The pneumococcal response to oxidative stress includes a role for Rgg

Streptococcus pneumoniae resides in the oxygen-rich environment of the upper respiratory tract, and therefore the ability to survive in the presence of oxygen is an important aspect of its in vivo survival. To investigate how S. pneumoniae adapts to oxygen, we determined the global gene expression profile of the micro-organism in aerobiosis and anaerobiosis. It was found that exposure to aerobiosis elevated the expression of 54 genes, while the expression of 15 genes was downregulated. Notably there were significant changes in putative genome plasticity and hypothetical genes. In addition, increased expression of rgg, a putative transcriptional regulator, was detected. To test the role of Rgg in the pneumococcal oxidative stress response, an isogenic mutant was constructed. It was found that the mutant was sensitive to oxygen and paraquat, but not to H2O2. In addition, the absence of Rgg strongly reduced the biofilm-forming ability of an unencapsulated pneumococcus. Virulence studies showed that the median survival time of mice infected intranasally with the rgg mutant was significantly longer than that of the wild-type-infected group, and the animals infected with the mutant developed septicaemia later than those infected intranasally with the wild-type

Pathotyping the Zoonotic Pathogen Streptococcus suis: Novel Genetic Markers To Differentiate Invasive Disease-Associated Isolates from Non-Disease-Associated Isolates from England and Wales.

Streptococcus suis is one of the most important zoonotic bacterial pathogens of pigs, causing significant economic losses to the global swine industry. S. suis is also a very successful colonizer of mucosal surfaces, and commensal strains can be found in almost all pig populations worldwide, making detection of the S. suis species in asymptomatic carrier herds of little practical value in predicting the likelihood of future clinical relevance. The value of future molecular tools for surveillance and preventative health management lies in the detection of strains that genetically have increased potential to cause disease in presently healthy animals. Here we describe the use of genome-wide association studies to identify genetic markers associated with the observed clinical phenotypes (i) invasive disease and (ii) asymptomatic carriage on the palatine tonsils of pigs on UK farms. Subsequently, we designed a multiplex PCR to target three genetic markers that differentiated 115 S. suis isolates into disease-associated and non-disease-associated groups, that performed with a sensitivity of 0.91, a specificity of 0.79, a negative predictive value of 0.91, and a positive predictive value of 0.79 in comparison to observed clinical phenotypes. We describe evaluation of our pathotyping tool, using an out-of-sample collection of 50 previously uncharacterized S. suis isolates, in comparison to existing methods used to characterize and subtype S. suis isolates. In doing so, we show our pathotyping approach to be a competitive method to characterize S. suis isolates recovered from pigs on UK farms and one that can easily be updated to incorporate global strain collections.This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) Knowledge Transfer Network CASE studentship co-funded by Zoetis (previously Pfizer Animal Health UK) and with significant contribution from BQP Ltd (Award Reference: BB/L502479/1). Funding bodies provided scholarship support but had no part in study design, data collection, analysis and interpretation of data or in writing the manuscript. AWT is supported by a BBSRC Longer and Larger (LoLa) grant (Award Reference: BB/G019274/1). LAW is supported by a Dorothy Hodgkin Fellowship funded by the Royal Society (Grant Number: DH140195) and a Sir Henry Dale Fellowship co-funded by the Royal Society and Wellcome Trust (Grant Number: 109385/Z/15/Z)

The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering.

Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.This work was supported by a Longer and Larger (LoLa) grant from the UK Biotechnology and Biological Sciences Research Council (grant nos BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G003203/1) and The Wellcome Trust (grant no. 102979/Z/13/Z)

Gut Microbial Gene Expression in Mother-Fed and Formula-Fed Piglets

Effects of diet on the structure and function of gut microbial communities in newborn infants are poorly understood. High-resolution molecular studies are needed to definitively ascertain whether gut microbial communities are distinct in milk-fed and formula-fed infants.Pyrosequencing-based whole transcriptome shotgun sequencing (RNA-seq) was used to evaluate community wide gut microbial gene expression in 21 day old neonatal piglets fed either with sow's milk (mother fed, MF; n = 4) or with artificial formula (formula fed, FF; n = 4). Microbial DNA and RNA were harvested from cecal contents for each animal. cDNA libraries and 16S rDNA amplicons were sequenced on the Roche 454 GS-FLX Titanium system. Communities were similar at the level of phylum but were dissimilar at the level of genus; Prevotella was the dominant genus within MF samples and Bacteroides was most abundant within FF samples. Screened cDNA sequences were assigned functional annotations by the MG-RAST annotation pipeline and based upon best-BLASTX-hits to the NCBI COG database. Patterns of gene expression were very similar in MF and FF animals. All samples were enriched with transcripts encoding enzymes for carbohydrate and protein metabolism, as well as proteins involved in stress response, binding to host epithelium, and lipopolysaccharide metabolism. Carbohydrate utilization transcripts were generally similar in both groups. The abundance of enzymes involved in several pathways related to amino acid metabolism (e.g., arginine metabolism) and oxidative stress response differed in MF and FF animals.Abundant transcripts identified in this study likely contribute to a core microbial metatranscriptome in the distal intestine. Although microbial community gene expression was generally similar in the cecal contents of MF and FF neonatal piglets, several differentially abundant gene clusters were identified. Further investigations of gut microbial gene expression will contribute to a better understanding of normal and abnormal enteric microbiology in animals and humans

A Functional Genomics Approach to Establish the Complement of Carbohydrate Transporters in Streptococcus pneumoniae

The aerotolerant anaerobe Streptococcus pneumoniae is part of the normal nasopharyngeal microbiota of humans and one of the most important invasive pathogens. A genomic survey allowed establishing the occurrence of twenty-one phosphotransferase systems, seven carbohydrate uptake ABC transporters, one sodium∶solute symporter and a permease, underlining an exceptionally high capacity for uptake of carbohydrate substrates. Despite high genomic variability, combined phenotypic and genomic analysis of twenty sequenced strains did assign the substrate specificity only to two uptake systems. Systematic analysis of mutants for most carbohydrate transporters enabled us to assign a phenotype and substrate specificity to twenty-three transport systems. For five putative transporters for galactose, pentoses, ribonucleosides and sulphated glycans activity was inferred, but not experimentally confirmed and only one transport system remains with an unknown substrate and lack of any functional annotation. Using a metabolic approach, 80% of the thirty-two fermentable carbon substrates were assigned to the corresponding transporter. The complexity and robustness of sugar uptake is underlined by the finding that many transporters have multiple substrates, and many sugars are transported by more than one system. The present work permits to draw a functional map of the complete arsenal of carbohydrate utilisation proteins of pneumococci, allows re-annotation of genomic data and might serve as a reference for related species. These data provide tools for specific investigation of the roles of the different carbon substrates on pneumococcal physiology in the host during carriage and invasive infection

Pneumococcal 6-Phospho-β-Glucosidase (BglA3) Is Involved in Virulence and Nutrient Metabolism.

For the generation of energy, the important human pathogen Streptococcus pneumoniae relies on host-derived sugars, including β-glucoside analogs. The catabolism of these nutrients involves the action of 6-phospho-β-glucosidase to convert them into usable monosaccharaides. In this study, we characterized a 6-phospho-β-glucosidase (BglA3) encoded by SPD_0247. We found that this enzyme has a cell membrane localization and is active only against a phosphorylated substrate. A mutated pneumococcal ΔSPD0247 strain had reduced 6-phospho-glucosidase activity and was attenuated in growth on cellobiose and hyaluronic acid compared to the growth of wild-type D39. ΔSPD0247-infected mice survived significantly longer than the wild-type-infected cohort, and the colony counts of the mutant were lower than those of the wild type in the lungs. The expression of SPD_0247 in S. pneumoniae harvested from infected tissues was significantly increased relative to its expression in vitro on glucose. Additionally, ΔSPD0247 is severely impaired in its attachment to an abiotic surface. These results indicate the importance of β-glucoside metabolism in pneumococcal survival and virulence

Soil nitrous oxide emissions from grassland: Potential inhibitor effect of hippuric acid

In grassland systems, cattle and sheep urine patches are recognized as nitrous oxide (N₂O) emission hot spots due to the high urinary nitrogen (N) concentrations. Hippuric acid (HA) is one of the constituents of ruminant urine that has been reported as a natural inhibitor of soil N₂O emissions. The aim of this study was to examine the potential for elevated ruminant urine HA concentrations to reduce N₂O emissions, in situ, on an acidic heavy clay soil under poorly drained conditions (WFPS > 85%). A randomized complete block design experiment with three replications and four treatments was conducted using the closed-static-flux chamber methodology. The four treatments were applied inside the chambers: control with no artificial urine application (C), control artificial urine (U), and enriched artificial urine with two rates of HA (55.8 and 90 mM, U+HA1, U+HA2). Soil inorganic-N, soil dissolved organic carbon (DOC), soil pH as well as N₂O and methane (CH₄ ) fluxes were monitored over a 79-d period. Although N₂O emissions were not affected by the HA enriched urine treatments, U+HA2 positively affected the retention of N as NH⁺₄ until day 3, when the soil pH dropped to values < 5. Subsequently, as a consequence of rainfall events and soil acidification, it is likely that leaching or sorption onto clay reduced the efficacy of HA, masking any treatment differential effect on N₂O emissions. Moreover, CH₄ fluxes as well as DOC results reflected the soil anaerobic conditions which did not favour nitrification processes. Further research is needed to determine the fate of HA into the soil which might clarify the lack of an in situ effect of this compound

Influência de diferentes ângulos articulares obtidos na posição inicial do exercício pressão de pernas e final do exercício puxada frontal sobre os valores de 1RM Influencia en los diferentes ángulos articulares obtenidos en la posición inicial del ejercicio de presión de piernas y al final del ejercicio de puje frontal sobre los valores de 1RM The influence of different joint angles obtained in the starting position of leg press exercise and at the end of the frontal pull exercise on the values of 1RM

O teste de uma repetição máxima (1RM) tem sido aplicado sobre várias circunstâncias e em diversos objetivos, sendo que variáveis potencialmente influenciadoras deste teste têm sido constantemente estudadas. Este estudo buscou avaliar a influência de diferentes ângulos na posição inicial de execução dos exercícios pressão de pernas e posição final do exercício puxada frontal sobre os resultados de 1RM. Para tal foram mensurados no teste de 1RM dos exercícios pressão de pernas e puxada frontal 20 sujeitos voluntários do sexo masculino (médias de idade 24,5 anos, estatura 1,75 metros e massa corporal de 72,0kg). Após consentimento de participação e a adaptação ao treinamento resistido com pesos, foi aplicado o teste de 1RM no exercício pressão de pernas em três diferentes ângulos de testagem na posição inicial (80º, 90º e 100º de flexão do joelho) e também no exercício puxada frontal posição final (60º, 70º e 80º de flexão do cotovelo), sendo que cada ângulo foi testado em dias diferentes, porém com os dois exercícios. Os resultados indicam que as médias de 1RM para o exercício pressão de pernas são estatisticamente diferentes (F = 30,199; p = 0,000) entre si (post hoc de Tukey). Já para o exercício puxada frontal, embora existam diferenças, estas não foram estatisticamente significativas (F = 1,330; p = 0,281). Conclui-se que diferentes técnicas de execução dos exercícios que envolvam angulações diversas, principalmente nas posições iniciais destes, devem ser rigorosamente controladas, pois podem afetar a quilagem levantada.<br>El test de Repetición Máxima (1RM) ha sido aplicado en varias circunstancias y en diversos objetivos siendo que variable potencialmente influyentes en este test vienen siendo constantemente estudiadas. Este estudio buscó evaluar la influencia de diferentes ángulos en la posición final del ejercicio de presión de piernas y de puje frontal sobre los resultados de 1RM. De tal manera que fueron medidos en el test de 1RM los ejercicios de presión de piernas y puje frontal de 20 sujetos voluntarios del sexo masculino (con medias de edad 24,5 años, estatura 1,75 metros y masa corporal de 72,0 kg). Despues de consentimiento de participación y de adaptación al entrenamiento resistido con pesos fué aplicado al test de 1RM en el ejercício de presión de piernas en tres diferentes ángulos de testado en la posición inicial (80º, 90º y 100º de flexión de rodilla) y tambien en el ejercicio de puje frontal en posición final (60º, 70º y 80º de flexión de codo), siendo que cada ángulo fué testado en días diferentes pueden, con los dos ejercicios. Los resultados indican que las medias de 1RM para el ejercicio de presión de piernas son estadísticamente diferentes (F = 30,199; p = 0,000) entre si (post hoc de Tukey). Si para el ejercicio de puje frontal, existen hoy diferencias estas no fueron estadísticamente significativas (F = 1,330; p = 0,281). Se concluye que diferentes técnicas de ejecución de los ejercicios que involucran ángulos diferentes principalmente en las posiciones iniciales de estos deben ser rigurosamente controladas pués pueden afectar el quilaje levantado.<br>The Maximum Repetition test (1RM) has been applied under various circumstances and with diverse objectives, and variables that might potentially influence this test have been constantly studied. This study sought to evaluate the influence of different angles in the initial position of the leg press exercises and in the final position of the frontal pull exercise on the results of the 1RM. Twenty male volunteers (with an average age of 24.5 years, height of 1.75 meters and weight of 72 kg) were measured in the 1RM test for the leg press exercise and the frontal pull exercise. After obtaining their consent to participate in and adapting to the weight-resistance training, the 1RM test was applied in the leg press exercise in three different test angles in the initial position (80º, 90º and 100º degrees of knee flexion) and in the final position of the frontal pull exercise (60º, 70º and 80º degrees of elbow flexion), thus each angle was tested on three different days for each of the exercises. The results indicate that the averages of the 1RM for the leg press exercises are statistically different (F = 30, 199; p = 0.000) amongst themselves (post hoc of Tukey). Although there were differences in the frontal pull exercise, they were not statistically significant (F = 1.330; p = 0.281). It can be concluded that different techniques used in the execution of exercises that involve different angles, mainly in their initial positions, must be rigorously controlled, since they can affect the amount of weight lifted