RESUMEN
We have developed a novel molecular methodology that utilizes stool samples containing intact sloughed epithelial cells to quantify intestinal gene expression profiles in the developing human neonate. Since nutrition exerts a major role in regulating neonatal intestinal development and function, our goal was to identify gene sets (combinations) that are differentially regulated in response to infant feeding. For this purpose, fecal mRNA was isolated from exclusively breast-fed (n = 12) and formula-fed (n = 10) infants at 3 mo of age. Linear discriminant analysis was successfully used to identify the single genes and the two- to three-gene combinations that best distinguish the feeding groups. In addition, putative "master" regulatory genes were identified using coefficient of determination analysis. These results support our premise that mRNA isolated from stool has value in terms of characterizing the epigenetic mechanisms underlying the developmentally regulated transcriptional activation/repression of genes known to modulate gastrointestinal function. As larger data sets become available, this methodology can be extended to validation and, ultimately, identification of the main nutritional components that modulate intestinal maturation and function.
Asunto(s)
Células Epiteliales/metabolismo , Heces/química , Tracto Gastrointestinal/crecimiento & desarrollo , Fenómenos Fisiológicos Nutricionales del Lactante , Adulto , Lactancia Materna , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Fórmulas Infantiles , Recién Nacido , Masculino , Análisis por Micromatrices , Embarazo , ARN Mensajero/metabolismoRESUMEN
Lactobacillus rhamnosus GG (LGG), a probiotics, ameliorates intestinal and other organ inflammation in infant rats. The hypothesis is that live and heat-killed LGG have similar effects on decreasing the inflammatory response induced by E. coli lipopolysaccharide (LPS) in the infant rat. Using a gastrostomy-fed rat model, 7-d-old rat pups were gastrostomy fed with or without live LGG (10(8) or 10(12) cfu x L(-1) x kg(-1) x d(-1)) for 6 d. In a separate experiment, LPS was administered to rat pups with or without live or heat-killed LGG (10(8) cfu x L(-1) x kg(-1) x d(-1)). Cytokine/chemokine proteins were determined by ELISA or multiplex assay. Both live and heat-killed LGG decreased LPS-induced cytokine-induced neutrophil chemoattractant-1 (CINC-1) production in liver and plasma (p < 0.05) and also showed a trend (p = 0.09) in lungs. Live and heat-killed LGG ameliorated LPS-suppressed IL-10 level in lungs (p < 0.05). Both forms of LGG decreased IL-1b production in liver. There was no difference between low and high doses of live LGG in the production of CINC-1, TNF-alpha, and myeloperoxidase (MPO). There was a trend of increase of claudin-1 in both live and heat-killed groups (p = 0.08). In conclusion, both live and heat-killed LGG provided by the enteral route decrease LPS-induced proinflammatory mediators and increase anti-inflammatory mediators.
Asunto(s)
Quimiocinas/inmunología , Citocinas/inmunología , Gastrostomía , Lacticaseibacillus rhamnosus/inmunología , Probióticos , Animales , Animales Recién Nacidos , Quimiocina CXCL1/metabolismo , Claudina-1 , Humanos , Recién Nacido , Mucosa Intestinal/metabolismo , Intestinos/citología , Proteínas de la Membrana/metabolismo , Ocludina , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Supplementation of infant formulas with prebiotic ingredients continues the effort to mimic functional properties of human milk. In this double-blind, controlled, 28-day study, healthy term infants received control formula (control group; n = 25) or control formula supplemented with polydextrose (PDX) and galactooligosaccharide (GOS) (4 g/liter) (PG4 group; n = 27) or with PDX, GOS, and lactulose (LOS) (either 4 g/liter [PGL4 group; n = 27] or 8 g/liter [PGL8 group; n = 25]). A parallel breast-fed group (BF group) (n = 30) was included. Stool characteristics, formula tolerance, and adverse events were monitored. Fecal bacterial subpopulations were evaluated by culture-based selective enumeration (Enterobacteriaceae), quantitative real-time PCR (Clostridium clusters I, XI, and XIV, Lactobacillus, and Bifidobacterium), and fluorescence in situ hybridization (FISH) (Bifidobacterium). Fecal bacterial community profiles were examined by using 16S rRNA gene PCR-denaturing gradient gel electrophoresis. The daily stool consistency was significantly softer or looser in the BF group than in all of the groups that received formula. The formulas were well tolerated, and the incidences of adverse events did not differ among feeding groups. Few significant changes in bacterial subpopulations were observed at any time point. The bacterial communities were stable; individual profiles tended to cluster by subject rather than by group. Post hoc analysis, however, demonstrated that the bacterial community profiles for subjects in the BF, PG4, PGL4, and PGL8 groups that first received formula at a younger age were less stable than the profiles for subjects in the same groups that received formula at an older age, but there was no difference for the control group. These data indicate that formulas containing PDX, GOS, and LOS blends are more likely to influence gut microbes when administration is begun in early infancy and justify further investigation of the age-related effects of these blends on fecal microbiota.
Asunto(s)
Bacterias/clasificación , Recuento de Colonia Microbiana , Suplementos Dietéticos/efectos adversos , Heces/microbiología , Tracto Gastrointestinal/fisiología , Fórmulas Infantiles/administración & dosificación , Fenómenos Fisiológicos Nutricionales del Lactante , Método Doble Ciego , Electroforesis/métodos , Humanos , Hibridación Fluorescente in Situ , Lactante , Recién Nacido , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genéticaRESUMEN
Freezing and lyophilization are common methods used for preservation and storage of microorganisms during the production of concentrated starter cultures destined for industrial fermentations or product formulations. The compatible solute trehalose has been widely reported to protect bacterial, yeast and animal cells against a variety of environmental stresses, particularly freezing and dehydration. Analysis of the Lactobacillus acidophilus NCFM genome revealed a putative trehalose utilization locus consisting of a transcriptional regulator, treR; a trehalose phosphoenolpyruvate transferase system (PTS) transporter, treB; and a trehalose-6-phosphate hydrolase, treC. The objective of this study was to characterize the tre locus in L. acidophilus and determine whether or not intracellular uptake of trehalose contributes to cryoprotection. Cells subjected to repeated freezing and thawing cycles were monitored for survival in the presence of various concentrations of trehalose. At 20% trehalose a 2-log increase in survival was observed. The trehalose PTS transporter and trehalose hydrolase were disrupted by targeted plasmid insertions. The resulting mutants were unable to grow on trehalose, indicating that both trehalose transport into the cell via a PTS and hydrolysis via a trehalose-6-phosphate hydrolase were necessary for trehalose fermentation. Trehalose uptake was found to be significantly reduced in the transporter mutant but unaffected in the hydrolase mutant. Additionally, the cryoprotective effect of trehalose was reduced in these mutants, suggesting that intracellular transport and hydrolysis contribute significantly to cryoprotection.
Asunto(s)
Genes Bacterianos , Lactobacillus acidophilus/genética , Lactobacillus acidophilus/metabolismo , Trehalosa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Crioprotectores/metabolismo , Crioprotectores/farmacología , ADN Bacteriano/genética , Disacaridasas/genética , Disacaridasas/metabolismo , Fermentación , Lactobacillus acidophilus/efectos de los fármacos , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Trehalosa/farmacologíaRESUMEN
Two-component regulatory systems are one primary mechanism for environmental sensing and signal transduction. Annotation of the complete genome sequence of the probiotic bacterium Lactobacillus acidophilus NCFM revealed nine two-component regulatory systems. In this study, the histidine protein kinase of a two-component regulatory system (LBA1524HPK-LBA1525RR), similar to the acid-related system lisRK from Listeria monocytogenes (P. D. Cotter et al., J. Bacteriol. 181:6840-6843, 1999), was insertionally inactivated. A whole-genome microarray containing 97.4% of the annotated genes of L. acidophilus was used to compare genome-wide patterns of transcription at various pHs between the control and the histidine protein kinase mutant. The expression pattern of approximately 80 genes was affected by the LBA1524HPK mutation. Putative LBA1525RR target loci included two oligopeptide-transport systems present in the L. acidophilus genome, other components of the proteolytic system, and a LuxS homolog, suspected of participating in synthesis of the AI-2 signaling compound. The mutant exhibited lower tolerance to acid and ethanol in logarithmic-phase cells and poor acidification rates in milk. Supplementation of milk with Casamino Acids essentially restored the acid-producing ability of the mutant, providing additional evidence for a role of this two component system in regulating proteolytic activity in L. acidophilus.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Lactobacillus acidophilus/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Péptido Hidrolasas/metabolismo , Transducción de Señal , Proteínas Bacterianas/genética , Secuencia de Bases , Medios de Cultivo , Perfilación de la Expresión Génica , Histidina Quinasa , Concentración de Iones de Hidrógeno , Lactobacillus acidophilus/genética , Lactobacillus acidophilus/crecimiento & desarrollo , Lactobacillus acidophilus/metabolismo , Mutación , Péptido Hidrolasas/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismoRESUMEN
A custom microarray was developed to study the temporal gene expression of the two groups of phages infecting the Gram-positive lactic acid bacterium Streptococcus thermophilus. The complete genomic sequence of the virulent cos-type phage DT1 (34,815 bp) and the pac-type phage 2972 (34,704 bp) were used for the construction of the microarray. Gene expression was measured at nine time intervals (0, 2, 7, 12, 17, 22, 27, 32 and 37 min) during phage infection and an expression curve was determined for each gene. Each phage gene was then classified into one of the three traditional transcription classes and these data were used to generate the complete transcriptional map of DT1 and 2972. Phage DT1 possesses 18 early genes, 12 middle genes and 12 late-expressed genes whereas 2972 has 16 early, 11 middle and 14 late genes. The trends of the phage gene expression profiles were also confirmed by slot blot hybridizations. Significant differences were observed when comparing the transcriptional maps of DT1 and 2972 with those already available for the S. thermophilus phages Sfi19 and Sfi21. To our knowledge, this report presents the first complete transcription analysis of bacteriophages infecting Gram-positive bacteria using the DNA microarray technology.
Asunto(s)
Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Fagos de Streptococcus/crecimiento & desarrollo , Streptococcus thermophilus/virología , Secuencia de Bases , Cartilla de ADN , Sondas de ADN , Regulación Viral de la Expresión Génica , Cinética , Lisogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Virales/genéticaRESUMEN
Lactobacillus acidophilus NCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R/M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism. In silico analyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus- and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence of L. acidophilus are likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota.
Asunto(s)
Genoma Bacteriano , Lactobacillus acidophilus/genética , Adhesión Bacteriana/genética , Bacteriocinas/genética , Bacteriófagos/genética , Bacteriófagos/patogenicidad , Metabolismo de los Hidratos de Carbono , Biología Computacional , Lactobacillus acidophilus/metabolismo , Lactobacillus acidophilus/virología , Datos de Secuencia Molecular , Péptido Hidrolasas/genética , Profagos/genética , Análisis de Secuencia de ADN , Transposasas/genéticaRESUMEN
Primers designed on the basis of nucleotide sequences conserved in DnaK and GroEL from Gram-positive organisms were used to PCR amplify internal regions of the cognate genes from the anaerobic ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1. Genome walking was then utilized to elucidate the remainder of the sequences in addition to upstream and downstream regions. The full sequence of the gene encoding the GroES protein (groES) was found directly upstream from groEL. The deduced amino acid sequence of the groEL gene showed the highest homology with the amino acid sequence of the Clostridium thermocellum GroEL protein (72% amino acid identity). Similarly, translation of the groES nucleotide sequence showed highest homology to the C. thermocellum GroES protein (61% amino acid identity). Analysis of the upstream region of this chaperonin operon revealed a CIRCE regulatory element 45 bp upstream from the putative start of the groES ORF. The deduced amino acid sequence of the putative dnaK gene showed the highest homology with the amino acid sequence of the Clostridium acetobutylicum DnaK protein (68% amino acid identity). Phylogenetic analyses based on the translated sequences reiterate this relationship between R. flavefaciens and the Clostridia. However, when the nucleotide sequences of Gram-positive organisms are analyzed, a different topology occurs of the relationship between high- and low-G+C Gram-positive organisms to the 16S rRNA interpretation.