Acyl chains of phospholipase d transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry

Background

Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.

Methodology/Principal findings

Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.

Conclusions

MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.     

Distribution and radiosensitizing effect of cholesterol-coupled Dbait molecule in rat model of glioblastoma

Background

Glioma is the most aggressive tumor of the brain and the most efficient treatments are based on radiotherapy. However, tumors are often resistant to radiotherapy due to an enhanced DNA repair activity. Short and stabilized DNA molecules (Dbait) have recently been proposed as an efficient strategy to inhibit DNA repair in tumor.

Methodology/Principal Findings

The distribution of three formulations of Dbait, (i) Dbait alone, (ii) Dbait associated with polyethylenimine, and (iii) Dbait linked with cholesterol (coDbait), was evaluated one day after intratumoral delivery in an RG2 rat glioma model. Dbait molecule distribution was assessed in the whole organ with 2D-FRI and in brain sections. CoDbait was chosen for further studies given its good retention in the brain, cellular localization, and efficacy in inducing the activation of DNA repair effectors. The radiosensitizing effect of coDbait was studied in four groups of rats bearing RG2-glioma: no treatment, radiotherapy only, coDbait alone, and CoDbait with radiotherapy. Treatment started 7 days after tumor inoculation and consisted of two series of treatment in two weeks: coDbait injection followed by a selective 6-Gy irradiation of the head. We evaluated the radiosensitizing effect using animal survival, tumor volume, cell proliferation, and vasculature characteristics with multiparametric MRI. CoDbait with radiotherapy improved the survival of rats bearing RG2-glioma by reducing tumor growth and cell proliferation without altering tumor vasculature.

Conclusion/Significance

coDbait is therefore a promising molecular therapy to sensitize glioma to radiotherapy.     

Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice

Bacteriophages have been known to be present in the gut for many years, but studies of relationships between these viruses and their hosts in the intestine are still in their infancy. We isolated three bacteriophages specific for an enteroaggregative O104:H4 Escherichia coli (EAEC) strain responsible for diarrhoeal diseases in humans. We studied the replication of these bacteriophages in vitro and in vivo in a mouse model of gut colonization. Each bacteriophage was able to replicate in vitro in both aerobic and anaerobic conditions. Each bacteriophage individually reduced biofilms formed on plastic pegs and a cocktail of the three bacteriophages was found to be more efficient. The cocktail was also able to infect bacterial aggregates formed on the surface of epithelial cells. In the mouse intestine, bacteriophages replicated for at least 3 weeks, provided the host was present, with no change in host levels in the faeces. This model of stable and continuous viral replication provides opportunities for studying the long-term coevolution of virulent bacteriophages with their hosts within a mammalian polymicrobial ecosystem.     

Proteomic studies highlight outer‐membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer‐membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer‐membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB‐dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT‐PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.     

12/15-Lipoxygenase regulates the inflammatory response to bacterial products in vivo

The peritoneal macrophage (Mphi) is the site of greatest 12/15-lipoxygenase (12/15-LOX) expression in the mouse; however, its immunoregulatory role in this tissue has not been explored. Herein, we show that 12/15-LOX is expressed by 95% of resident peritoneal CD11b(high) cells, with the remaining 5% being 12/15-LOX(-). 12/15-LOX(+) cells are phenotypically defined by high F4/80, SR-A, and Siglec1 expression, and enhanced IL-10 and G-CSF generation. In contrast, 12/15-LOX(-) cells are a dendritic cell population. Resident peritoneal Mphi numbers were significantly increased in 12/15-LOX(-/-) mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mphi from 12/15-LOX(-/-) mice exhibit multiple abnormalities in the regulation of cytokine/growth factor production both basally and after stimulation with Staphylococcus epidermidis cell-free supernatant. Resident adherent cells from 12/15-LOX(-/-) mice generate more IL-1, IL-3, GM-CSF, and IL-17, but less CCL5/RANTES than do cells from wild-type mice, while Staphylococcus epidermidis cell-free supernatant-elicited 12/15-LOX(-/-) adherent cells release less IL-12p40, IL-12p70, and RANTES, but more GM-CSF. This indicates a selective effect of 12/15-LOX on peritoneal cell cytokine production. In acute sterile peritonitis, 12/15-LOX(+) cells and LOX products were cleared, then reappeared during the resolution phase. The peritoneal lavage of 12/15-LOX(-/-) mice showed elevated TGF-beta1, along with increased immigration of monocytes/Mphi, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-alpha. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MPhi population and regulates both the recruitment of monocytes/Mphi and cytokine response to bacterial products in vivo.     

IL-6, in synergy with IL-7 or IL-15, stimulates TCR-independent proliferation and functional differentiation of CD8+ T lymphocytes

Recent reports have shown that IL-21, in synergy with IL-15, stimulates proliferation of CD8(+) T lymphocytes in the absence of signaling via the TCR. In this study, we show that IL-6, which induces phosphorylation of STAT3 similarly to IL-21, also can stimulate proliferation of CD8(+) T cells in synergy with IL-7 or IL-15. IL-6 displays a stronger synergy with IL-7 than with IL-15 to stimulate naive CD8(+) T cells. Concomitant stimulation by IL-6 or IL-21 augments phosphorylation and DNA-binding activity of STAT5 induced by IL-7 or IL-15. Like IL-21, IL-6 reduces the TCR signaling threshold required to stimulate CD8(+) T cells. Prior culture of P14 TCR transgenic CD8 T cells with IL-6 or IL-21 in the presence of IL-7 or IL-15 augments their proliferation and cytolytic activity upon subsequent stimulation by Ag. Furthermore, cytokine stimulation induces quantitatively and qualitatively distinct phenotypic changes on CD8(+) T cells compared with those induced by TCR signaling. We propose that the ability of IL-6 to induce TCR-independent activation of CD8(+) T cells in synergy with IL-7 or IL-15 may play an important role in the transition from innate to adaptive immunity.     

Characterization of 15 microsatellite loci in the pulmonate snail Pseudosuccinea columella (Mollusca, Gastropoda)

We characterized 15 new variable microsatellites in the freshwater snail Pseudosuccinea (Lymnaea) columella, as well as conditions for multiplexing and simultaneously genotyping sets of loci. Two to six alleles were detected per locus over the six populations studied. Gene diversity ranged from 0.000 to 0.498, but essentially no heterozygous individuals were observed. This resulted in extremely high F(IS) estimates, and therefore high selfing rates. The F(ST) estimates ranged from 0.18 to 1 among populations, but was generally high. These markers will constitute efficient tools for investigating the population structure of this invasive species. Cross-species amplification was on the whole unsuccessful.