Effector proteins of the bacterial pathogen Pseudomonas syringae alter the extracellular proteome of the host plant, Arabidopsis thaliana

In plants, potential pathogenic bacteria do not enter the host cell. Therefore, a large portion of the molecular interaction between microbial pathogen and host occurs in the extracellular space. To investigate potential mechanisms of disease resistance and susceptibility, we analyzed changes in the extracellular proteome, or secretome, using the Arabidopsis-Pseudomonas syringae pathosystem. This system provides the possibility to directly compare interactions resulting in basal resistance, susceptibility, and gene-specific resistance by using different genotypes of Pseudomonas on the same host. After infecting suspension-cultured cells of Arabidopsis with the Pseudomonas strain of interest, we isolated protein from the cell culture medium representing the secretome. After one-dimensional gel separation and in-gel digestion of proteins, we used iTRAQ (isobaric tags for relative and absolute quantitation) labeling in conjunction with LC-MS/MS to perform relative quantitative comparisons of the secretomes from each of these interactions. We obtained quantitative information from 45 Arabidopsis proteins that were present in all three biological experiments. We observed complex patterns of accumulation, ranging from proteins that decreased in abundance in the presence of all three bacterial strains to proteins that specifically increased or decreased during only one of the interactions. A particularly intriguing result was that the virulent bacteria (e.g. a susceptible interaction) caused the extracellular accumulation of a specific subset of host proteins lacking traditional signal peptides. These results indicate that the pathogen may manipulate host secretion to promote the successful invasion of plants.     

Ecological barcoding of corallivory by second internal transcribed spacer sequences: hosts of coralliophiline gastropods detected by the cnidarian DNA in their stomach

The second internal transcribed spacer (ITS2) of the nuclear ribosomal RNA cluster (rDNA) is significantly smaller in the Cnidaria (120–260 bp) than in the rest of the Metazoa. ITS2 is one of the fastest evolving DNA regions among those commonly used in molecular systematics and has been proposed as a possible barcoding gene for Cnidaria to replace the currently problematic mitochondrial sequences used. We have reviewed the intraspecific and interspecific variation of ITS2 rRNA sequences in the Anthozoa. We have observed that the lower limits of the interspecific DNA divergence ranges very often overlap with intraspecific ranges, and identical sequences from individuals of different species are not rare. This finding can result in problems similar to those encountered with the mitochondrial COI, and we conclude that ITS2 does not prove significantly better than COI for standard taxonomic DNA barcoding in Anthozoa. However, ITS2 appears to be a promising gene in the ecological DNA barcoding of corallivory, where taxonomic accuracy at genus or even family level may represent a significant improvement of current knowledge. We have successfully amplified and sequenced ITS2 from template DNA extracted from foot muscle and from stomach contents of corallivorous gastropods, and from their anthozoan hosts. The small size of cnidarian ITS2 makes it a very easy and efficient tool for ecological barcoding of associations. Ecological barcoding of corallivory is an indispensable approach to the study of the associations in deep water, where direct observation is severely limited by logistics and costs.     

A rapid and sensitive method for measuring cell adhesion

We have adapted the CyQuant® assay to provide a simple, rapid, sensitive and highly reproducible method for measuring cell adhesion. The modified CyQuant® assay eliminates the requirement for labour intensive fluorescent labelling protocols prior to experimentation and has the sensitivity to measure small numbers (>1000) of adherent cells.     

Temporal and Individual Variation in Offspring Provisioning by Tree Swallows: A New Method of Automated Nest Attendance Monitoring

Studies of the ecology and evolution of avian nesting behavior have been limited by the difficulty and expense of sampling nest attendance behavior across entire days or throughout a substantial portion of the nestling period. Direct observation of nesting birds using human observers and most automated devices requires sub-sampling of the nestling period, which does not allow for the quantification of the duration of chick-feeding by parents within a day, and may also inadequately capture temporal variation in the rate at which chicks are fed. Here I describe an inexpensive device, the Automated Perch Recorder (APR) system, which collects accurate, long-term data on hourly rates of nest visitation, the duration of a pair''s workday, and the total number of visits the pair makes to their nest across the entire period for which it is deployed. I also describe methods for verifying the accuracy of the system in the field, and several examples of how these data can be used to explore the causes of variation in and tradeoffs between the rate at which birds feed their chicks and the total length of time birds spend feeding chicks in a day.     

Ants,Cataglyphis cursor,Use Precisely Directed Rescue Behavior to Free Entrapped Relatives

Although helping behavior is ubiquitous throughout the animal kingdom, actual rescue activity is particularly rare. Nonetheless, here we report the first experimental evidence that ants, Cataglyphis cursor, use precisely directed rescue behavior to free entrapped victims; equally important, they carefully discriminate between individuals in distress, offering aid only to nestmates. Our experiments simulate a natural situation, which we often observed in the field when collecting Catagyphis ants, causing sand to collapse in the process. Using a novel experimental technique that binds victims experimentally, we observed the behavior of separate, randomly chosen groups of 5 C. cursor nestmates under one of six conditions. In five of these conditions, a test stimulus (the “victim”) was ensnared with nylon thread and held partially beneath the sand. The test stimulus was either (1) an individual from the same colony; (2) an individual from a different colony of C cursor; (3) an ant from a different ant species; (4) a common prey item; or, (5) a motionless (chilled) nestmate. In the final condition, the test stimulus (6) consisted of the empty snare apparatus. Our results demonstrate that ants are able to recognize what, exactly, holds their relative in place and direct their behavior to that object, the snare, in particular. They begin by excavating sand, which exposes the nylon snare, transporting sand away from it, and then biting at the snare itself. Snare biting, a behavior never before reported in the literature, demonstrates that rescue behavior is far more sophisticated, exact and complexly organized than the simple forms of helping behavior already known, namely limb pulling and sand digging. That is, limb pulling and sand digging could be released directly by a chemical call for help and thus result from a very simple mechanism. However, it''s difficult to see how this same releasing mechanism could guide rescuers to the precise location of the nylon thread, and enable them to target their bites to the thread itself.     

Allogeneic Non-Adherent Bone Marrow Cells Facilitate Hematopoietic Recovery but Do Not Lead to Allogeneic Engraftment

Background

Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4⁺, murine CD4^−/−, HLA-DR3⁺ mice.

Methodology/Principal Findings

Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45⁺ showed depletion of CD4⁺, CD8⁺, CD19⁺, and CD117⁺ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4⁺ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death.

Conclusions/Significance

NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.     

N-Cadherin Negatively Regulates Osteoblast Proliferation and Survival by Antagonizing Wnt,ERK and PI3K/Akt Signalling

Background

Osteoblasts are bone forming cells that play an essential role in osteogenesis. The elucidation of the mechanisms that control osteoblast number is of major interest for the treatment of skeletal disorders characterized by abnormal bone formation. Canonical Wnt signalling plays an important role in the control of osteoblast proliferation, differentiation and survival. Recent studies indicate that the cell-cell adhesion molecule N-cadherin interacts with the Wnt co-receptors LRP5/6 to regulate osteoblast differentiation and bone accrual. The role of N-cadherin in the control of osteoblast proliferation and survival remains unknown.

Methods and Principal Findings

Using murine MC3T3-E1 osteoblastic cells and N-cadherin transgenic mice, we demonstrate that N-cadherin overexpression inhibits cell proliferation in vitro and in vivo. The negative effect of N-cadherin on cell proliferation results from decreased Wnt, ERK and PI3K/Akt signalling and is restored by N-cadherin neutralizing antibody that antagonizes N-cadherin-LRP5 interaction. Inhibition of Wnt signalling using DKK1 or Sfrp1 abolishes the ability of N-cadherin blockade to restore ERK and PI3K signalling and cell proliferation, indicating that the altered cell growth in N-cadherin overexpressing cells is in part secondary to alterations in Wnt signalling. Consistently, we found that N-cadherin overexpression inhibits the expression of Wnt3a ligand and its downstream targets c-myc and cyclin D1, an effect that is partially reversed by N-cadherin blockade. We also show that N-cadherin overexpression decreases osteoblast survival in vitro and in vivo. This negative effect on cell survival results from inhibition of PI3K/Akt signalling and increased Bax/Bcl-2, a mechanism that is rescued by Wnt3a.

Conclusion

The data show that N-cadherin negatively controls osteoblast proliferation and survival via inhibition of autocrine/paracrine Wnt3a ligand expression and attenuation of Wnt, ERK and PI3K/Akt signalling, which provides novel mechanisms by which N-cadherin regulates osteoblast number.     

Sporadic Infantile Epileptic Encephalopathy Caused by Mutations in PCDH19 Resembles Dravet Syndrome but Mainly Affects Females

Dravet syndrome (DS) is a genetically determined epileptic encephalopathy mainly caused by de novo mutations in the SCN1A gene. Since 2003, we have performed molecular analyses in a large series of patients with DS, 27% of whom were negative for mutations or rearrangements in SCN1A. In order to identify new genes responsible for the disorder in the SCN1A-negative patients, 41 probands were screened for micro-rearrangements with Illumina high-density SNP microarrays. A hemizygous deletion on chromosome Xq22.1, encompassing the PCDH19 gene, was found in one male patient. To confirm that PCDH19 is responsible for a Dravet-like syndrome, we sequenced its coding region in 73 additional SCN1A-negative patients. Nine different point mutations (four missense and five truncating mutations) were identified in 11 unrelated female patients. In addition, we demonstrated that the fibroblasts of our male patient were mosaic for the PCDH19 deletion. Patients with PCDH19 and SCN1A mutations had very similar clinical features including the association of early febrile and afebrile seizures, seizures occurring in clusters, developmental and language delays, behavioural disturbances, and cognitive regression. There were, however, slight but constant differences in the evolution of the patients, including fewer polymorphic seizures (in particular rare myoclonic jerks and atypical absences) in those with PCDH19 mutations. These results suggest that PCDH19 plays a major role in epileptic encephalopathies, with a clinical spectrum overlapping that of DS. This disorder mainly affects females. The identification of an affected mosaic male strongly supports the hypothesis that cellular interference is the pathogenic mechanism.     

Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA⁺/VacA⁺ H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.