RESUMO
In this research, we examined the production of hyaluronic acid (HA) by Streptococcus zooepidemicus strain MW26985 using different substrates and potato peel waste (PPW) as an affordable substrate. First, culture medium components, including carbon and nitrogen sources, were optimized for bacterial HA production. Five different carbon sources (glucose, sucrose, lactose, sago starch, and potato starch, at a concentration of 30 g/L) and three distinct nitrogen sources (peptone, yeast extract, and ammonium sulfate, at a concentration of 10 g/L) were investigated. Glucose, among the carbon sources, and yeast extract, among nitrogen sources, produced the most HA which was determined as 1.41 g/L. Afterward, potato peel sugars were extracted by dilute acid and enzymatic hydrolysis and then employed as a cost-effective carbon source for the growth of S. zooepidemicus. Based on the results, the fermentation process yielded 0.59 g/L HA from potato peel sugars through acid hydrolysis and 0.92 g/L HA from those released by enzymatic hydrolysis. The supplementation of both hydrolyzates with glucose as an additional carbon source enhanced HA production to 0.95 g/L and 1.18 g/L using acidic and enzymatic hydrolyzates, respectively. The cetyltrimethylammonium bromide (CTAB) turbidimetric method was used to evaluate the concentration of HA in the fermentation broth using the colorimetric method. Also, the peaks observed by Fourier transform infrared (FTIR) spectroscopy confirmed that the exopolysaccharide (EPS) was composed of HA. These observations demonstrate that potato peel residues can be a novel alternative as a carbon source for the economical production of HA by S. zooepidemicus.
Assuntos
Ácido Hialurônico , Solanum tuberosum , Streptococcus equi , Ácido Hialurônico/biossíntese , Streptococcus equi/metabolismo , Streptococcus equi/crescimento & desenvolvimento , Hidrólise , Fermentação , Meios de Cultura/química , Carbono/metabolismoRESUMO
The present study is focused on systematic process and kinetic investigation of hyaluronic acid (HA) production strategy unraveling the role of dissolved oxygen (DO) and N-acetyl glucosamine (GlcNAc) towards the enhancement of HA titer and its molecular weight. Maintaining excess DO levels (10-40% DO) through DO-stat control and the substitution of GlcNAc at a range (5-20 g/L) with glucose (Glc) critically influenced HA production. DO-stat control strategy yielded a promising HA titer (2.4 g/L) at 40% DO concentration. Controlling DO level at 20% (DO-stat) was observed to be optimum resulting in a significant HA production (2.1 g/L) and its molecular weight ranging 0.98-1.45 MDa with a consistent polydispersity index (PDI) (1.57-1.69). Substitution of GlcNAc with Glc at different proportions explicitly addressed the metabolic trade-off between HA titer and its molecular weight. GlcNAc substitution positively influenced the molecular weight of HA. The highest HA molecular weight (2.53 MDa) of two-fold increase compared with glucose as sole carbon substrate and narrower PDI (1.35 ± 0.18) was achieved for the 10:20 (Glc:GlcNAc) proportion. A novice attempt on modeling the uptake of dual substrates (Glc and GlcNAc) by Streptococcus zooepidemicus for HA production was successfully accomplished using double Andrew's growth model and the kinetic parameters were estimated reliably.
Assuntos
Acetilglucosamina/metabolismo , Ácido Hialurônico/biossíntese , Oxigênio/metabolismo , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/metabolismo , Biomassa , Fermentação , Glucose/metabolismo , Cinética , Peso MolecularRESUMO
Streptococcus zooepidemicus is an important opportunistic pathogen of several species including humans. This organism is also well-known as the main producing strain in industrial production of hyaluronic acid (HA), which is the component of its capsule polysaccharide. How its virulence and capsule polysaccharide production is regulated remains poorly understood. Intercellular chemical signaling among bacteria provides communities of microbes the opportunity to coordinate gene expression to facilitate group behavior, such as pathogenicity, capsule polysaccharide production, etc. Yet no conserved cell-to-cell signaling system has been elucidated in S. zooepidemicus. Encoded within the genome of S. zooepidemicus is one Rgg regulator encoding gene (rgg) with low similarity to both rgg2 and rgg3 from Streptococcus pyogenes. A small ORF (named as shp) encoding a novel short hydrophobic peptide (SHP) was found in the vicinity of rgg. We found that the active form of pheromone is short and hydrophobic (LLLLKLA), corresponding to the C terminal 7 amino acids of the pre-peptide Shp, which shows divergent sequence to all peptide pheromones reported in streptococci. In response to active SHP, Rgg functions as a transcriptional activator to induce the expression of shp, forming a positive feedback circuit. Bacteria social behaviors, such as capsule polysaccharide production and biofilm formation, were significantly affected when the rgg-shp pathway was inactivated. These data provide the first demonstration that Rgg/Shp signaling pathway comprises an active quorum sensing system in S. zooepidemicus.
Assuntos
Cápsulas Bacterianas/metabolismo , Biofilmes/crescimento & desenvolvimento , Feromônios/metabolismo , Percepção de Quorum , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/metabolismo , Regulação Bacteriana da Expressão Gênica , Transdução de Sinais , Streptococcus equi/genética , Transcrição GênicaRESUMO
After strangles outbreaks, Streptococcus equi ssp. equi (S. equi) can persist in clinically normal silent carriers for months to years. Two naturally occurring outbreaks of strangles with 53 and 100% morbidity, respectively, were followed longitudinally to assess occurrence of carrier state and optimal detection methods Outbreak A involved 98 yearling warmbloods, and outbreak B 38 mature Icelandic horses. Fully recovered horses were sampled at least 6 months after index cases using nasal swabs (one sampling occasion only) nasopharyngeal lavage and guttural pouch visualisation and lavages for culture and qPCR to S. equi. Any horse with at least a single sample positive was deemed a carrier. Descriptive statistics and sensitivity and negative predictive values were calculated. Comparisons were made with McNemars and Fishers exact tests. Carrier rates in outbreak A were 3% based on culture and 15% based on qPCR and for outbreak B 13% based on culture and 37% based on qPCR. All culture positives were also qPCR positive. One carrier culture negative sampled after an additional 8 months was culture positive to S. equi, indicating that qPCR positives should be suspected to carry live bacteria. Findings indicate that reliance on guttural pouch sampling and appearance does not capture all silent carriers. All culture positives were identified by qPCR and even horses positive by qPCR but culture negative should be suspected carriers of live bacteria.
Assuntos
Portador Sadio/veterinária , Orelha Média/microbiologia , Doenças dos Cavalos/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Infecções Estreptocócicas/veterinária , Streptococcus equi/isolamento & purificação , Animais , Portador Sadio/diagnóstico , Portador Sadio/epidemiologia , Surtos de Doenças/veterinária , Feminino , Doenças dos Cavalos/microbiologia , Cavalos , Estudos Longitudinais , Masculino , Lavagem Nasal/veterinária , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/epidemiologia , Streptococcus equi/crescimento & desenvolvimentoRESUMO
BACKGROUND: Streptococcus equi represents a common hazard to equids worldwide. Environmental contamination with bacteria shed from an infected horse may represent a significant source of contagion and further knowledge of ex vivo bacterial survival under different conditions is important for disinfection and isolation protocols. OBJECTIVES: To determine the potential duration of survival and vigour of growth of S. equi inoculated onto surfaces relevant to equine veterinary practice and stabling in summer and winter. STUDY DESIGN: Repeat sampling of environmental inocula of S. equi. METHODS: Cultures of S. equi were inoculated onto wood, a shoe sole, cotton overalls, inside a nasogastric tube, inside a dental rasp, in a wet plastic bucket and onto a fence post both in the summer and winter seasons. Frequent resampling and culture from the inoculated sites was conducted until no viable bacteria were found. Bacterial viability was determined by both duration (time to first negative culture) and vigour of growth (growth score over the first 3 days of culture) and compared between inoculated sites and times of year. RESULTS: Bacterial viability was enhanced by a wet local environment and by the winter season. Survival tended to be short in the summer (up to 9 days in wet sites and up to 2 days in dry sites) but much longer in the winter (up to 34 days in wet sites and up to 13 days in dry sites). Vigour of bacterial growth was also greater in the winter than in the summer as judged by 3-day-growth scores. MAIN LIMITATIONS: Direct comparison with the variable size and nature of naturally shed infectious material is difficult. CONCLUSIONS: Veterinarians and personnel handling horses should be aware that S. equi may survive in an equine environment for longer than previously found, especially when protected by wet and cold conditions.
Assuntos
Microbiologia Ambiental , Streptococcus equi/crescimento & desenvolvimento , Animais , Temperatura Baixa , Fibra de Algodão/microbiologia , Instrumentos Odontológicos/microbiologia , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/microbiologia , Cavalos , Intubação Gastrointestinal/instrumentação , Plásticos , Estações do Ano , Sapatos , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/veterinária , Microbiologia da Água , Madeira/microbiologiaRESUMO
Hyaluronidase (hyase) is a glycosidase enzyme that predominantly degrades hyaluronic acid (HA) having important applications in many biotechnological processes and therapeutics. Several assay methods have been proposed to screen hyase producing microorganisms; however, they rely on unique reagents and sophisticated instruments, which are expensive and could be unavailable in general laboratories. In the present studies, a rapid, simple, sensitive, highly reproducible, and cost-effective qualitative plate assay has been developed for the screening of hyase producing microorganisms. The routinely used plate assay method of Richman and Baer requires a special chemical cetylpyridinium chloride and long incubation period of 20 h; but still, the zones of clearance are not very clear and distinct. While, the present method requires an incubation period of only 1 h and the distinct zones of clearance appear with Gram's iodine within 1 min of time. This method does not require any special medium, unlike previously reported methods. Moreover, use of commonly available Gram's iodine makes this method suitable for many researchers. The results of the assay method were validated by TLC, zymographic analysis and determining the growth of isolates in minimal medium containing HA as a sole carbon source.
Assuntos
Ensaios Enzimáticos/métodos , Hialuronoglucosaminidase/isolamento & purificação , Streptococcus equi/enzimologia , Meios de Cultura/química , Ensaios Enzimáticos/economia , Humanos , Ácido Hialurônico/isolamento & purificação , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Iodo , Sensibilidade e Especificidade , Sefarose , Streptococcus equi/química , Streptococcus equi/crescimento & desenvolvimento , Streptococcus mitis/enzimologiaRESUMO
Swine streptococcosis is a significant threat to the Chinese pig industry, and Streptococcus equi ssp. zooepidemicus (SEZ) is one of the major pathogens. SEZ ATCC35246 is a classical virulent strain, while SEZ ST171 is a Chinese attenuated vaccine strain. In this study, we employed stable isotope labeling by amino acids in cell culture and liquid chromatography-mass spectrometry (LC-MS) to determine the differential response of macrophages to infection by these two strains. Eighty-seven upregulated proteins and 135 downregulated proteins were identified. The proteomic results were verified by real-time polymerase chain reaction for 10 chosen genes and Western blotting for three proteins. All differentially abundant proteins were analyzed for their Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations. Certain downregulated proteins were associated with immunity functions, and the upregulated proteins were related to cytomembrane and cytoskeleton regulation. The phagocytosis rate and cytokine genes transcription in Raw264.7 cells during SEZ ATCC35246 and ST171 infection were detected to confirm the bioinformatics results. These results showed that different effects on macrophage phagocytosis and cytokine expression might explain the different phenotypes of SEZ ATCC35246 and ST171 infection. This research provided clues to the mechanisms of host immunity responses to SEZ ST171and SEZ ATCC35246, which could identify potential therapy and vaccine development targets.
Assuntos
Citocinas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/imunologia , Fagocitose , Proteoma/imunologia , Streptococcus equi/patogenicidade , Animais , Linhagem Celular , Cromatografia Líquida , Citocinas/genética , Regulação da Expressão Gênica/imunologia , Ontologia Genética , Marcação por Isótopo , Macrófagos/microbiologia , Camundongos , Anotação de Sequência Molecular , Proteoma/genética , Especificidade da Espécie , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Vacinas Estreptocócicas/imunologia , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/isolamento & purificação , Suínos , Doenças dos Suínos/microbiologia , Doenças dos Suínos/patologia , Espectrometria de Massas em Tandem , VirulênciaRESUMO
AIMS: To compare the rate of growth of four microbial strains that cause disease in the horse, on four commonly used types of bedding. The moisture-holding capacity of each bedding type was also tested. METHODS AND RESULTS: Microbial strains included Streptococcus equi, Streptococcus zooepidemicus, Fusobacterium necrophorum, Dichelobacter nodosus and Dermatophilus congolensis. The bedding types tested were Pinus sylvestris (Scots pine shavings), Pinus nigra (Corsican pine shavings), Picea sitchensis (Sitka spruce shavings), Cannabis sativa (hemp) and chopped wheat straw. A suspension of each microbial strain was spread in triplicate on agar media and incubated in its optimal growth conditions. The viable count (colony-forming unit per ml) was determined for each bacterial strain for the five different bedding types. Pinus sylvestris bedding resulted in significantly less (P = 0·001) bacterial growth of all strains tested. CONCLUSIONS: Factors resulting in the inhibition of bacterial growth include the antibacterial effects reported in the Pinacea family and the physical properties of the bedding substrate. Research is currently focussed on the diagnosis and management of disease. Prevention of disease is also important for matters of biosecurity. Strategies should include the provision of a hygienic environment and the use of specific types of bedding. SIGNIFICANCE AND IMPACT OF THE STUDY: Bedding choice has implications for global equine health and disease prevention as well as potential benefits in other animal species.
Assuntos
Actinobacteria/crescimento & desenvolvimento , Infecções Bacterianas/veterinária , Microbiologia Ambiental , Fusobacterium/crescimento & desenvolvimento , Doenças dos Cavalos/microbiologia , Abrigo para Animais , Streptococcus/crescimento & desenvolvimento , Actinobacteria/classificação , Actinobacteria/fisiologia , Animais , Infecções Bacterianas/microbiologia , Infecções Bacterianas/prevenção & controle , Infecções Bacterianas/transmissão , Cannabis , Fusobacterium/fisiologia , Doenças dos Cavalos/prevenção & controle , Doenças dos Cavalos/transmissão , Cavalos , Higiene , Pinus , Streptococcus/classificação , Streptococcus/fisiologia , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/fisiologia , TriticumRESUMO
The mariner-based Himar1 system has been utilized for creating mutant libraries of many Gram-positive bacteria. Streptococcus suis serotype 2 (SS2) and Streptococcus equi ssp. zooepidemicus (SEZ) are primary pathogens of swine that threaten the swine industry in China. To provide a forward-genetics technology for finding virulent phenotype-related genes in these two pathogens, we constructed a novel temperature-sensitive suicide shuttle plasmid, pMar4s, which contains the Himar1 system transposon, TnYLB-1, and the Himar1 C9 transposase from pMarA and the repTAs temperature-sensitive fragment from pSET4s. The kanamycin (Kan) resistance gene was in the TnYLB-1 transposon. Temperature sensitivity and Kan resistance allowed the selection of mutant strains and construction of the mutant library. The SS2 and SEZ mutant libraries were successfully constructed using the pMar4s plasmid. Inverse-Polymerase Chain Reaction (Inverse-PCR) results revealed large variability in transposon insertion sites and that the library could be used for phenotype alteration screening. The thiamine biosynthesis gene apbE was screened for its influence on SS2 anti-phagocytosis; likewise, the sagF gene was identified to be a hemolytic activity-related gene in SEZ. pMar4s was suitable for mutant library construction, providing more information regarding SS2 and SEZ virulence factors and illustrating the pathogenesis of swine streptococcosis.
Assuntos
Mutação , Plasmídeos/genética , Streptococcus equi/crescimento & desenvolvimento , Streptococcus suis/crescimento & desenvolvimento , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/genética , China , Elementos de DNA Transponíveis , Genes Transgênicos Suicidas , Sorogrupo , Streptococcus equi/genética , Streptococcus equi/patogenicidade , Streptococcus suis/genética , Streptococcus suis/patogenicidade , Suínos , TemperaturaRESUMO
The biosynthetic pathway for hyaluronic acid (HA) has been proposed; however, a thorough genetic and functional analysis is required to further elucidate the roles of genes involved in HA production. Previously, we developed a markerless gene-deletion system for Streptococcus zooepidemicus and confirmed that hasA is essential for HA synthesis. Here, we constructed a comprehensive set of deletion mutants and investigated the roles of ten additional predicted genes in the HA synthetic pathway. Phenotypic assays revealed that all ten genes play a role in cell growth and/or HA synthesis. As expected, the deletion of hasA or hasB abolished HA production with little effect on growth, while the deletion of genes that are also required for peptidoglycan biosynthesis (hasE, glmM, and glmS) significantly reduced cell growth and HA production. Either of the glmU homologues (hasD and gcaD) was sufficient for optimal growth and the mucoid phenotype, while no double mutant could be isolated. Of the two UDP-glucose pyrophosphorylase (UGPase) paralogues, the operon-encoded hasC1 was responsible for 65 % of the activity, while hasC2 was responsible for the remaining 35 %. The deletion of hasC1 had no effect on cell growth and caused only a moderate decrease in the UDP-glucose level and HA production. The deletion of both hasC1 and hasC2 resulted in a severe growth defect and negligible UDP-glucose accumulation, HA production, and pyrophosphorylase activity. Of the two phosphoglucomutase paralogues, pgm1 and pgm2, the former is responsible for around 10 % of activity, while the latter is responsible for 90 %. The deletion of pgm1 showed no apparent effect on HA synthesis and growth, while the deletion of pgm2 resulted in the abolishment of HA synthesis and a significantly slower growth. These results should guide the metabolic engineering of S. zooepidemicus to improve HA productivity and quality.
Assuntos
Proteínas de Bactérias/genética , Ácido Hialurônico/biossíntese , Streptococcus equi/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Óperon , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/metabolismoRESUMO
Thrombolytic therapy for acute myocardial infarction standardly makes use of the medications streptokinase (SK) and tissue plasminogen activator (tPA). In this study, the potential of silica-coated magnetic nanoparticles (SiO2-MNPs) as nanocarriers clinical thrombolytic therapy was investigated. SiO2-MNPs for use in targeted therapeutic delivery of tPA and SK were prepared using a combined technique incorporating controlled precipitation and hydrothermal methods. Response surface methodology (RSM) was employed to evaluate the efficiency of the SiO2-MNPs. The production of SK secreted from Streptococcus equi was enhanced using random mutagenesis. The tPA and SK A were encapsulated by means of a silanizing agent with a surface rich in 3-aminopropyltrimethoxysilane layered around the SiO2-MNPs. Blood clot lysis assays and fibrin-containing agarose plates were used to carry out in vitro thrombolysis testing. The optimum conditions for producing MNPs were found to be at pH=13 and at a temperature of 75°C for 45 min. Culture conditions of 2.75% NaCl concentration at initial pH=7.5 for 90 s under UV resulted in maximum SK activity. The tPA/SK-conjugated SiO2-MNPs (SiO2-MNP-tPA-SK) increased operating stability in whole blood and storage stability in a buffer by 92%. More effective thrombolysis using magnetic targeting was indicated by a 38% reduction in blood clot lysis time achieved with SiO2-MNP-tPA-SK compared to administering the SiO2-MNPs without guidance. The silica-coated magnetic nanocarriers developed in this study show potential for improved clinical thrombolytic therapy.
Assuntos
Portadores de Fármacos , Nanopartículas de Magnetita/química , Estreptoquinase/administração & dosagem , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/administração & dosagem , Doenças Vasculares/tratamento farmacológico , Química Farmacêutica/métodos , Estabilidade de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Tamanho da Partícula , Propilaminas/química , Silanos/química , Dióxido de Silício/química , Streptococcus equi/enzimologia , Streptococcus equi/crescimento & desenvolvimento , Estreptoquinase/farmacologia , Propriedades de Superfície , Temperatura , Ativador de Plasminogênio Tecidual/farmacologiaRESUMO
Endometritis in horses caused by Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) may be underdiagnosed due to traditional diagnostic methods lacking sensitivity and specificity. We serendipitously identified a bacterial growth medium (bActivate) that appeared capable of inducing growth of dormant S. zooepidemicus, which subsequently allowed detection by standard diagnostics. To assess the effect of bActivate we compared its ability to activate dormant S. zooepidemicus in a group of potentially infected subfertile mares with phosphate-buffered saline (PBS). All mares had to test negative for S. zooepidemicus on a low-volume uterine lavage, be negative on endometrial cytology and without clinical signs of endometritis to be included in the investigation. The mares were instilled with bActivate or PBS in the uterus. Growth of S. zooepidemicus was induced by bActivate in 64% (16/25) and PBS in 8% (1/12) of the mares, respectively (p<0.002). In vitro studies supported that some strains of S. zooepidemicus were able to form persister cells tolerating 32-times of the minimal inhibitory concentration of penicillin compared to normal growing cells. Persister cells had not acquired penicillin resistance, but seemed to tolerate the antimicrobial due to dormancy. This is, to our knowledge, the first description of controlled growth induction of dormant bacteria from a subclinical infection. Moreover we demonstrated how endometritis can origin from a reservoir of dormant bacteria residing within the endometrium, and not only as an ascending infection. Further studies should aim at determining the prevalence of dormant S. zooepidemicus, impact of activation on diagnostic and treatment efficacy, uterine health and mare fertility.
Assuntos
Endometrite/veterinária , Doenças dos Cavalos/diagnóstico , Infecções Estreptocócicas/veterinária , Streptococcus equi/crescimento & desenvolvimento , Animais , Infecções Assintomáticas , Endometrite/diagnóstico , Endometrite/microbiologia , Endométrio/microbiologia , Feminino , Doenças dos Cavalos/microbiologia , Cavalos , Testes de Sensibilidade Microbiana/veterinária , Sensibilidade e Especificidade , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/microbiologia , Streptococcus equi/isolamento & purificaçãoRESUMO
Infection with Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) can cause septicemia, meningitis, and mastitis in domesticated species. Identification of this organism's virulence factors is an effective way of clarifying its pathogenic mechanism. We employed in vivo-induced antigen technology (IVIAT) to find bacterial genes that were only expressed or upregulated in an infected host (IVI genes). Convalescent-phase sera from pigs infected with SEZ were pooled, adsorbed against in vitro antigens, and used to screen SEZ genomic expression libraries. This analysis identified 43 genes as IVI genes. Six of these 43 genes were verified via real-time PCR. Following the analysis, we were able to assign a putative function to 36 of the 43 proteins. These proteins included those involved in virulence and adaptation; formation of intermediary products; gene replication, transcription and expression; energy metabolism; transport and also various proteins of unknown function. The relationship between sagD gene and bacterial virulence was confirmed. This study provides new molecular data for the study of streptococcal disease in swine and is important for identifying the pathogenic mechanisms of SEZ.
Assuntos
Perfilação da Expressão Gênica , Genes Bacterianos , Interações Hospedeiro-Patógeno , Infecções Estreptocócicas/veterinária , Streptococcus equi/crescimento & desenvolvimento , Fatores de Virulência/biossíntese , Animais , Infecções Estreptocócicas/microbiologia , Streptococcus equi/genética , Suínos , Fatores de Virulência/genéticaRESUMO
AIM: Selection of high-mucoid morphotype of Streptococcus equi subsp. zooepidemicus (Streptococcus zooepidemicus) and study of its morphological, physiological, biochemical and technological characteristics for providing increased secretion of hyaluronic acid (HA). MATERIALS AND METHODS: Submerged cultivation was performed in 100 ml glass flasks without baffles or in 1.5 or 10 1 laboratory bioreactors. LB and MRS media were used for cultivation. Mutagenesis was carried out by UV exposure with consequent selection of mucoid phenotype. HA was determined by carbazole method or after exhaustive acid hydrolysis by reaction of N-acetylglucosamine with Morgan-Elson reagent. Total hyaluronidase activity was evaluated by viscosimeter. Determination of cell and capsule size, ability to ferment carbohydrates and other microbiological, physiological and biochemical tests were performed by standard techniques. RESULTS: Instability of capsule phenotype of S. zooepidemicus B-8014 strain was revealed that is explained most probably by formation under certain conditions of bacterial hyaluronidase. This is confirmed by a reduction of HA concentration in cultural medium at pre- and stationary growth phases. Mucoid strain S. zooepidemicus KB-04 was obtained by mutagenesis with subsequent selection that is characterized by increased capsules. The strain was studied for HA formation. Optimization of growth medium composition, physical-chemical conditions and modes of cultivation allowed to significantly increase HA yield. CONCLUSION: The studies of morphologic, physiologic, biochemical and technological characteristics of the high-mucoid S. zooepidemicus KB-04 strain obtained by mutagenesis with consequent selection were performed, conditions of its cultivation and composition of growth mediu by carbon source and content of bivalent metal ions were optimized.
Assuntos
Cápsulas Bacterianas/efeitos da radiação , Ácido Hialurônico/biossíntese , Streptococcus equi/metabolismo , Cápsulas Bacterianas/ultraestrutura , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Cálcio/metabolismo , Meios de Cultura , Fermentação , Glucose/metabolismo , Ácido Hialurônico/isolamento & purificação , Hialuronoglucosaminidase/metabolismo , Magnésio/metabolismo , Mutagênese , Seleção Genética , Streptococcus equi/genética , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/efeitos da radiação , Raios UltravioletaRESUMO
Here we show that mast cells (MCs) express the metalloproteases of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, and that ADAMTS expression is influenced by MC activation. Co-culture of MCs with live Gram-positive bacteria caused a profound induction of ADAMTS-9 and -6, as well as down-regulated expression of ADAMTS-5. Similar patterns were also seen after MC activation with calcium ionophore and by immunoglobulin E receptor crosslinking. Moreover, ADAMTS-5, -6 and -9 were all induced by activation of terminally differentiated murine peritoneal MCs and in a human MC line. ADAMTS-9 up-regulation in response to immunoglobulin E receptor crosslinking was strongly dependent on Gö6976-sensitive protein kinase C and partly dependent on nuclear factor of activated T cells and nuclear factor kappa-light-chain-enhancer of activated B cells, respectively. The expression of ADAMTS-5, -6 and -9 was closely linked to MC maturation, as shown by their strong induction during the differentiation of bone marrow precursor cells into mature MCs. ADAMTS family members have been shown to possess aggrecanase activity. Accordingly, MCs were shown to express aggrecanase activity. Finally, ADAMTS-5 protein was detected in MCs by immunocytochemistry. Taken together, the present study reveals ADAMTS expression by MCs and that MC activation regulates the expression of these proteases, thus implicating the ADAMTS family of proteases in MC function.
Assuntos
Proteínas ADAM/biossíntese , Proteínas ADAM/genética , Mastócitos/citologia , Mastócitos/enzimologia , Proteínas ADAM/metabolismo , Proteína ADAMTS5 , Proteína ADAMTS9 , Animais , Linhagem Celular , Técnicas de Cocultura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/fisiologiaRESUMO
The cashew fruit (Anacardium occidentale L.) has been used as a promising agricultural resource for the production of low-molecular weight (M(W)) hyaluronic acid (HA) (10(4)-10(5) Da). The cashew juice is a rich source of vitamin C containing, 1.2-2.0 g L(-1). This work explores the effects of the initial concentration of the ascorbate on the solid fermentation of the juice-moisturized bagasse from the cashew apple fruit. The results show that the M(W) reduction of HA is proportional to the initial ascorbate concentration. The presence of ascorbate did not influence the Streptococcus zooepidemicus metabolism. However, the HA productivity was increased from 0.18 to 0.28 mg g(-1) h(-1) when the ascorbate concentration ranged from 1.7 to 10 mg mL(-1). These findings contribute to the controlled production of HA in a low M(W) range, which is important in cell signalization, angiogenesis and nanoparticles production.
Assuntos
Anacardium/metabolismo , Ácido Ascórbico/metabolismo , Celulose/metabolismo , Ácido Hialurônico/metabolismo , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/metabolismo , Anacardium/química , Celulose/química , Fermentação , Ácido Hialurônico/química , Hidrólise , Peso Molecular , OxirreduçãoRESUMO
Streptococcus equi is the etiologic agent of a highly infectious upper respiratory disease of horses known as strangles. Bacterial culture methods and polymerase chain reaction (PCR) of nasopharyngeal washes and guttural pouch lavages are used routinely to test clinical and carrier animals for the presence of S. equi but no definitive or gold standard test method has been shown to be optimal. We hypothesized that (i) a flocked swab submerged in ten-fold serial dilution suspensions of S. equi prepared in 0.9% NaCl would detect more colony forming units (CFU) than a rayon swab when used to inoculate a blood agar plate, (ii) centrifugation of a 1 ml aliquot of each suspension would improve the limit of detection (LOD) by bacterial culture and PCR compared to the culture or PCR of submerged swab samples, (iii) PCR of the centrifuged samples from each suspension would be more sensitive than aerobic culture alone, and (iv) PCR of a 1 ml aliquot directly from a sample would be more sensitive than PCR of a sample following submersion of a flocked swab in 1 ml saline. Using 7 ten-fold serial dilutions of S. equi in 0.9% NaCl, the LOD for 4 bacterial culture methods and 3 PCR methods were compared. The LOD of direct PCR and flocked swab culture was determined at 1 cfu/ml. All PCR methods were equivalent to each other and were more sensitive than any of the culture methods at the lower dilutions. At higher cell densities (>100 cfu/ml) flocked swab culture was not statistically better than rayon swab culture, but it was superior to all other methods tested.
Assuntos
Técnicas Bacteriológicas/métodos , Doenças dos Cavalos/microbiologia , Linfadenite/veterinária , Reação em Cadeia da Polimerase/métodos , Infecções Estreptocócicas/veterinária , Streptococcus equi/isolamento & purificação , Animais , Técnicas Bacteriológicas/veterinária , Portador Sadio/diagnóstico , Portador Sadio/microbiologia , Portador Sadio/veterinária , Doenças dos Cavalos/diagnóstico , Cavalos , Linfadenite/diagnóstico , Linfadenite/microbiologia , Nasofaringe/microbiologia , Reação em Cadeia da Polimerase/veterinária , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/microbiologia , Infecções Respiratórias/veterinária , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/microbiologia , Streptococcus equi/genética , Streptococcus equi/crescimento & desenvolvimentoRESUMO
The effect of phosphatidylcholine on the molecular weight properties of hyaluronic acid (HA) was studied in batch culture of Streptococcus zooepidemicus by adding phosphatidylcholine at the early stage of exponential phase. With the addition of 80 mg/L of phosphatidylcholine, maximum HA yield (2.47 g/L) and weight-average molecular weight (902.60 KDa) were achieved, increased by 17.4% and 67.1%, respectively, as compared to the control. Metabolic flux analysis was employed to study the mechanism of phosphatidylcholine on the molecular weight of HA. The normalized flux distribution maps based on fermentation data at phosphatidylcholine addition indicated that phosphatidylcholine resulted in higher flux flowing to the HA pathway and lower flux flowing to the glycolysis and biomass synthesis pathway, coupling with higher level of UDPNAG generation and extra regeneration of ATP. The GC-MS analysis of fatty acids in the plasma membrane showed that the addition of phosphatidylcholine could promote the mobility and permeability of the cell membrane, making the HA chain pass through the membrane more easily, thus decreasing the energy consumption. All these results led to higher molecular weight of hyaluronic acid.
Assuntos
Ácido Hialurônico/biossíntese , Ácido Hialurônico/química , Fosfatidilcolinas/farmacologia , Streptococcus equi/efeitos dos fármacos , Streptococcus equi/metabolismo , Acetatos/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ácidos Graxos/análise , Fermentação/efeitos dos fármacos , Glucose/farmacologia , Ácido Láctico/metabolismo , Lipídeos de Membrana/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Peso Molecular , Streptococcus equi/citologia , Streptococcus equi/crescimento & desenvolvimentoRESUMO
Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the (8)F1(9)F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to (8)F1(9)F1 and that UR binding to (8)F1 is likely to occur through anti-parallel ß-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem ß-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.
Assuntos
Fibronectinas/metabolismo , Gelatina/metabolismo , Proteínas Recombinantes/metabolismo , Streptococcus equi/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Fibronectinas/química , Fibronectinas/genética , Gelatina/química , Gelatina/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Streptococcus equi/genética , Streptococcus equi/crescimento & desenvolvimentoRESUMO
The objective of the present work was to evaluate the metabolic effects induced by the initial glucose concentration (IGC) on the cultivation of Streptococcus zooepidemicus for the production of hyaluronic acid (HA). These effects were monitored along non-controlled pH cultivations, carried out in 250-mL Erlenmeyer flasks (natural aeration) and in a 3-L bioreactor (forced aeration) as well. Effects of the IGC were observed with focus on the main metabolites, cell growth, production, and average molecular weight of HA. The absence of glucose resulted in a mixed acid metabolism independent of the oxygen supply, while, for IGCs ranging from 5 to 90 g L(-1), the homolactic metabolism was prevalent. The IGC had no influence on the amounts of either biomass or HA produced in the cultivations carried out in flasks; however, cultivations in 3-L bioreactor were found to be strongly dependent on it. The highest concentration of HA (1.21 g L(-1)) was obtained from 25 g L(-1) IGC, the only cultivation where the conversion of glucose to HA was higher than the one of glucose to biomass. Average molecular weight of HA increased concomitant with the IGC, independently of aeration; nevertheless, it decreased along cultivation under forced aeration, due to the shear imparted by stirring.