RESUMO
Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.
Assuntos
Biofilmes/crescimento & desenvolvimento , Boca/microbiologia , Polissacarídeos/metabolismo , Streptococcus mutans , Streptococcus oralis , Animais , Humanos , Concentração de Íons de Hidrogênio , Streptococcus mutans/crescimento & desenvolvimento , Streptococcus mutans/metabolismo , Streptococcus mutans/patogenicidade , Streptococcus oralis/crescimento & desenvolvimento , Streptococcus oralis/metabolismo , Streptococcus oralis/patogenicidade , Fatores de Virulência/metabolismoRESUMO
BACKGROUND: Thyrotoxic periodic paralysis (TPP) is a rare condition characterized by muscle paralysis, thyrotoxicosis, and hypokalemia. It presents with paralysis of both proximal and distal musculature in upper and lower limbs and may affect respiratory musculature and the cardiac conduction system. Early diagnosis is essential, as the condition is potentially reversible by oral or intravenous potassium treatment, leading to rapid resolution without lasting weakness. Overlooking the diagnosis may result in respiratory failure and cardiac arrhythmias including QT prolongation, Torsades de points, and ventricular arrhythmias. CASE PRESENTATION: A 19-year-old Caucasian man was admitted acutely with paralysis in upper and lower limbs and tachycardia. Over several months, he had experienced anxiousness, sweating more than usual, had daily palpitations, shortness of breath on exertion, and loose stools, and had lost 21 kg over the last year. Initial blood gas showed very low potassium of 1.4 mM, and blood tests showed decreased Thyroid-stimulating hormone (TSH) < 0.01 × 10- 3 IU/L, elevated free thyroxine (fT4) of 63.5 pM (reference interval (RI): 12.0-22.0 pM), and elevated total triiodothyronine (T3) of 8.2 nM (RI: 1.0-2.6 nM). He was diagnosed with TPP and treated with liquid oral potassium chloride (30 mmol every 30 minutes) and propylthiouracil (initial dose of 400 mg followed by 200 mg three times daily). TSH-receptor antibodies (TRAB) and thyroid-peroxidase antibodies (TPO-ab) were highly elevated. Thyroid ultrasound showed a normal-sized gland and color Doppler sonography showed increased vascularity throughout the gland, compatible with Graves' disease. He was discharged on day 4 with a normal potassium level and followed in the outpatient clinic where he received standard care for Graves' disease. Genetic testing using whole-genome sequencing found no genetic variants in genes previously associated with TPP. CONCLUSION: TPP is very rare in Caucasians but more often affects young men in East Asian populations. The case presents a Caucasian man with TPP where genetic testing of CACNA1S, KCNJ18, SCN4A, KCNJ2, KCNE3, and ABCC8 shows no pathogenic variants in genes previously associated with TPP.
RESUMO
Animal models of human diseases are invaluable and inevitable elements in identifying and testing novel treatments for serious diseases, including severe infections. Planning and conducting investigator-initiated human trials are generally accepted as being enormously challenging. In contrast, it is often underestimated how much planning, including background and modifying experiments, is needed to establish a relevant infectious disease animal model. However, representative animal infectious models, well designed to test generated hypotheses, are useful to improve our understanding of pathogenesis, virulence factors and host response and to identify novel treatment candidates and therapeutic strategies. Such results can subsequently proceed to clinical testing if suitable. The present review aims at presenting all the pulmonary Pseudomonas aeruginosa infectious models we have knowledge of and the detailed descriptions of established animal models in our laboratory focusing on macrolide therapy are presented.
Assuntos
Fibrose Cística , Infecções por Pseudomonas , Animais , Antibacterianos/uso terapêutico , Fibrose Cística/tratamento farmacológico , Modelos Animais de Doenças , Humanos , Pulmão , Macrolídeos/farmacologia , Macrolídeos/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/fisiologiaRESUMO
The angiotensin II (AngII) type 1 receptor (AT(1)R) has been shown to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) through G proteins or G protein-independently through beta-arrestin2 in cellular expression systems. As activation mechanisms may greatly influence the biological effects of ERK1/2 activity, differential activation of the AT(1)R in its native cellular context could have important biological and pharmacological implications. To examine if AT(1)R activates ERK1/2 by G protein-independent mechanisms in the heart, we used the [Sar(1), Ile(4), Ile(8)]-AngII ([SII] AngII) analogue in native preparations of cardiac myocytes and beating hearts. We found that [SII] AngII does not activate G(q)-coupling, yet stimulates the beta-arrestin2-dependent ERK1/2. The G(q)-activated pool of ERK1/2 rapidly translocates to the nucleus, while the beta-arrestin2-scaffolded pool remains in the cytosol. Similar biased agonism was achieved in Langendorff-perfused hearts, where both agonists elicit ERK1/2 phosphorylation, but [SII] AngII induces neither inotropic nor chronotropic effects.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Receptor Tipo 1 de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Arrestinas/metabolismo , Núcleo Celular/enzimologia , Células Cultivadas , Circulação Coronária/efeitos dos fármacos , Citosol/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Perfusão , Ratos , Ratos Sprague-Dawley , Ratos Wistar , beta-ArrestinasRESUMO
The angiotensin II (AngII) type 1 receptor (AT(1)R) is a seven-transmembrane receptor well established to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) by discrete G protein-dependent and beta-arrestin2-dependent pathways. The biological importance of this, however, remains obscure. Application of the modified analogue [Sar(1), Ile(4), Ile(8)]-AngII ([SII] AngII) allowed us to dissect the two pathways of ERK1/2 activation in native cardiac myocytes. Although cytosol-retained, the beta-arrestin2-bound pool of ERK1/2 represents an active signalling component that phosphorylates p90 Ribosomal S6 Kinase, a ubiquitous and versatile mediator of ERK1/2 signal transduction. Moreover, the beta-arrestin2-dependent ERK1/2 signal supports intact proliferation of cardiac myocytes. In contrast to G(q)-activated ERK1/2, and in keeping with its failure to translocate to the nucleus, the beta-arrestin2-scaffolded pool of ERK1/2 does not phosphorylate the transcription factor Elk-1, induces no increased transcription of the immediate-early gene c-Fos, and does not entail myocyte hypertrophy. These results clearly demonstrate the biological significance of differential signalling by the AT(1)R. The opportunity to separate desirable cardiac myocyte division from detrimental hypertrophy holds promise that novel pharmacological approaches will allow targeting of pathway-specific actions.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Miócitos Cardíacos/enzimologia , Receptor Tipo 1 de Angiotensina/fisiologia , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Western Blotting , Proliferação de Células , Células Cultivadas , Sistema de Sinalização das MAP Quinases , Miócitos Cardíacos/efeitos dos fármacos , Fenótipo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Redistribution technology is a cell-based assay technology that uses protein translocation as the primary readout for the activity of cellular signaling pathways and other intracellular events. Protein targets are labeled with the green fluorescent protein, and stably transfected cell lines are generated. The assays are read using a high-throughput, optical microscope-based instrument, several of which have become available commercially. Protein translocation assays can be formatted as agonist assays, in which compounds are tested for their ability to promote protein translocation, or as antagonist assays, in which compounds are tested for their ability to inhibit protein translocation caused by a known agonist. Protein translocation assays are high-content, high-throughput assays primarily used for profiling of lead series, primary screening of compound libraries, and as readouts for gene-silencing studies using siRNAs. This chapter describes two novel high-content Redistribution assay technologies: (1) The p53:hdm2 GRIP interaction assay, in which one high-content image feature is used for detection of primary hits, whereas a different feature is used to deselect compounds with unwanted mode of action, and (2) application of siRNAs to Redistribution assays, exemplified by knockdown of Akt isoforms in a FKHR translocation assay reporting on the PI3 kinase signaling pathway.
Assuntos
Bioquímica/instrumentação , Bioquímica/métodos , Microscopia de Fluorescência/métodos , Transporte Proteico , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/instrumentação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Green fluorescent protein-assisted readout for interacting proteins (GRIP) is a universal protein interaction discovery system that can be used to generate truly high throughput screening-compatible cellular assays to be used to screen for inhibitors of protein-protein interactions. The technology uses a "bait and prey" principle based on the distinct translocation behavior of the human cyclic AMP phosphodiesterase 4A4. Here we use the p53-Hdm2 Redistribution assay (Fisher BioImage ApS, Søborg, Denmark) as an example to describe the GRIP technology. The p53-Hdm2 Redistribution assay is a high content imaging assay based on the GRIP technology that is designed to measure the interaction between Hdm2 and the tumor suppressor p53. Hdm2 regulates p53 and inhibits its function by modulating its transcriptional activity and stability. Activation of p53 in tumor cells through inhibition of its physical interaction with Hdm2 is therefore a focus of cancer drug discovery. We have performed a pilot screen by screening 3,165 compounds from a diverse small-molecule library for inhibitors of the p53-Hdm2 interaction by using the p53-Hdm2 Redistribution assay. Here we show that by taking advantage of the translocation behavior of nonbound p53, it is possible to identify true inhibitors of the p53-Hdm2 interaction by extracting high content information from the acquired images.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Transporte ProteicoRESUMO
G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Animais , Bioensaio , Neoplasias Ósseas , Brefeldina A/farmacologia , Células COS , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Quimiocinas CXC/farmacologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Osteossarcoma , Receptores CXCR4/agonistas , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
The mechanisms underlying targeted sorting of endocytosed receptors for recycling to the plasma membrane or degradation in lysosomes are poorly understood. In this report, the C-terminal tails of the five dopamine receptors (D1-D5) were expressed as glutathione S-transferase (GST) fusion proteins and studied for their interaction with ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) and N-ethylmaleimide-sensitive factor (NSF), which are known to be involved in post-endocytic recycling of receptors back to the plasma membrane, and with sorting nexin 1 (SNX1), known to be involved in targeting receptors to lysosomal degradation. EBP50 did not bind any of the dopamine receptor tails. NSF bound strongly to D1 and D5 and only weakly to D2, D3 and D4. However, SNX1 clearly distinguished between D1 and D5, as only D5 bound strongly to this protein. This report shows that there are distinct interaction patterns for NSF and SNX1 to the various dopamine receptor subtypes.
Assuntos
Proteínas de Transporte/metabolismo , Endocitose/fisiologia , Fosfoproteínas/metabolismo , Receptores Dopaminérgicos/metabolismo , Trocadores de Sódio-Hidrogênio , Proteínas de Transporte Vesicular , Animais , Proteínas de Transporte/genética , Escherichia coli/genética , Glutationa Transferase/metabolismo , Humanos , Proteínas Sensíveis a N-Etilmaleimida , Fosfoproteínas/genética , Isoformas de Proteínas , Ratos , Receptores Dopaminérgicos/química , Receptores Dopaminérgicos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Radioisótopos de EnxofreRESUMO
Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg(+)) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching off mechanism has been mapped to the algT/U locus, and the molecular basis for this conversion was partially attributed to mutations in the algT/U gene itself. To further characterize molecular changes resulting in the unstable phenotype, an isogenic PAO1 derivative that is constitutively Alg(+) due to the replacement of the mucA with mucA22 (PDO300) was used. The mucA22 allele is common in mucoid CF isolates. Thirty-four spontaneous nonmucoid variants, or sap (suppressor of alginate production) mutants, of PDO300 were isolated under low oxygen tension. About 40% of the sap mutants were rescued by a plasmid carrying algT/U (Group A). The remaining sap mutants were not (Group B). The members of Group B fall into two subsets: one similar to PAO1, and another comparable to PDO300. Sequence analysis of the algT/U and mucA genes in Group A shows that mucA22 is intact, whereas algT/U contains mutations. Genetic complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism for alginate synthesis suppression.
Assuntos
Alginatos/metabolismo , Proteínas de Bactérias/genética , Pseudomonas aeruginosa/genética , Fator sigma/genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Fator sigma/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition (1, 2). In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci (3). The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate (3). Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research. The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously (4, 5). Moreover, it allows temporal evaluation, inclusion of various microbial species (5) and assessment of the effects of distinct experimental conditions (e.g. treatments (6); comparison of knockout mutants vs. parental strain (5); carbohydrates availability (7)). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.
Assuntos
Biofilmes , Perfilação da Expressão Gênica/métodos , Boca/microbiologia , Streptococcus mutans/fisiologia , Imagem Molecular/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Polissacarídeos Bacterianos/análise , Polissacarídeos Bacterianos/metabolismo , Streptococcus mutans/genética , Streptococcus mutans/metabolismoRESUMO
Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one module. Galpha proteins modified in both modules are significantly more efficacious in channeling non-G(q) -selective receptors to G(q)-mediated signaling events compare with those containing each module alone. Additive effects of both modules were observed even if individual modules lacked an effect on GPCR-to-effector specificity. Dually modified Galpha proteins were also superior in conferring high-affinity agonist sites onto a coexpressed GPCR in the absence, but not in the presence, of guanine nucleotides. Together, our data suggest that receptor-G protein coupling selectivity involves cooperative interactions between the extreme C terminus and linker I of Galpha proteins and that distinct determinants of selectivity exist for individual receptors.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Glicina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Cálcio/metabolismo , Membrana Celular/metabolismo , Chlorocebus aethiops , Sequência Conservada , DNA/biossíntese , DNA/genética , Ensaio de Imunoadsorção Enzimática , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Fosfatos de Inositol/metabolismo , Ligantes , Dados de Sequência Molecular , Transdução de Sinais , TransfecçãoRESUMO
The structural organization of microbial communities is influenced by many factors, e.g. nutrient composition, shear stress and temperature. This paper presents a general method for quantitative comparison of biofilm structures and assessment of experimental reproducibility between independent biofilm experiments. By using a novel computer program, COMSTAT, biofilm structures of Pseudomonas aeruginosa and an isogenic rpoS mutant were quantified. The strains were tagged with the green fluorescent protein (GFP) and grown in flow chambers with a defined minimal medium as substrate. Three independent rounds of biofilm experiments were performed and in each round, each of the two variants was grown in two separate channels. Nine image stacks were acquired in each channel 146 h after inoculation. An analysis of variance model incorporating the factors experiment round, bacterial strain, channel number and image stack number was used to analyse the data calculated by COMSTAT. Experimental reproducibility was verified by estimating the magnitude of the variance of the effects round (sigma(2)R) and the interaction between bacterial strain and round (sigma(2)BR). Mean thickness of the wild-type and rpoS mutant biofilms was estimated at 6.31 microm (SE 0.81 microm) and 16.85 microm (SE 0.87 microm), respectively.
Assuntos
Biofilmes , Processamento de Imagem Assistida por Computador/métodos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Software , Análise de Variância , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Meios de Cultura , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Pseudomonas aeruginosa/genética , Reprodutibilidade dos Testes , Fator sigma/genética , Fator sigma/metabolismoRESUMO
We analyzed metabolic interactions and the importance of specific structural relationships in a benzyl alcohol-degrading microbial consortium comprising two species, Pseudomonas putida strain R1 and Acinetobacter strain C6, both of which are able to utilize benzyl alcohol as their sole carbon and energy source. The organisms were grown either as surface-attached organisms (biofilms) in flow chambers or as suspended cultures in chemostats. The numbers of CFU of P. putida R1 and Acinetobacter strain C6 were determined in chemostats and from the effluents of the flow chambers. When the two species were grown together in chemostats with limiting concentrations of benzyl alcohol, Acinetobacter strain C6 outnumbered P. putida R1 (500:1), whereas under similar growth conditions in biofilms, P. putida R1 was present in higher numbers than Acinetobacter strain C6 (5:1). In order to explain this difference, investigations of microbial activities and structural relationships were carried out in the biofilms. Insertion into P. putida R1 of a fusion between the growth rate-regulated rRNA promoter rrnBP1 and a gfp gene encoding an unstable variant of the green fluorescent protein made it possible to monitor the physiological activity of P. putida R1 cells at different positions in the biofilms. Combining this with fluorescent in situ hybridization and scanning confocal laser microscopy showed that the two organisms compete or display commensal interactions depending on their relative physical positioning in the biofilm. In the initial phase of biofilm development, the growth activity of P. putida R1 was shown to be higher near microcolonies of Acinetobacter strain C6. High-pressure liquid chromatography analysis showed that in the effluent of the Acinetobacter strain C6 monoculture biofilm the metabolic intermediate benzoate accumulated, whereas in the biculture biofilms this was not the case, suggesting that in these biofilms the excess benzoate produced by Acinetobacter strain C6 leaks into the surrounding environment, from where it is metabolized by P. putida R1. After a few days, Acinetobacter strain C6 colonies were overgrown by P. putida R1 cells and new structures developed, in which microcolonies of Acinetobacter strain C6 cells were established in the upper layer of the biofilm. In this way the two organisms developed structural relationships allowing Acinetobacter strain C6 to be close to the bulk liquid with high concentrations of benzyl alcohol and allowing P. putida R1 to benefit from the benzoate leaking from Acinetobacter strain C6. We conclude that in chemostats, where the organisms cannot establish in fixed positions, the two strains will compete for the primary carbon source, benzyl alcohol, which apparently gives Acinetobacter strain C6 a growth advantage, probably because it converts benzyl alcohol to benzoate with a higher yield per time unit than P. putida R1. In biofilms, however, the organisms establish structured, surface-attached consortia, in which heterogeneous ecological niches develop, and under these conditions competition for the primary carbon source is not the only determinant of biomass and population structure.
Assuntos
Acinetobacter/fisiologia , Antibiose , Biofilmes , Pseudomonas putida/fisiologia , Biofilmes/crescimento & desenvolvimentoRESUMO
Reversible phase variation between the rugose and smooth colony variants is predicted to be important for the survival of Vibrio cholerae in natural aquatic habitats. Microarray expression profiling studies of the rugose and smooth variants of the same strain led to the identification of 124 differentially regulated genes. Further expression profiling experiments showed how these genes are regulated by the VpsR and HapR transcription factors, which, respectively, positively and negatively regulate production of VPS(El Tor), a rugose-associated extracellular polysaccharide. The study of mutants of rpoN and rpoS demonstrated the effects of these alternative sigma factors on phase variation-specific gene expression. Bioinformatics analysis of these expression data shows that 'rugosity' and 'smoothness' are determined by a complex hierarchy of positive and negative regulators, which also affect the biofilm, surface hydrophobicity and motility phenotypes of the organism.
Assuntos
Adaptação Fisiológica , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Vibrio cholerae O1/genética , Vibrio cholerae O1/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/fisiologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Interações Hidrofóbicas e Hidrofílicas , Movimento , Mutação , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/metabolismo , RNA Polimerase Sigma 54 , Regulon , Proteínas Repressoras/fisiologia , Fator sigma/genética , Fator sigma/fisiologia , Transdução de Sinais , Transativadores/fisiologiaRESUMO
In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp(1) promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 microm wide in colony biofilms and 30 microm wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 microm into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.
Assuntos
Biofilmes/crescimento & desenvolvimento , Pseudomonas aeruginosa/fisiologia , Anaerobiose , Reatores Biológicos , Carbenicilina/farmacologia , Meios de Cultura , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Microscopia Eletrônica , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/ultraestrutura , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
The structural organization of four microbial communities was analysed by a novel computer program, COMSTAT, which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria, Pseudomonas: putida, P. aureofaciens, P. fluorescens and P. aeruginosa, tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure - mean thickness, roughness, substratum coverage and surface to volume ratio - showed that the four Pseudomonas: strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies, which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum, and formed flat, uniform biofilms. P. aureofaciens resembled P. aeruginosa, but had a stronger tendency to form micro-colonies. Finally, the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0.03 mM, 0.1 mM or 0.5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover, biofilm roughness increased with lower citrate concentrations, whereas surface to volume ratio increased with higher citrate concentrations.
Assuntos
Biofilmes , Processamento de Imagem Assistida por Computador/métodos , Pseudomonas/crescimento & desenvolvimento , Software , Biofilmes/crescimento & desenvolvimento , Meios de Cultura/química , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal/métodos , Filogenia , Pseudomonas/genéticaRESUMO
Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, the isolation of random mini-Tn5 insertion mutants of B. cepacia H111 defective in biofilm formation on an abiotic surface is reported. It is demonstrated that one of these mutants no longer produces N-acylhomoserine lactones (AHLs) due to an inactivation of the cepR gene. cepR and the cepI AHL synthase gene together constitute the cep quorum-sensing system of B. cepacia. By using a gene replacement method, two defined mutants, H111-I and H111-R, were constructed in which cepI and cepR, respectively, had been inactivated. These mutants were used to demonstrate that biofilm formation by B. cepacia H111 requires a functional cep quorum-sensing system. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains suggested that the quorum-sensing system is not involved in the regulation of initial cell attachment, but rather controls the maturation of the biofilm. Furthermore, it is shown that B. cepacia is capable of swarming motility, a form of surface translocation utilized by various bacteria to rapidly colonize appropriate substrata. Evidence is provided that swarming motility of B. cepacia is quorum-sensing-regulated, possibly through the control of biosurfactant production. Complementation of the cepR mutant H111-R with different biosurfactants restored swarming motility while biofilm formation was not significantly increased. This result suggests that swarming motility per se is not essential for biofilm formation on abiotic surfaces.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Burkholderia cepacia/fisiologia , Ligases , Aderência Bacteriana/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Sequência de Bases , Infecções por Burkholderia/microbiologia , Burkholderia cepacia/genética , Burkholderia cepacia/patogenicidade , Fibrose Cística/microbiologia , DNA Bacteriano/genética , Genes Bacterianos , Humanos , Pulmão/microbiologia , Movimento , Mutagênese Insercional , Infecções Oportunistas/microbiologia , Fenótipo , Propriedades de SuperfícieRESUMO
Biofilm formation by Gfp-tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time-lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild-type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild-type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild-type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.
Assuntos
Biofilmes/crescimento & desenvolvimento , Fímbrias Bacterianas/metabolismo , Flagelos/metabolismo , Mutação , Pseudomonas aeruginosa/fisiologia , Citratos/metabolismo , Meios de Cultura , Fímbrias Bacterianas/genética , Flagelos/genética , Proteínas de Fluorescência Verde , Processamento de Imagem Assistida por Computador , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , SoftwareRESUMO
Four strains of Pseudomonas aeruginosa (wild type, Delta(pil)HIJK mutant, lasI mutant, and rpoS mutant) were genetically tagged with the green fluorescent protein, and the development of flow chamber-grown biofilms by each of them was investigated by confocal laser scanning microscopy. The structural developments of the biofilms were quantified by the computer program COMSTAT (A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, Microbiology 146:2395-2407, 2000). Two structural key variables, average thickness and roughness, formed the basis for an analysis of variance model comprising the four P. aeruginosa strains, five time points (55, 98, 146, 242, and 314 h), and three independent rounds of biofilm experiments. The results showed that the wild type, the Delta(pil)HIJK mutant, and the rpoS mutant display conspicuously different types of temporal biofilm development, whereas the lasI mutant was indistinguishable from the wild type at all time points. The wild type and the lasI mutant formed uniform, densely packed biofilms. The rpoS mutant formed densely packed biofilms that were significantly thicker than those of the wild type, whereas the Delta(pil)HIJK mutant formed distinct microcolonies that were regularly spaced and almost uniform in size. The results are discussed in relation to the current model of P. aeruginosa biofilm development.