under simulated gastric, intestinal and vaginal conditions was studied. The strain exhibited strong autoaggregation phenotype and coaggregation with other Lactic acid bacteria (LAB) and E. coli. The adhesion studies of NCDC 252 to pig's intestinal epithelial cells showed its adhesive ability. Aggregation and adhesiveness were related through cell surface proteins as removal/extraction of surface proteins resulted in altered aggregation and no adhesiveness. Cell surface proteins were analysed by SDS-PAGE and also in silico analysed from its genome. SDS-PAGE analysis of cell surface proteins of NCDC 252 revealed two potential proteins of approximately 74.3 and 53.6 kDa to be involved in host-probiotic interaction. Removal of cell surface proteins by LiCl-treatment (5 mol l-1) resulted in loss of aggregation and adhesiveness. Further survival of NCDC 252 under simulated gastrointestinal and vaginal conditions in terms of high viable counts confirmed its efficacy for its survival under gut and urogenital conditions. These observations suggest that it can be used further in functional foods, nutraceuticals and in combating urogenital infections. As NCDC 252 was able to survive in intestinal conditions, interaction of its cell surface proteins with intestinal mucins was studied in silico by docking. Highest affinity of adhesion was observed for MUC3B. In conclucion, NCDC 252, exhibited aggregation phenotype and adhesion capability. Survivability of NCDC 252 under simulated conditions and its interaction with human mucins confirms its efficacy to be used as probiotic.


Assuntos
Aderência Bacteriana/fisiologia , Pediococcus acidilactici/fisiologia , Probióticos/metabolismo , Animais , Suplementos Nutricionais , Células Epiteliais/microbiologia , Feminino , Trato Gastrointestinal/microbiologia , Humanos , Lactobacillales/fisiologia , Proteínas de Membrana , Viabilidade Microbiana , Simulação de Acoplamento Molecular , Mucinas , Vagina/microbiologia
11.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31570554

RESUMO

Serotype 4821 (ST-4821) clonal complex (cc4821) Neisseria meningitidis strains are divided into two groups (groups I and II) according to the core genome-based phylogenetic analysis. Group I contains the greater number of invasive disease isolates. However, the differences in pathogenicity between the two groups are unclear. In this study, the pathogenicity of cc4821 isolates (n = 28) belonging to group I and group II (each containing eight invasive isolates and six isolates from healthy carriers) was investigated, including adhesion, invasion, and induction of interleukin-6 (IL-6) and interleukin-8 (IL-8) release from host cells (Hep2 and A549). The invasive isolates had higher adhesion and invasion capabilities than the carried isolates in both groups. The carried cc4821 isolates in group I had stronger invasion capability than those in group II. Invasive isolates induced more IL-6 and IL-8 secretion than carried isolates in both groups. The carried cc4821 isolates stimulated higher levels of IL-8 in group I than in group II. The isolates were defined as hyperadherent and hypoadherent groups according to their adhesion ability and as hyperinvasive and hypoinvasive groups based on their invasion ability. The hyperadherent and hyperinvasive isolates mediated more IL-6 and IL-8 release than the hypoadherent and hypoinvasive isolates. There was no difference in the level of cytokine release when cc4821 isolates lost their adhesion and invasion capability after lysis. The results revealed that differences in pathogenicity existed between the two groups and that the differences were mainly determined by differences in adhesion and invasion capabilities.


Assuntos
Aderência Bacteriana/fisiologia , Interleucina-6/imunologia , Interleucina-8/imunologia , Meningite Meningocócica/patologia , Neisseria meningitidis/patogenicidade , Células A549 , Linhagem Celular , China , Humanos , Meningite Meningocócica/microbiologia , Neisseria meningitidis/isolamento & purificação , Sorogrupo , Virulência
12.
Microb Pathog ; 137: 103748, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31521802

RESUMO

Salmonellosis is a serious threat to human and animal health. Salmonella adhesion to the host cell is an initial and most crucial step in the pathogenesis of salmonellosis. Many factors are involved in the adhesion process of Salmonella infection. Fimbriae are one of the most important factors in the adhesion of Salmonella. The Salmonella fimbriae are assembled in three types of assembly pathways: chaperon-usher, nucleation-precipitation, and type IV fimbriae. These assembly pathways lead to multiple types of fimbriae. Salmonella fimbriae bind to host cell receptors to initiate adhesion. So far, many receptors have been identified, such as Toll-like receptors. However, several receptors that may be involved in the adhesive mechanism of Salmonella fimbriae are still un-identified. This review aimed to summarize the types of Salmonella fimbriae produced by different assembly pathways and their role in adhesion. It also enlisted previously discovered receptors involved in adhesion. This review might help readers to develop a comprehensive understanding of Salmonella fimbriae, their role in adhesion, and recently developed strategies to counter Salmonella infection.


Assuntos
Adesinas Bacterianas/fisiologia , Aderência Bacteriana/fisiologia , Fímbrias Bacterianas/fisiologia , Salmonella/fisiologia , Adesinas Bacterianas/genética , Animais , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/fisiologia , Genes Bacterianos , Humanos , Salmonella/genética , Infecções por Salmonella , Receptores Toll-Like
13.
mBio ; 10(5)2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31506311

RESUMO

Bacterial adhesion is accompanied by altered gene expression, leading to "emergent" properties of biofilm bacteria that are alien to planktonic ones. With the aim of revealing the role of environmental adhesion forces in emergent biofilm properties, genes in Streptococcus mutans UA159 and a quorum-sensing-deficient mutant were identified that become expressed after adhesion to substratum surfaces. Using atomic force microscopy, adhesion forces of initial S. mutans colonizers on four different substrata were determined and related to gene expression. Adhesion forces upon initial contact were similarly low across different substrata, ranging between 0.2 and 1.2 nN regardless of the strain considered. Bond maturation required up to 21 s, depending on the strain and substratum surface involved, but stationary adhesion forces also were similar in the parent and in the mutant strain. However, stationary adhesion forces were largest on hydrophobic silicone rubber (19 to 20 nN), while being smallest on hydrophilic glass (3 to 4 nN). brpA gene expression in thin (34 to 48 µm) 5-h S. mutans UA159 biofilms was most sensitive to adhesion forces, while expression of gbpB and comDE expressions was weakly sensitive. ftf, gtfB, vicR, and relA expression was insensitive to adhesion forces. In thicker (98 to 151 µm) 24-h biofilms, adhesion-force-induced gene expression and emergent extracellular polymeric substance (EPS) production were limited to the first 20 to 30 µm above a substratum surface. In the quorum-sensing-deficient S. mutans, adhesion-force-controlled gene expression was absent in both 5- and 24-h biofilms. Thus, initial colonizers of substratum surfaces sense adhesion forces that externally trigger emergent biofilm properties over a limited distance above a substratum surface through quorum sensing.IMPORTANCE A new concept in biofilm science is introduced: "adhesion force sensitivity of genes," defining the degree up to which expression of different genes in adhering bacteria is controlled by the environmental adhesion forces they experience. Analysis of gene expression as a function of height in a biofilm showed that the information about the substratum surface to which initially adhering bacteria adhere is passed up to a biofilm height of 20 to 30 µm above a substratum surface, highlighting the importance and limitations of cell-to-cell communication in a biofilm. Bacteria in a biofilm mode of growth, as opposed to planktonic growth, are responsible for the great majority of human infections, predicted to become the number one cause of death in 2050. The concept of adhesion force sensitivity of genes provides better understanding of bacterial adaptation in biofilms, direly needed for the design of improved therapeutic measures that evade the recalcitrance of biofilm bacteria to antimicrobials.


Assuntos
Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Streptococcus mutans/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Liases de Carbono-Enxofre/genética , Matriz Extracelular de Substâncias Poliméricas , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Mutação , Percepção de Quorum/genética , Streptococcus mutans/metabolismo
14.
Nat Commun ; 10(1): 4387, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558724

RESUMO

Despite the importance of nanoparticle's multipods topology in multivalent-interactions enhanced nano-bio interactions, the precise manipulation of multipods surface topological structures is still a great challenge. Herein, the surface-kinetics mediated multi-site nucleation strategy is demonstrated for the fabrication of mesoporous multipods with precisely tunable surface topological structures. Tribulus-like tetra-pods Fe3O4@SiO2@RF&PMOs (RF = resorcinol-formaldehyde resin, PMO = periodic mesoporous organosilica) nanocomposites have successfully been fabricated with a centering core@shell Fe3O4@SiO2@RF nanoparticle, and four surrounding PMO nanocubes as pods. By manipulating the number of nucleation sites through mediating surface kinetics, a series of multipods mesoporous nanocomposites with precisely controllable surface topological structures are formed, including Janus with only one pod, nearly plane distributed dual-pods and tri-pods, three-dimensional tetrahedral structured tetra-pods, etc. The multipods topology endows the mesoporous nanocomposites enhanced bacteria adhesion ability. Particularly, the tribulus-like tetra-pods mesoporous nanoparticles show ~100% bacteria segregation and long-term inhibition over 90% after antibiotic loading.


Assuntos
Aderência Bacteriana/fisiologia , Formaldeído/química , Nanocompostos/química , Nanopartículas/química , Resorcinóis/química , Escherichia coli/fisiologia , Escherichia coli/ultraestrutura , Cinética , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Nanocompostos/ultraestrutura , Nanopartículas/ultraestrutura , Tamanho da Partícula , Porosidade , Propriedades de Superfície
15.
Colloids Surf B Biointerfaces ; 183: 110432, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31421403

RESUMO

Staphylococcus aureus is an important opportunistic pathogen that causes a broad range of infections due to the bacteria capacity to form biofilms on medical devices. This work is aimed at inhibiting the biofilm formation by S. aureus on solid substrates using a simple surface biofunctionalization strategy. We previously found that surface biofunctionalization with structural perturbed albumin inhibited the initial stage of S. aureus adhesion. The current work extends this strategy with other plasma protein, fibrinogen, which in addition can be bond specifically to the cell wall-anchored proteins of S. aureus. The study of fibrinogen adsorption indicates that the fraction of surface-perturbed molecules is enlarged at long adsorption times and low protein concentration. In these conditions, a significant diminution of ca.60% of alive adhered bacteria were observed after 40 min and the biofilm formation was completely prevented. Thus, it seems that the inhibition of bacterial adhesion on substrates with surface-perturbed proteins represents a general trend even when specific interactions are present. On this basis, we developed a simple strategy to inhibit the formation of S. aureus biofilm, using thermally treated albumin or fibrinogen molecules prior to the substrate biofunctionalization. This strategy shows an excellent performance since the alive adhered bacteria diminishes ca. 90% at short incubation time, followed by the fully inhibition of biofilm formation. This novel and simple resource represents a change of the usual notion in avoiding post-surgery infections, mostly related to the use of medical devices.


Assuntos
Aderência Bacteriana/fisiologia , Biofilmes/crescimento & desenvolvimento , Parede Celular/metabolismo , Fibrinogênio/metabolismo , Staphylococcus aureus/fisiologia , Adsorção , Aderência Bacteriana/efeitos dos fármacos , Fibrinogênio/química , Fibrinogênio/farmacologia , Ligação Proteica , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Propriedades de Superfície
16.
Mol Microbiol ; 112(5): 1403-1422, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31419359

RESUMO

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)-encoded type one secretion system (T1SS) and the SPI1-encoded type three secretion system (T3SS-1). The SPI4-encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure-function relationships in SiiA, a non-canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA-PD). The crystal structure of SiiA-PD displays homology to that of MotB and other peptidoglycan (PG)-binding domains. SiiA-PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA-MotB homology is paralleled by a predicted SiiB-MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.


Assuntos
Elonguina/metabolismo , Infecções por Salmonella/patologia , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo I/metabolismo , Adesinas Bacterianas/metabolismo , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Relação Estrutura-Atividade , Sistemas de Secreção Tipo III/metabolismo
17.
Microsc Res Tech ; 82(11): 1869-1877, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31373738

RESUMO

Laser irradiation has been proposed as a preventive method against dental caries since it is capable to inhibit enamel demineralization by reducing carbonate and modifying organic matter, yet it can produce significant morphological changes. The purpose of this study was to evaluate the influence of Er:YAG laser irradiation on superficial roughness of deciduous dental enamel and bacterial adhesion. Fifty-four samples of deciduous enamel were divided into three groups (n = 18 each). G1_control (nonirradiated); G2_100 (7.5 J/cm2 ) and G3_100 (12.7 J/cm2 ) were irradiated with Er:YAG laser at 7.5 and 12.7 J/cm2 , respectively, under water irrigation. Surface roughness was measured before and after irradiation using a profilometer. Afterwards, six samples per group were used to measure bacterial growth by XTT cell viability assay. Adhered bacteria were observed using confocal laser scanning microscopy (CLSM) and a scanning electron microscopy (SEM). Paired t-, one-way analysis of variance (ANOVA), Kruskal-Wallis and pairwise Mann-Whitney U tests were performed to analyze statistical differences (p < .05). Before treatment, samples showed homogenous surface roughness, and after Er:YAG laser irradiation, the surfaces showed a significant increase in roughness values (p < .05). G3_100 (12.7 J/cm2 ) showed the highest amount of Streptococcus mutans adhered (p < .05). The increase in the roughness of the tooth enamel surfaces was proportional to the energy density used; the increase in surface roughness caused by laser irradiation did not augment the adhesion of Streptococcus sanguinis; only the use of the energy density of 12.7 J/cm2 favored significantly the adhesion of S. mutans.


Assuntos
Aderência Bacteriana/efeitos da radiação , Cárie Dentária/prevenção & controle , Esmalte Dentário/efeitos da radiação , Lasers de Estado Sólido/uso terapêutico , Streptococcus mutans/fisiologia , Streptococcus/fisiologia , Aderência Bacteriana/fisiologia , Humanos , Microscopia Eletrônica de Varredura , Streptococcus/crescimento & desenvolvimento , Streptococcus/efeitos da radiação , Streptococcus mutans/crescimento & desenvolvimento , Streptococcus mutans/efeitos da radiação , Propriedades de Superfície/efeitos da radiação
18.
PLoS Pathog ; 15(8): e1008031, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31465434

RESUMO

Enterohemorrhagic E. coli (EHEC) is a human intestinal pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. No vaccines or specific therapies are currently available to prevent or treat these infections. EHEC tightly attaches to the intestinal epithelium by injecting the intimin receptor Tir into the host cell via a type III secretion system (T3SS). In this project, we identified a camelid single domain antibody (nanobody), named TD4, that recognizes a conserved Tir epitope overlapping the binding site of its natural ligand intimin with high affinity and stability. We show that TD4 inhibits attachment of EHEC to cultured human HeLa cells by preventing Tir clustering by intimin, activation of downstream actin polymerization and pedestal formation. Furthermore, we demonstrate that TD4 significantly reduces EHEC adherence to human colonic mucosa in in vitro organ cultures. Altogether, these results suggest that nanobody-based therapies hold potential in the development of much needed treatment and prevention strategies against EHEC infection.


Assuntos
Aderência Bacteriana/fisiologia , Colo/metabolismo , Escherichia coli Êntero-Hemorrágica/fisiologia , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Anticorpos de Domínio Único/farmacologia , Sequência de Aminoácidos , Animais , Aderência Bacteriana/efeitos dos fármacos , Sítios de Ligação , Camelus , Colo/microbiologia , Colo/patologia , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Homologia de Sequência , Anticorpos de Domínio Único/imunologia
19.
mBio ; 10(4)2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289181

RESUMO

Neisseria gonorrhoeae is a significant threat to global health for which a vaccine and novel treatment options are urgently needed. Glycans expressed by human cells are commonly targeted by pathogens to facilitate interactions with the host, and thus characterization of these interactions can aid identification of bacterial receptors that can be exploited as vaccine and/or drug targets. Using glycan array analysis, we identified 247 specific interactions between N. gonorrhoeae and glycans representative of those found on human cells. Interactions included those with mannosylated, fucosylated, and sialylated glycans, glycosaminoglycans (GAGs), and glycans terminating with galactose (Gal), N-acetylgalactosamine (GalNAc), and N-acetylglucosamine (GlcNAc). By investigating the kinetics of interactions with selected glycans, we demonstrate that whole-cell N. gonorrhoeae has a high affinity for mannosylated glycans (dissociation constant [KD ], 0.14 to 0.59 µM), which are expressed on the surface of cervical and urethral epithelial cells. Using chromatography coupled with mass spectrometric (MS) analysis, we identified potential mannose-binding proteins in N. gonorrhoeae Pretreatment of cells with mannose-specific lectin (concanavalin A) or free mannose competitor (α-methyl-d-mannopyranoside) substantially reduced gonococcal adherence to epithelial cells. This suggests that N. gonorrhoeae targets mannosyl glycans to facilitate adherence to host cells and that mannosides or similar compounds have the potential to be used as a novel treatment option for N. gonorrhoeae IMPORTANCE Multidrug-resistant strains of Neisseria gonorrhoeae are emerging worldwide, and novel treatment and prevention strategies are needed. Glycans are ubiquitously expressed by all human cells and can be specifically targeted by pathogens to facilitate association with host cells. Here we identify and characterize the N. gonorrhoeae host-glycan binding profile (glycointeractome), which revealed numerous interactions, including high-affinity binding to mannosyl glycans. We identify gonococcal potential mannose-binding proteins and show that N. gonorrhoeae uses mannosyl glycans expressed on the surface of cervical and urethral epithelia to facilitate adherence. Furthermore, a mannose-binding lectin or a mannoside compound was able to reduce this adherence. By characterizing the glycointeractome of N. gonorrhoeae, we were able to elucidate a novel mechanism used by this important pathogen to interact with human cells, and this interaction could be exploited to develop novel therapeutics to treat antibiotic-resistant gonorrhea.


Assuntos
Aderência Bacteriana/fisiologia , Colo do Útero/citologia , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Neisseria gonorrhoeae/metabolismo , Polissacarídeos/metabolismo , Uretra/citologia , Aderência Bacteriana/efeitos dos fármacos , Células Cultivadas , Concanavalina A/farmacologia , Células Epiteliais/efeitos dos fármacos , Feminino , Gonorreia/microbiologia , Humanos , Masculino , Lectina de Ligação a Manose/metabolismo , Metilglicosídeos/farmacologia , Análise em Microsséries , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/patogenicidade
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