Functional Divergence in Shrimp Anti-Lipopolysaccharide Factors (ALFs): From Recognition of Cell Wall Components to Antimicrobial Activity

Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded β-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a heterologous expression system. Group D ALFs were found to have impaired LPS-binding activities and only limited antimicrobial activity compared to Group B ALFs. Interestingly, all four ALF groups were shown to be simultaneously expressed in an individual shrimp and to follow different patterns of gene expression in response to a microbial infection. Group B was by far the more expressed of the ALF genes. From our results, nucleotide sequence variations in shrimp ALFs result in functional divergence, with significant differences in LPS-binding and antimicrobial activities. To our knowledge, this is the first functional characterization of the sequence diversity found in the ALF family.     

Stretched to the limit; can a short pelagic larval duration connect adult populations of an Indo‐Pacific diadromous fish (Kuhlia rupestris)?

Freshwater species on tropical islands face localized extinction and the loss of genetic diversity. Their habitats can be ephemeral due to variability in freshwater run‐off and erosion. Even worse, anthropogenic effects on these ecosystems are intense. Most of these species are amphidromous or catadromous (i.e. their life cycle includes a marine larval phase), which buffers them against many of these effects. A long pelagic larval duration (PLD) was thought to be critical to ensure the colonization and persistence in tropical islands, but recent findings indicated that several species with short PLDs are successful in those ecosystems. To test the potential of a short PLD in maintaining genetic connectivity and forestalling extirpation, we studied Kuhlia rupestris, a catadromous fish species with an extensive distribution in the western Pacific and Indian Oceans. Using a combination of molecular genetic markers (13 microsatellite loci and two gene regions from mtDNA) and modelling of larval dispersal, we show that a short PLD constrains genetic connectivity over a wide geographical range. Molecular markers showed that the short PLD did not prevent genetic divergence through evolutionary time and speciation has occurred or is occurring. Modelling of larvae dispersal suggested limited recent connectivity between genetically homogeneous populations across the Coral Sea. However, a short PLD can maintain connectivity on a subocean basin scale. Conservation and management of tropical diadromous species needs to take into account that population connectivity may be more limited than previously suspected in those species.     

Identification of a novel sn-glycerol-3-phosphate acyltransferase isoform, GPAT4, as the enzyme deficient in Agpat6-/- mice

Elucidation of the metabolic pathways of triacylglycerol (TAG) synthesis is critical to the understanding of chronic metabolic disorders such as obesity, cardiovascular disease, and diabetes. sn-Glycerol-3-phosphate acyltransferase (GPAT) and sn-1-acylglycerol-3-phosphate acyltransferase (AGPAT) catalyze the first and second steps in de novo TAG synthesis. AGPAT6 is one of eight AGPAT isoforms identified through sequence homology, but the enzyme activity for AGPAT6 has not been confirmed. We found that in liver and brown adipose tissue from Agpat6-deficient (Agpat6(-/-)) mice, N-ethylmaleimide (NEM)-sensitive GPAT specific activity was 65% lower than in tissues from wild-type mice, but AGPAT specific activity was similar. Overexpression of Agpat6 in Cos-7 cells increased an NEM-sensitive GPAT specific activity, but AGPAT specific activity was not increased. Agpat6 and Gpat1 overexpression in Cos-7 cells increased the incorporation of [(14)C]oleate into diacylglycerol (DAG) or into DAG and TAG, respectively, suggesting that the lysophosphatidic acid, phosphatidic acid, and DAG intermediates initiated by each of these isoforms lie in different cellular pools. Together, these data show that "Agpat6(-/-) mice" are actually deficient in a novel NEM-sensitive GPAT, GPAT4, and indicate that the alterations in lipid metabolism in adipose tissue, liver, and mammary epithelium of these mice are attributable to the absence of GPAT4.     

The Leishmania infantum cytosolic SIR2-related protein 1 (LiSIR2RP1) is an NAD+ -dependent deacetylase and ADP-ribosyltransferase

Proteins of the SIR2 (Silent Information Regulator 2) family are characterized by a conserved catalytic domain that exerts unique NAD(+)-dependent deacetylase activity on histones and various other cellular substrates. Previous reports from us have identified a Leishmania infantum gene encoding a cytosolic protein termed LiSIR2RP1 (Leishmania infantum SIR2-related protein 1) that belongs to the SIR2 family. Targeted disruption of one LiSIR2RP1 gene allele led to decreased amastigote virulence, in vitro as well as in vivo. In the present study, attempts were made for the first time to explore and characterize the enzymatic functions of LiSIR2RP1. The LiSIR2RP1 exhibited robust NAD(+)-dependent deacetylase and ADP-ribosyltransferase activities. Moreover, LiSIR2RP1 is capable of deacetylating tubulin, either in dimers or, when present, in taxol-stabilized microtubules or in promastigote and amastigote extracts. Furthermore, the immunostaining of parasites revealed a partial co-localization of alpha-tubulin and LiSIR2RP1 with punctate labelling, seen on the periphery of both promastigote and amastigote stages. Isolated parasite cytoskeleton reacted with antibodies showed that part of LiSIR2RP1 is associated to the cytoskeleton network of both promastigote and amastigote forms. Moreover, the Western blot analysis of the soluble and insoluble fractions of the detergent of promastigote and amastigote forms revealed the presence of alpha-tubulin in the insoluble fraction, and the LiSIR2RP1 distributed in both soluble and insoluble fractions of promastigotes as well as amastigotes. Collectively, the results of the present study demonstrate that LiSIR2RP1 is an NAD(+)-dependent deacetylase that also exerts an ADP-ribosyltransferase activity. The fact that tubulin could be among the targets of LiSIR2RP1 may have significant implications during the remodelling of the morphology of the parasite and its interaction with the host cell.     

Phylogenetic diversity of stress signalling pathways in fungi

Background  

Microbes must sense environmental stresses, transduce these signals and mount protective responses to survive in hostile environments. In this study we have tested the hypothesis that fungal stress signalling pathways have evolved rapidly in a niche-specific fashion that is independent of phylogeny. To test this hypothesis we have compared the conservation of stress signalling molecules in diverse fungal species with their stress resistance. These fungi, which include ascomycetes, basidiomycetes and microsporidia, occupy highly divergent niches from saline environments to plant or mammalian hosts.     

An updated 18S rRNA phylogeny of tunicates based on mixture and secondary structure models

Background  

Tunicates have been recently revealed to be the closest living relatives of vertebrates. Yet, with more than 2500 described species, details of their evolutionary history are still obscure. From a molecular point of view, tunicate phylogenetic relationships have been mostly studied based on analyses of 18S rRNA sequences, which indicate several major clades at odds with the traditional class-level arrangements. Nonetheless, substantial uncertainty remains about the phylogenetic relationships and taxonomic status of key groups such as the Aplousobranchia, Appendicularia, and Thaliacea.     

GMDD: a database of GMO detection methods

Background  

Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed.