The SdiA-Regulated Gene srgE Encodes a Type III Secreted Effector

Salmonella enterica serovar Typhimurium is a food-borne pathogen that causes severe gastroenteritis. The ability of Salmonella to cause disease depends on two type III secretion systems (T3SSs) encoded in two distinct Salmonella pathogenicity islands, 1 and 2 (SPI1 and SPI2, respectively). S. Typhimurium encodes a solo LuxR homolog, SdiA, which can detect the acyl-homoserine lactones (AHLs) produced by other bacteria and upregulate the rck operon and the srgE gene. SrgE is predicted to encode a protein of 488 residues with a coiled-coil domain between residues 345 and 382. In silico studies have provided conflicting predictions as to whether SrgE is a T3SS substrate. Therefore, in this work, we tested the hypothesis that SrgE is a T3SS effector by two methods, a β-lactamase activity assay and a split green fluorescent protein (GFP) complementation assay. SrgE with β-lactamase fused to residue 40, 100, 150, or 300 was indeed expressed and translocated into host cells, but SrgE with β-lactamase fused to residue 400 or 488 was not expressed, suggesting interference by the coiled-coil domain. Similarly, SrgE with GFP S11 fused to residue 300, but not to residue 488, was expressed and translocated into host cells. With both systems, translocation into host cells was dependent upon SPI2. A phylogenetic analysis indicated that srgE is found only within Salmonella enterica subspecies. It is found sporadically within both typhoidal and nontyphoidal serovars, although the SrgE protein sequences found within typhoidal serovars tend to cluster separately from those found in nontyphoidal serovars, suggesting functional diversification.     

Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss)

The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpo B gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpo B-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpo B-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus , and Shewanella marinus . In contrast to 16S rRNA gene-TTGE, rpo B-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpo B leads to a single band in TTGE, the rpo B gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.     

Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis

The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (–) RNA synthesis have been identified in the 3′ non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (–) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3′-end of the (–) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.     

Lasuén's pterodactyl: An early use of a pterosaur in plastic arts

Among the sculptures adorning the former building of the Faculties of Medicine and Sciences (1893) in Saragossa (Spain) is one that represents a pterosaur. It is a work by D. Lasuén based on one of the first, but little known, restorations of these animals, drawn and engraved by T. Susemihl more than half a century before. Although it seems out of place, this sculpture was simply seen as a symbol of zoology. We suggest that it may have several significations. Besides embodying the animal kingdom, it may have been a surrogate of the dragon and thus a reference to the House of Aragon through its dragon-slaying holy patron, Saint George.     

Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin

Transmission of the phytopathogenic mollicutes, spiroplasmas, and phytoplasmas by their insect vectors mainly depends on their ability to pass through gut cells, to multiply in various tissues, and to traverse the salivary gland cells. The passage of these different barriers suggests molecular interactions between the plant mollicute and the insect vector that regulate transmission. In the present study, we focused on the interaction between Spiroplasma citri and its leafhopper vector, Circulifer haematoceps. An in vitro protein overlay assay identified five significant binding activities between S. citri proteins and insect host proteins from salivary glands. One insect protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as actin. Confocal microscopy observations of infected salivary glands revealed that spiroplasmas colocated with the host actin filaments. An S. citri actin-binding protein of 44 kDa was isolated by affinity chromatography and identified by LC-MS/MS as phosphoglycerate kinase (PGK). To investigate the role of the PGK-actin interaction, we performed competitive binding and internalization assays on leafhopper cultured cell lines (Ciha-1) in which His₆-tagged PGK from S. citri or purified PGK from Saccharomyces cerevisiae was added prior to the addition of S. citri inoculum. The results suggested that exogenous PGK has no effect on spiroplasmal attachment to leafhopper cell surfaces but inhibits S. citri internalization, demonstrating that the process leading to internalization of S. citri in eukaryotic cells requires the presence of PGK. PGK, regardless of origin, reduced the entry of spiroplasmas into Ciha-1 cells in a dose-dependent manner.Phloem-feeding leafhoppers transmit plant pathogenic mollicutes, spiroplasmas, and phytoplasmas from plant to plant in a persistent propagative manner (26, 43). These phytopathogenic mollicutes are restricted to phloem and to certain vector tissues; thus, their vectors are phloem sap-feeding specialists. After being ingested from plant phloem by their insect vectors, they traverse the insect gut wall, move into the hemolymph, where they multiply, and invade the salivary glands (20, 33, 34, 36). During their movements in the insect vector until its transmission to a new host plant, spiroplasmas and phytoplasmas must traverse two major physical barriers, namely, the insect intestine and the salivary gland (35, 53). Until now, little was known about the molecular and cellular interactions contributing to the crossing of these physical barriers. Several lines of evidence suggest that host-pathogen interactions could be a prerequisite for invasion and colonization of insect vector organs (2, 48, 53). For human and animal pathogenic mollicutes, it is well established that successful colonization of the host cells requires adhesion as the first step. This event is mediated by surface proteins, and among these proteins adhesins play an important role (8, 44). Recently, it was reported that an antigenic membrane protein (Amp) of onion yellow phytoplasma interacts with the insect microfilament complex and that interaction plays an important role in determining the insect vector specificity (48). Several other immunodominant membrane proteins from various phytoplasmas have been mentioned in the literature as candidates for involvement in host-phytoplasma interactions (29, 30).Spiroplasma citri, the first phytopathogenic mollicute available in culture (45), has emerged as an outstanding model for studying spiroplasma interactions with its two hosts: the periwinkle plant and the insect vector Circulifer haematoceps. Following observations of membrane-bound cytoplasmic vesicles of midgut epithelium and salivary gland cells, S. citri was hypothesized to cross these physical barriers by receptor-mediated cell endocytosis (3, 33, 39). Several S. citri protein candidates have been identified as involved in transmission and, for a few of them, in an interaction with leafhopper vector proteins. Spiralin, the most abundant membrane protein, was suspected to be involved in the transmission for two reasons: (i) a S. citri spiralinless mutant was less effective in its transmissibility (19); (ii) spiralin acted in vitro as a lectin able to bind to glycoproteins of insect vectors and therefore might function as a ligand able to interact with leafhopper receptors (32). In addition, the ability of S. citri to be transmitted by C. haematoceps is clearly affected by disruption of a gene predicted to encode a lipoprotein with homology to a solute-binding protein of an ABC transporter (14). The proteome of nontransmissible S. citri strains specifically lacks adhesion-related proteins (ScARPs) and the membrane-associated protein P32 present in the proteome of transmissible strains (12, 13, 31). These proteins are encoded by plasmids pSci1 to -6 (46), which are present only in transmissible strains, and ScARPs share strong similarities with the adhesion-related protein SARP1 of S. citri strain BR3, in which the presence has been correlated to the ability for the spiroplasma to adhere to insect cells in vitro (9, 55). The specific interactions of S. citri with eukaryotic cells remain to be elucidated, but a combination of the effects of several proteins or a complex would be necessary to explain the invasion of a variety of host cell types by S. citri (33).Nevertheless, in the last sequence of events involved in insect vector transmission, the first contact and recognition for the efficient penetration of the salivary gland cells represents an essential step. In the present study, confocal images of infected salivary glands show the localization of S. citri cells along the actin filaments. We report the results of the first attempt to decipher the role of the spiroplasma''s phosphoglycerate kinase (PGK) in the internalization of S. citri in its insect vector''s cells.     

Analysis of skeletal muscle metabolome: Evaluation of extraction methods for targeted metabolite quantification using liquid chromatography tandem mass spectrometry

Functional metabolomics of skeletal muscle involves the simultaneous identification and quantification of a large number of metabolites. For this purpose, the extraction of metabolites from animal tissues is a crucial technical step that needs to be optimized. In this work, five extraction methods for skeletal muscle metabolome analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) were tested. Bird skeletal muscles sampled postmortem and quenched in liquid nitrogen were used. Three replicates of the same sample were extracted using the following solvent systems of varying polarity: boiling water (BW, +100 °C), cold pure methanol (CPM, −80 °C), methanol/chloroform/water (MCW, −20 °C), boiling ethanol (BE, +80 °C), and perchloric acid (PCA, −20 °C). Three injections by extraction were performed. The BW extraction showed the highest recovery of metabolites with the lowest variability (<10%) except for creatine-phosphate (creatine-P). Considering yield (area of the peaks), reproducibility, and ease, the current experiment drew a scale for the muscle metabolome extraction starting from the best to the least convenient: BW > MCW > CPM > PCA ? BE. In addition, the semiquantification of metabolites in two muscles showing different metabolic and contractile properties was carried out after BW extraction and showed expected differences in metabolite contents, thereby validating the technique for biological investigations. In conclusion, the BW extraction is recommended for analysis of skeletal muscle metabolome except for creatine-P, which was poorly recovered with this technique.     

Characterisation of root amino acid exudation in white clover (Trifolium repens L.)

Lateral roots are crucial for the plasticity of root responses to environmental conditions in soil. The bacterivorous microfauna has been shown to increase root branching and to foster auxin producing soil bacteria. However, information on modifications of plant internal auxin content by soil bacteria and bacterivores is missing. Therefore, the effects of a rhizosphere bacterial community and a common soil amoeba (Acanthamoeba castellanii) on root branching and on auxin (indole-3-acetic acid) metabolism in Lepidium sativum and Arabidopsis thaliana were investigated. In a first experimental series, bacteria increased conjugated auxin concentrations in L. sativum shoots, but did not alter free bioactive auxin content nor root branching. In contrast, in presence of soil bacteria plus amoebae free auxin concentrations in shoots and root branching increased, demonstrating that effects of bacteria on auxin metabolism in plants were strongly modified by the bacterivorous amoebae. In a second experiment, A. thaliana reporter plants for auxin (DR5) and cytokinin (ARR5) responded similarly with increased root branching in the presence of amoebae. Surprisingly, in reporter plants cytokinin but not auxin responses were detectable, accompanied by higher soil nitrate concentrations in the presence of amoebae. Likely, increased nitrate concentrations in the rhizosphere led to an accumulation of cytokinin and interactions with free auxin in plants and finally to increased root growth in the presence of amoebae. Altogether, the results show that mutual control mechanisms exist between plant hormone metabolism and microbial signalling, and that effects on hormonal concentrations of plants by free-living bacteria are strongly influenced by bacterial grazers like amoebae.     

A Bayesian method to estimate the optimal threshold of a longitudinal biomarker

The objective of this study was to develop methods to estimate the optimal threshold of a longitudinal biomarker and its credible interval when the diagnostic test is based on a criterion that reflects a dynamic progression of that biomarker. Two methods are proposed: one parametric and one non‐parametric. In both the cases, the Bayesian inference was used to derive the posterior distribution of the optimal threshold from which an estimate and a credible interval could be obtained. A numerical study shows that the bias of the parametric method is low and the coverage probability of the credible interval close to the nominal value, with a small coverage asymmetry in some cases. This is also true for the non‐parametric method in case of large sample sizes. Both the methods were applied to estimate the optimal prostate‐specific antigen nadir value to diagnose prostate cancer recurrence after a high‐intensity focused ultrasound treatment. The parametric method can also be applied to non‐longitudinal biomarkers.