RESUMO
Ureaplasma species (spp.) are considered commensals of the adult genitourinary tract, but have been associated with chorioamnionitis, preterm birth, and invasive infections in neonates, including meningitis. Data on mechanisms involved in Ureaplasma-driven neuroinflammation are scarce. The present study addressed brain inflammatory responses in preterm lambs exposed to Ureaplasma parvum (UP) in utero. 7 days after intra-amniotic injection of UP (n = 10) or saline (n = 11), lambs were surgically delivered at gestational day 128-129. Expression of inflammatory markers was assessed in different brain regions using qRT-PCR and in cerebrospinal fluid (CSF) by multiplex immunoassay. CSF was analyzed for UP presence using ureB-based real-time PCR, and MRI scans documented cerebral white matter area and cortical folding. Cerebral tissue levels of atypical chemokine receptor (ACKR) 3, caspases 1-like, 2, 7, and C-X-C chemokine receptor (CXCR) 4 mRNA, as well as CSF interleukin-8 protein concentrations were significantly increased in UP-exposed lambs. UP presence in CSF was confirmed in one animal. Cortical folding and white matter area did not differ among groups. The present study confirms a role of caspases and the transmembrane receptors ACKR3 and CXCR4 in Ureaplasma-driven neuroinflammation. Enhanced caspase 1-like, 2, and 7 expression may reflect cell death. Increased ACKR3 and CXCR4 expression has been associated with inflammatory central nervous system (CNS) diseases and impaired blood-brain barrier function. According to these data and previous in vitro findings from our group, we speculate that Ureaplasma-induced caspase and receptor responses affect CNS barrier properties and thus facilitate neuroinflammation.
Assuntos
Corioamnionite , Nascimento Prematuro , Recém-Nascido , Gravidez , Humanos , Feminino , Ovinos , Animais , Doenças Neuroinflamatórias , Ureaplasma/metabolismo , Caspases/metabolismo , Líquido Amniótico/metabolismoRESUMO
Ureaplasma diversum is a member of the Mollicutes class responsible for urogenital tract infection in cattle and small ruminants. Studies indicate that the process of horizontal gene transfer, the exchange of genetic material among different species, has a crucial role in mollicute evolution, affecting the group's characteristic genomic reduction process and simplification of metabolic pathways. Using bioinformatics tools and the STRING database of known and predicted protein interactions, we constructed the protein-protein interaction network of U. diversum and compared it with the networks of other members of the Mollicutes class. We also investigated horizontal gene transfer events in subnetworks of interest involved in purine and pyrimidine metabolism and urease function, chosen because of their intrinsic importance for host colonization and virulence. We identified horizontal gene transfer events among Mollicutes and from Ureaplasma to Staphylococcus aureus and Corynebacterium, bacterial groups that colonize the urogenital niche. The overall tendency of genome reduction and simplification in the Mollicutes is echoed in their protein interaction networks, which tend to be more generalized and less selective. Our data suggest that the process was permitted (or enabled) by an increase in host dependence and the available gene repertoire in the urogenital tract shared via horizontal gene transfer.
Assuntos
Proteínas de Bactérias/metabolismo , Transferência Genética Horizontal , Genoma Bacteriano , Mapas de Interação de Proteínas , Tenericutes/genética , Ureaplasma/genética , Animais , Proteínas de Bactérias/genética , Bovinos , Corynebacterium/genética , Evolução Molecular , Tamanho do Genoma , Genômica , Redes e Vias Metabólicas , Purinas/metabolismo , Pirimidinas/metabolismo , Staphylococcus aureus/genética , Tenericutes/classificação , Tenericutes/metabolismo , Ureaplasma/classificação , Ureaplasma/metabolismo , VirulênciaRESUMO
BackgroundTo characterize the influence of microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI) on the intensity of the fetal inflammatory response and the association between the presence of the fetal inflammatory response syndrome (FIRS) and short-term neonatal morbidity in the preterm prelabor rupture of membranes (PPROM) between the gestational ages of 34 and 37 weeks.MethodsOne hundred and fifty-nine women were included in the study. The umbilical cord blood interleukin (IL)-6 concentrations were determined using enzyme-linked immunosorbent assay kits. FIRS was defined based on the umbilical cord blood IL-6 concentration and the presence of funisitis and/or chorionic plate vasculitis.ResultsWomen with both MIAC and IAI had the highest median umbilical cord blood IL-6 concentrations and highest rates of FIRS. Women with FIRS had the higher rates of early-onset sepsis and intraventricular hemorrhage grades I and II when FIRS was characterized based on the umbilical cord blood IL-6 concentrations and the histopathological findings.ConclusionThe presence of both MIAC and IAI was associated with a higher fetal inflammatory response and a higher rate of FIRS. Different aspects of short-term neonatal morbidity were related to FIRS when defined by umbilical cord blood IL-6 concentrations and the histopathology of the placenta.
Assuntos
Líquido Amniótico/microbiologia , Corioamnionite/microbiologia , Ruptura Prematura de Membranas Fetais/microbiologia , Inflamação/microbiologia , Interleucina-6/sangue , Adulto , Chlamydia trachomatis , Ensaio de Imunoadsorção Enzimática , Feminino , Sangue Fetal/química , Idade Gestacional , Humanos , Recém-Nascido , Mycoplasma hominis , Placenta/patologia , Gravidez , Resultado da Gravidez , Estudos Prospectivos , Ureaplasma/metabolismo , Vasculite/microbiologiaRESUMO
Mycoplasma hominis is an opportunistic human mycoplasma. Two other pathogenic human species, M. genitalium and Ureaplasma parvum, reside within the same natural niche as M. hominis: the urogenital tract. These three species have overlapping, but distinct, pathogenic roles. They have minimal genomes and, thus, reduced metabolic capabilities characterized by distinct energy-generating pathways. Analysis of the M. hominis PG21 genome sequence revealed that it is the second smallest genome among self-replicating free living organisms (665,445 bp, 537 coding sequences (CDSs)). Five clusters of genes were predicted to have undergone horizontal gene transfer (HGT) between M. hominis and the phylogenetically distant U. parvum species. We reconstructed M. hominis metabolic pathways from the predicted genes, with particular emphasis on energy-generating pathways. The Embden-Meyerhoff-Parnas pathway was incomplete, with a single enzyme absent. We identified the three proteins constituting the arginine dihydrolase pathway. This pathway was found essential to promote growth in vivo. The predicted presence of dimethylarginine dimethylaminohydrolase suggested that arginine catabolism is more complex than initially described. This enzyme may have been acquired by HGT from non-mollicute bacteria. Comparison of the three minimal mollicute genomes showed that 247 CDSs were common to all three genomes, whereas 220 CDSs were specific to M. hominis, 172 CDSs were specific to M. genitalium, and 280 CDSs were specific to U. parvum. Within these species-specific genes, two major sets of genes could be identified: one including genes involved in various energy-generating pathways, depending on the energy source used (glucose, urea, or arginine) and another involved in cytadherence and virulence. Therefore, a minimal mycoplasma cell, not including cytadherence and virulence-related genes, could be envisaged containing a core genome (247 genes), plus a set of genes required for providing energy. For M. hominis, this set would include 247+9 genes, resulting in a theoretical minimal genome of 256 genes.
Assuntos
Arginina/metabolismo , Genes Bacterianos , Genoma Bacteriano , Mycoplasma hominis/genética , Arginina/análogos & derivados , Metabolismo dos Carboidratos/genética , Adesão Celular/genética , Transferência Genética Horizontal , Humanos , Redes e Vias Metabólicas/genética , Modelos Biológicos , Dados de Sequência Molecular , Mycoplasma genitalium/genética , Mycoplasma genitalium/metabolismo , Mycoplasma hominis/crescimento & desenvolvimento , Mycoplasma hominis/metabolismo , Ureaplasma/genética , Ureaplasma/metabolismo , Virulência/genéticaRESUMO
The conserved Lipoprotein-17 domain of membrane-associated protein Q9PRA0_UREPA from Ureaplasma parvum was selected for structure determination by the Northeast Structural Genomics Consortium, as part of the Protein Structure Initiative's program on structure-function analysis of protein domains from large domain sequence families lacking structural representatives. The 100-residue Lipoprotein-17 domain is a "domain of unknown function" (DUF) that is a member of Pfam protein family PF04200, a large domain family for which no members have characterized biochemical functions. The three-dimensional structure of the Lipoprotein-17 domain of protein Q9PRA0_UREPA was determined by both solution NMR and by X-ray crystallography at 2.5 Å. The two structures are in good agreement with each other. The domain structure features three α-helices, α1 through α3, and five ß-strands. Strands ß1/ß2, ß3/ß4, ß4/ß5 are anti-parallel to each other. Strands ß1and ß2 are orthogonal to strands ß3, ß4, ß5, while helix α3 is formed between the strands ß3 and ß4. One-turn helix α2 is formed between the strands ß1 and ß2, while helix α1 occurs in the N-terminal polypeptide segment. Searches of the Protein Data Bank do not identify any other protein with significant structural similarity to Lipoprotein-17 domain of Q9PRA0_UREPA, indicating that it is a novel protein fold.
Assuntos
Lipoproteínas/química , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Ureaplasma/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Mycoplasma/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , SoluçõesRESUMO
Ureaplasma spp. is detected in the urogenital tract, including the vagina, cervix, chorioamnion, and placenta. Their colonization is associated with histologic chorioamnionitis (CAM), often observed in placentas from preterm delivery. We isolated Ureaplasma spp. from 63 preterm placentas among 151 specimens, which were delivered at <32 wk of gestation. Of the 63 placentas, 52 (83%) revealed CAM in cultures positive for Ureaplasma spp., however, CAM was observed only in 30% (26/88) of cultures negative for Ureaplasma spp. (p < 0.01). Colonization by Ureaplasma spp. was an independent risk factor for CAM (OR, 11.27; 95% CI, 5.09-24.98). Characteristic neutrophil infiltration was observed in the amnion and subchorion (bistratified pattern) in cultures positive for Ureaplasma spp. FISH analysis of CAM placenta with male infant pregnancy indicated that bistratified infiltrated neutrophils showed the XX karyotype and umbilical vein infiltrated neutrophils showed XY karyotype. The distribution of sulfoglycolipid, the receptor of Ureaplasma spp., was mainly detected in the amnion. Ureaplasmal urease D protein and ureB gene were both detected in the amnion, indicating direct colonization by Ureaplasma spp.
Assuntos
Corioamnionite/microbiologia , Placenta/microbiologia , Nascimento Prematuro/microbiologia , Infecções por Ureaplasma/microbiologia , Ureaplasma/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estudos de Casos e Controles , Corioamnionite/genética , Corioamnionite/imunologia , Cromossomos Humanos X , Cromossomos Humanos Y , Feminino , Idade Gestacional , Glicolipídeos/metabolismo , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Modelos Logísticos , Masculino , Infiltração de Neutrófilos , Razão de Chances , Placenta/imunologia , Reação em Cadeia da Polimerase , Gravidez , Nascimento Prematuro/genética , Nascimento Prematuro/imunologia , Medição de Risco , Fatores de Risco , Ureaplasma/genética , Ureaplasma/metabolismo , Infecções por Ureaplasma/complicações , Infecções por Ureaplasma/genética , Infecções por Ureaplasma/imunologia , Urease/genética , Urease/metabolismoRESUMO
Around 40% of preterm births are attributed to ascending intrauterine infection, and Ureaplasma parvum (UP) is commonly isolated in these cases. Here we present a mouse model of ascending UP infection that resembles human disease, using vaginal inoculation combined with mild cervical injury induced by a common spermicide (Nonoxynol-9, as a surrogate for any mechanism of cervical epithelial damage). We measure bacterial load in a non-invasive manner using a luciferase-expressing UP strain, and post-mortem by qPCR and bacterial titration. Cervical exposure to Nonoxynol-9, 24 h pre-inoculation, facilitates intrauterine UP infection, upregulates pro-inflammatory cytokines, and increases preterm birth rates from 13 to 28%. Our results highlight the crucial role of the cervical epithelium as a barrier against ascending infection. In addition, we expect the mouse model will facilitate further research on the potential links between UP infection and preterm birth.
Assuntos
Colo do Útero/lesões , Inflamação/metabolismo , Complicações Infecciosas na Gravidez , Ureaplasma/metabolismo , Animais , Proliferação de Células , Colo do Útero/microbiologia , Colo do Útero/patologia , Citocinas , Modelos Animais de Doenças , Feminino , Humanos , Recém-Nascido , Camundongos , Camundongos Endogâmicos C57BL , Nonoxinol , GravidezRESUMO
Ureaplasma respiratory tract colonization stimulates prolonged, dysregulated inflammation in the lungs of preterm infants, contributing to bronchopulmonary dysplasia (BPD) pathogenesis. Surfactant protein-A (SP-A), a lung collectin critical for bacterial clearance and regulating inflammation, is deficient in the preterm lung. To analyze the role of SP-A in modulating Ureaplasma-mediated lung inflammation, SP-A deficient (SP-A-/-) and WT mice were inoculated intratracheally with a mouse-adapted U. parvum isolate and indices of inflammation were sequentially assessed up to 28 d postinoculation. Compared with infected WT and noninfected controls, Ureaplasma-infected SP-A-/- mice exhibited an exaggerated inflammatory response evidenced by rapid influx of neutrophils and macrophages into the lung, and higher bronchoalveolar lavage TNF-alpha, mouse analogue of human growth-related protein alpha (KC), and monocyte chemotactic factor (MCP-1) concentrations. However, nitrite generation in response to Ureaplasma infection was blunted at 24 h and Ureaplasma clearance was delayed in SP-A-/- mice compared with WT mice. Coadministration of human SP-A with the Ureaplasma inoculum to SP-A-/- mice reduced the inflammatory response, but did not improve the bacterial clearance rate. SP-A deficiency may contribute to the prolonged inflammatory response in the Ureaplasma-infected preterm lung, but other factors may contribute to the impaired Ureaplasma clearance.
Assuntos
Inflamação , Pulmão , Pneumonia/microbiologia , Pneumonia/fisiopatologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Ureaplasma/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/microbiologia , Citocinas/metabolismo , Humanos , Recém-Nascido , Inflamação/microbiologia , Inflamação/patologia , Pulmão/citologia , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Pneumonia/patologia , Proteína A Associada a Surfactante Pulmonar/administração & dosagem , Proteína A Associada a Surfactante Pulmonar/genéticaRESUMO
Background: The multiple banded antigen (MBA), a surface-exposed lipoprotein, is a proposed virulence factor of Ureaplasma spp. We previously demonstrated that the number of Ureaplasma parvum MBA size variants in amniotic fluid was inversely proportional to the severity of chorioamnionitis in experimentally infected pregnant sheep. However, the effect of ureaplasma MBA size variation on inflammation in human pregnancies has not been reported. Methods: Ureaplasmas isolated from the chorioamnion of pregnant women from a previous study (n = 42) were speciated/serotyped and MBA size variation was demonstrated by PCR and western blot. Results were correlated with the severity of chorioamnionitis and cord blood cytokines. In vitro, THP-1-derived macrophages were exposed to recombinant-MBA proteins of differing sizes and NF-κB activation and cytokine responses were determined. Results: MBA size variation was identified in 21/32 (65.6%) clinical isolates (in 10 clinical isolates MBA size variation was unable to be determined). Any size variation (increase/decrease) of the MBA (regardless of Ureaplasma species or serovar) was associated with mild or absent chorioamnionitis (P = 0.023) and lower concentrations of cord blood cytokines IL-8 (P = 0.04) and G-CSF (P = 0.008). In vitro, recombinant-MBA variants elicited different cytokine responses and altered expression of NF-κB p65. Conclusion: This study demonstrates that size variation of the ureaplasma MBA protein modulates the host immune response in vivo and in vitro.
Assuntos
Variação Antigênica/genética , Variação Antigênica/imunologia , Proteínas de Bactérias/imunologia , Inflamação , Placenta/imunologia , Ureaplasma/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Western Blotting , Corioamnionite/imunologia , Corioamnionite/microbiologia , Citocinas/sangue , DNA Bacteriano , Escherichia coli/genética , Feminino , Sangue Fetal/imunologia , Interações Hospedeiro-Parasita , Humanos , Interleucina-8/sangue , NF-kappa B/metabolismo , Placenta/microbiologia , Reação em Cadeia da Polimerase , Gravidez , Nascimento Prematuro/imunologia , Nascimento Prematuro/microbiologia , Proteínas Recombinantes , Sorotipagem , Células THP-1/efeitos dos fármacos , Ureaplasma/genética , Ureaplasma/isolamento & purificação , Ureaplasma/patogenicidade , Fatores de VirulênciaRESUMO
Phase variation of the UU172 phase-variable element of Ureaplasma parvum is governed by a DNA inversion event that takes place at short inverted repeats. The putative tyrosine recombinase XerC of Ureaplasma has been suggested as a mediator in the proposed site-specific recombination event. Here, we provide evidence that XerC mediates DNA inversion at the inverted repeats located on a synthetic locus that was introduced into the model organism Escherichia coli. Synthetic loci were created by exchanging the genes UU171 and UU172 with the two reporter genes gfp (green fluorescent protein) and mrfp1 (monomeric red fluorescent protein 1) either containing or missing the inverted repeats of the UU172 phase-variable element. E. coli was transformed with these loci and also co-transformed with the expression vector pBAD24 that contained the xerC gene behind the arabinose inducible pBAD promoter. Upon XerC expression, DNA inversion was observed only in the locus that contained the inverted repeat regions. We also demonstrate that XerC can process the recombination event with both an N-terminal maltose binding protein tag and a C-terminal 6×His tag in E. coli. A XerC mutant, where the proposed catalytic tyrosine residue 228 was exchanged with an alanine, did not process the recombination event.
Assuntos
DNA Bacteriano/genética , Integrases/metabolismo , Sequências Repetidas Invertidas , Inversão de Sequência , Ureaplasma/genética , Ureaplasma/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ordem dos Genes , Vetores Genéticos/genética , Ligação ProteicaRESUMO
Some of the ammonia produced by hydrolysis of urea by Ureaplasma urealyticum is channelled into an anabolic pathway with resultant 'de novo' synthesis of citrulline. The organism appears to possess ornithine carbamoyltransferase and carbamoyl phosphate synthetase or some modified form of these enzymes.
Assuntos
Citrulina/biossíntese , Ureia/metabolismo , Ureaplasma/metabolismo , Amônia/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Hidrólise , Ornitina Carbamoiltransferase/metabolismoRESUMO
Antenatal inflammation may be associated with adverse neonatal outcomes in several organ systems. Bacteria and a few viruses have been detected in cases of microbial invasion of the amniotic cavity which is referred to as chorioamnionitis. Many aspects of this disease remain unclear such as the causes, time of onset and the fetal responses. Chorioamnionitis was therefore induced in pregnant sheep by injections of lipopolysaccharide (LPS) or Ureaplasma species into the amniotic cavity under ultrasound guidance. LPS-induced chorioamnionitis caused a cascade of organ injury, inflammation, and remodeling. The organ-specific changes were accompanied by systemic effects. The systemic effects after LPS-induced chorioamnionitis resulted in immune suppression against several Toll-like receptor agonists (cross-tolerance). Ureaplasma induced chorioamnionitis made changes in the fetal lung structure depending on the time of infection during pregnancy. The mechanisms of inflammation, structural damage and decreased expression of growth factors need to be further studied to determine therapeutic targets in suitable animal models.
Assuntos
Corioamnionite/terapia , Modelos Teóricos , Animais , Infecções Bacterianas/complicações , Corioamnionite/induzido quimicamente , Corioamnionite/etiologia , Corioamnionite/patologia , Modelos Animais de Doenças , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Feminino , Humanos , Lipopolissacarídeos , Gravidez , Ureaplasma/metabolismo , Ureaplasma/fisiologiaRESUMO
PURPOSE: The objective of the present study is to verify possible association between infections with mycoplasmas and ureaplasmas and the presence of HPV infections in women diagnosed with abnormal cervical cytology. MATERIAL/METHODS: The investigation included 387 non-pregnant women among whom: 62 were diagnosed with ASCUS, 167 with LSIL, 27 with HSIL, 49 with cervical carcinomas, and 82 females with normal cytology.The presence of HPV infection and identification of both ureaplasma and mycoplasma were confirmed by PCR using specific primers. RESULTS: HPV infections were demonstrated in 156 females (40%), with mycoplasmas and/or ureaplasmas were confirmed in 93 cases (24%). In HPV-positive patients, infections with mycoplasmas/ureaplasmas were more frequent, particularly for ureaplasmas (U. urealyticum p=0.004, U. parvum p=0.027). The percentage of females infected with U. urealyticum significantly increased in women diagnosed with cervical carcinoma as compared to controls.The statistical analysis demonstrated that the risk of HPV infection while already infected with any of the four analyzed species of Mycoplasmataceae increased two-fold. With concomitant of U. urealyticum infection, the risk of HPV infection was 4.7-fold greater than in the absence U. urealyticum infection. CONCLUSION: Since the presence of U. urealyticum associates significantly with the HPV infection, genotyping of the ureaplasma species should be recomended.
Assuntos
Colo do Útero/microbiologia , Colo do Útero/virologia , Mycoplasma/metabolismo , Papillomaviridae/metabolismo , Ureaplasma/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Transformação Celular Neoplásica , Técnicas Citológicas , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Infecções por Mycoplasma/diagnóstico , Reação em Cadeia da Polimerase/métodos , Infecções por Ureaplasma/diagnóstico , Neoplasias do Colo do Útero/microbiologia , Neoplasias do Colo do Útero/virologiaRESUMO
Intra-amniotic infection (IAI) is associated with preterm birth and perinatal mortality. To identify potential biomarkers, we performed a comprehensive survey of the cervical-vaginal fluid (CVF) proteome from a primate IAI model utilizing multidimensional protein identification technology (LC/LC-MS/MS) and MALDI-TOF-MS analyses. Analyses of CVF proteome identified 205 unique proteins and differential expression of 27 proteins in controls and IAI samples. Protein expression signatures and immunodetection of specific biomarkers identified can be employed for noninvasive detection of IAI.
Assuntos
Líquido Amniótico/microbiologia , Biomarcadores/química , Líquidos Corporais/metabolismo , Colo do Útero/metabolismo , Infecções/diagnóstico , Proteômica/métodos , Vagina/metabolismo , Animais , Líquidos Corporais/microbiologia , Colo do Útero/microbiologia , Feminino , Regulação da Expressão Gênica , Macaca mulatta , Gravidez , Prenhez , Nascimento Prematuro/prevenção & controle , Ureaplasma/metabolismo , Vagina/microbiologiaRESUMO
The crystal structure of the hypothetical protein MPN555 from Mycoplasma pneumoniae (gi|1673958) has been determined to a resolution of 2.8 Angstrom using anomalous diffraction data at the Se-peak wavelength. Structure determination revealed a mostly alpha-helical protein with a three-lobed shape. The three lobes or fingers delineate a central binding groove and additional grooves between lobes 1 and 3 and between lobes 2 and 3. For one of the molecules in the asymmetric unit, the central binding pocket was filled with a peptide from the uncleaved N-terminal affinity tag. The MPN555 structure has structural homology to two bacterial chaperone proteins: SurA and trigger factor from Escherichia coli. The structural data and the homology to other chaperone proteins suggests an involvement in protein folding as a molecular chaperone for MPN555.
Assuntos
Proteínas de Transporte/química , Proteínas de Escherichia coli/química , Mycoplasma/metabolismo , Peptidilprolil Isomerase/química , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Primers do DNA/química , Elétrons , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Chaperonas Moleculares , Dados de Sequência Molecular , Peptidilprolil Isomerase/metabolismo , Reação em Cadeia da Polimerase , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pseudomonas/metabolismo , Ureaplasma/metabolismo , Vibrio cholerae/metabolismo , Difração de Raios XRESUMO
Enzymes of the Embden-Meyerhof-Parnas pathway and hexose monophosphate shunt were examined in cytoplasmic extracts of three serovars of Ureaplasma urealyticum. We found no glucose-6-phosphate or 6-phosphogluconate dehydrogenase, hexokinase, phosphoglucose isomerase, aldolase, or lactic dehydrogenase activities. We failed to find cytochrome pigments in extracts and found no significant production of 14CO2 from [U-14C]glucose, nor did we find oxygen-dependent reduced nicotinamide adenine dinucleotide oxidase activity. Lactic acid was found only at trace levels in spent culture fluids. Ureaplasmas are apparently nonfermentative and are unlike all other mollicutes in that they have no detectable oxygen-dependent reduced nicotinamide adenine dinucleotide oxidase activity.
Assuntos
Ureaplasma/metabolismo , Ureaplasma/enzimologiaRESUMO
Continuous culture of Ureaplasma urealyticum is reported with a steady-state cell biomass of greater than 10(6) cells per ml. Thus, large cell numbers can be easily obtained; in addition, the system provides a powerful means for exploring what nutrients(s) limits the growth yield of this organism. Urea is shown not to be the growth-limiting nutrient in conventional media, although when provided in excess it appears to be completely hydrolyzed.
Assuntos
Ureaplasma/crescimento & desenvolvimento , Técnicas Bacteriológicas , Meios de Cultura , Cinética , Ureia/metabolismo , Ureaplasma/metabolismoRESUMO
The effect of urea on growth of Ureaplasma urealyticum type VIII was studied by cultivating the organisms in a dialysate broth, prepared from soy peptone and autoclaved yeast, supplemented with 5% dialyzed horse serum, 100 mM 2-(N-morpholino)ethane sulfonic acid buffer (pH 5.75), and defined amounts of urea. Without urea, growth did not occur. Total growth was directly related to urea concentration. The least amount of urea that supported growth was 0.032 mM, which resulted in 3 x 10(4) colony-forming units per ml. The maximum yield of organisms, 8.0 x 10(7) colony-forming units per ml, was observed at 32 mM urea. Growth was limited not only by urea concentration, but also by the buffer capacity of the medium. The maximum amount of 2-(N-morpholino)ethane sulfonic acid buffer that could be employed was 100 mM; at higher concentrations, growth was inhibited. The yield of U. urealyticum was small even in medium with 32 mM urea and 100 mM 2-(N-morpholino)ethane sulfonic acid buffer: 0.63 mg of protein per liter of culture containing 5 x 10(10) total colony-forming units. The molar growth yield was 20 mg of protein per mol of urea. The growth rate was also a function of urea concentration. Generation times ranged from 8 h at 0.032 mM urea to 1.6 h at 3.2 mM urea, where the substrate level was saturating. The K(s) value for growth was 2.0 x 10(-4) M urea. Thus, urea is a growth-limiting factor for U. urealyticum, but remarkably large amounts of this substrate are required.
Assuntos
Ureia/metabolismo , Ureaplasma/crescimento & desenvolvimento , Soluções Tampão , Meios de Cultura , Ácidos Sulfônicos , Ureaplasma/metabolismoRESUMO
The growth requirements of Ureaplasma urealyticum strains T-McA, T-Pi, T-27, and T-207 were studied. All strains grew in T-broth containing horse serum. Only strain T-McA grew in broth containing serum fraction A, a finding that confirmed that ureaplasmas are not homogeneous and suggested that they can be divided into two species on the basis of their requirement for horse serum. The growth factors required by T-McA are arginine, cystine, methionine, and unidentified metabolites in serum fraction A and trypticase soy broth. The vitamins in Eagle's minimal essential medium are not essential metabolites for T-McA but enhanced its growth. Therefore, we recommend that these vitamins be incorporated in ureaplasma media and that the effect of supplementing media with arginine, cystine, and methionine for the primary isolation of ureaplasmas be ascertained.
Assuntos
Ureaplasma/classificação , Arginina , Meios de Cultura , Cistina , Metionina , Ureaplasma/metabolismo , VitaminasRESUMO
Seven species of human T-mycoplasmas that grow in Fraction A and 20 mug urea/ml died when the urea was omitted. Two species would not grow in Fraction A broth containing 10 mug/urea/ml. The other five strains grew in broth containing 10 mug urea/ml and were adapted by serial passage in broth containing decreasing concentrations of urea to grow in broth containing 2.5 mug/ml urea, but not in broth containing 1.25 mug/ml. Therefore the minimal urea requirement is not the same for the growth of all strains of T-mycoplasmas. In exponential phase broth cultures, urease was detected only intracellularly, none being found in the medium.