Human probiotic bacteria attenuate Pseudomonas aeruginosa biofilm and virulence by quorum-sensing inhibition

Abstract

This work investigated chloroform extracts from culture supernatants of two human probiotic bacteria, Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730 for the production of virulence factors and quorum sensing (QS) interference against three Pseudomonas aeruginosa strains. Both extracts inhibited biofilm biomass (up to 50%), biofilm metabolic activity (up to 39%), the production of the enzyme elastase (up to 63%) and pyocyanin (up to 77%), and decreased QS, without presenting any antibacterial acgivity. In addition, the chloroform extracts of both strains disrupted preformed biofilms of the three strains of P. aeruginosa analyzed (up to 40%). GC-MS analysis revealed that the major compounds detected in the bioactive extracts were four diketopiperazines. This study suggests that the metabolites of L. casei and L. acidophilus could be a promising alternative to combat the pathogenicity of P. aeruginosa.     

A Foundation for Reliable Spatial Proteomics Data Analysis

Quantitative mass-spectrometry-based spatial proteomics involves elaborate, expensive, and time-consuming experimental procedures, and considerable effort is invested in the generation of such data. Multiple research groups have described a variety of approaches for establishing high-quality proteome-wide datasets. However, data analysis is as critical as data production for reliable and insightful biological interpretation, and no consistent and robust solutions have been offered to the community so far. Here, we introduce the requirements for rigorous spatial proteomics data analysis, as well as the statistical machine learning methodologies needed to address them, including supervised and semi-supervised machine learning, clustering, and novelty detection. We present freely available software solutions that implement innovative state-of-the-art analysis pipelines and illustrate the use of these tools through several case studies involving multiple organisms, experimental designs, mass spectrometry platforms, and quantitation techniques. We also propose sound analysis strategies for identifying dynamic changes in subcellular localization by comparing and contrasting data describing different biological conditions. We conclude by discussing future needs and developments in spatial proteomics data analysis.     

Sitting on a Sloping Seat Does Not Reduce the Strain Sustained by the Postural Chain

The objective of this study was to explore the effect of a forward sloping seat on posture and muscular activity of the trunk and lower limbs. To this aim, twelve asymptomatic participants were tested in six conditions varying seat slope (0°, 15° forward) and height (high, medium, low). Angular position of head, trunk and pelvis was assessed with an inertial orientation system, and muscular activity of 11 superficial postural muscles located in the trunk and lower limbs was estimated using normalized EMG. Results showed that a forward sloping seat, compared to a flat seat, induced a greater activity of the soleus (p<0.01), vastus lateralis (p<0.05) and vastus medialis (p<0.05), as well a lower hip flexion (p<0.01). In contrast, no significant variation of head, trunk and pelvis angular position was observed according to seat slope. It was concluded that forward sloping seats increase the load sustained by the lower limbs, without a systematic improvement of body posture.     

Sodium selenide toxicity is mediated by O2-dependent DNA breaks

Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H(2)Se/HSe(-/)Se(2-)). Among the genes whose deletion caused hypersensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O(2)-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The (?)OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O(2). Finally we showed that, in vivo, toxicity strictly depended on the presence of O(2). Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O(2)-dependent radical-based mechanism.     

Characterization of new microsatellite loci isolated from Santiria trimera (Burseraceae)

? Premise of the study: To study the genetic structure among three morphotypes of an African rainforest tree species, Santiria trimera, nuclear microsatellite markers were isolated and characterized. ? Methods and Results: Seven polymorphic loci were isolated using a pyrosequencing-based protocol and successfully amplified on three different morphotypes of S. trimera. For six of the seven loci, there is at least one private allele for one of the three morphotypes. The mean effective number of alleles is about four for each of the three morphotypes. ? Conclusions: These microsatellite markers are promising to explore the genetic delimitation among sympatric morphotypes found in Gabonese forests and to study the spatial genetic structure within each gene pool.     

The Decay of the Chromosomally Encoded ccdO157 Toxin–Antitoxin System in the Escherichia coli Species

The origin and the evolution of toxin–antitoxin (TA) systems remain to be uncovered. TA systems are abundant in bacterial chromosomes and are thought to be part of the flexible genome that originates from horizontal gene transfer. To gain insight into TA system evolution, we analyzed the distribution of the chromosomally encoded ccd_O157 system in 395 natural isolates of Escherichia coli. It was discovered in the E. coli O157:H7 strain in which it constitutes a genomic islet between two core genes (folA and apaH). Our study revealed that the folA–apaH intergenic region is plastic and subject to insertion of foreign DNA. It could be composed (i) of a repetitive extragenic palindromic (REP) sequence, (ii) of the ccd_O157 system or subtle variants of it, (iii) of a large DNA piece that contained a ccdA_O157 antitoxin remnant in association with ORFs of unknown function, or (iv) of a variant of it containing an insertion sequence in the ccdA_O157 remnant. Sequence analysis and functional tests of the ccd_O157 variants revealed that 69% of the variants were composed of an active toxin and antitoxin, 29% were composed of an active antitoxin and an inactive toxin, and in 2% of the cases both ORFs were inactive. Molecular evolution analysis showed that ccdB_O157 is under neutral evolution, suggesting that this system is devoid of any biological role in the E. coli species.     

Na+ -D-glucose cotransporter in the kidney of Leucoraja erinacea: molecular identification and intrarenal distribution

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-D-glucose cotransporter type 1 involved in renal D-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus laevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of D-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryosections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb d-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts.     

Genetic diversity of Costa Rican populations of the rice planthopper Tagosodes orizicolus (Homoptera: Delphacidae)

Tagosodes orizicolus (Homoptera: Delphacidae) is one of the main constraints of the rice production in the Neotropics. This planthopper produces severe damages as a phloem feeder, causes mechanical injury during oviposition and vectors the rice hoja blanca virus (RHBV). The main objective of this study was to determine the genetic diversity of T. orizicolus populations from three rice growing regions of Costa Rica, using RAPDs. Individuals from Guanacaste, Parrita, San Carlos and Cali-Colombia, as outgroup, were analyzed using the random primers. Phenetic relationships revealed that the Costa Rican populations were clearly separated from Cali-Colombia, sharing less than 25% similarity. Costa Rican populations were divided into two main branches separated at 30% similarity. The first branch included Guanacaste and San Carlos and the second displayed Parrita. In relation to similarity indexes within groups, the Guanacaste cluster showed the highest (over 50%) and Cali-Colombia was the most diverse (28%). The correspondence analysis confirmed the clusters of the phenogram and showed close interactions between the Parrita and San Carlos populations. The genetic separation observed could be the result of the geographic isolation among populations, but it could also be explained by the infection with the rickettsia Wolbachia pipientis. This bacterium causes cytoplasmic incompatibility in its host, which results in non-viable progeny when infected males mate with non-infected females, or when insects hosting different strains of Wolbachia mate. Then, a search for Wolbachia in previously described populations of T orizicolus was initiated. The presence of the bacteria was analyzed by PCR with 16S rDNA-specific primers for Wolbachia. The PCR analyses revealed infections of 86% in the population of San Carlos, 96% in Guanacaste, 37% in Parrita and 100% in Cali-Colombia. Crosses between individuals of T. orizicolus from Parrita and Guanacaste were performed for testing cytoplasmic incompatibility. When infected males were crossed with non-infected females within the same population, a significant reduction in progeny number was obtained as well as when crosses between infected individuals belonging to different populations were performed. These experiments showed cytoplasmic incompatibility not only caused by the presence of Wolbachia within the population, but also by the presence of different strains of the bacteria between populations.