The PhoB regulatory system modulates biofilm formation and stress response in El Tor biotype Vibrio cholerae

The PhoBR regulatory system is required for the induction of multiple genes under conditions of phosphate limitation. Here, we examine the role of PhoB in biofilm formation and environmental stress response in Vibrio cholerae of the El Tor biotype. Deletion of phoB or hapR enhanced biofilm formation in a phosphate-limited medium. Planktonic and redispersed biofilm cells of the Δ phoB mutant did not differ from wild type for the expression of HapR, suggesting that PhoB negatively affects biofilm formation through an HapR-independent pathway. The Δ phoB mutant exhibited elevated expression of exopolysaccharide genes vpsA and vpsL compared with the wild type. Deletion of hapR enhanced the expression of the positive regulator vpsT , but had no effect on the expression of vpsR . In contrast, deletion of phoB enhanced the expression of the positive regulator vpsR , but had no effect on the expression of hapR and vpsT . The Δ phoB mutant was more sensitive to hydrogen peroxide compared with the wild type and with an isogenic Δ rpoS mutant. Conversely, the Δ phoB mutant was more resistant to acidic conditions and high osmolarity compared with the wild type and with an isogenic Δ rpoS mutant. Taken together, our data suggest that phosphate limitation induces V. cholerae to adopt a free-swimming life style in which PhoB modulates environmental stress response in a manner that differs from the general stress response regulator RpoS.     

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida

Background

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure – the syncytium – which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium.

Results

The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure.

Conclusion

This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-923) contains supplementary material, which is available to authorized users.     

Weighted Interaction SNP Hub (WISH) network method for building genetic networks for complex diseases and traits using whole genome genotype data

Background

High-throughput genotype (HTG) data has been used primarily in genome-wide association (GWA) studies; however, GWA results explain only a limited part of the complete genetic variation of traits. In systems genetics, network approaches have been shown to be able to identify pathways and their underlying causal genes to unravel the biological and genetic background of complex diseases and traits, e.g., the Weighted Gene Co-expression Network Analysis (WGCNA) method based on microarray gene expression data. The main objective of this study was to develop a scale-free weighted genetic interaction network method using whole genome HTG data in order to detect biologically relevant pathways and potential genetic biomarkers for complex diseases and traits.

Results

We developed the Weighted Interaction SNP Hub (WISH) network method that uses HTG data to detect genome-wide interactions between single nucleotide polymorphism (SNPs) and its relationship with complex traits. Data dimensionality reduction was achieved by selecting SNPs based on its: 1) degree of genome-wide significance and 2) degree of genetic variation in a population. Network construction was based on pairwise Pearson's correlation between SNP genotypes or the epistatic interaction effect between SNP pairs. To identify modules the Topological Overlap Measure (TOM) was calculated, reflecting the degree of overlap in shared neighbours between SNP pairs. Modules, clusters of highly interconnected SNPs, were defined using a tree-cutting algorithm on the SNP dendrogram created from the dissimilarity TOM (1-TOM). Modules were selected for functional annotation based on their association with the trait of interest, defined by the Genome-wide Module Association Test (GMAT). We successfully tested the established WISH network method using simulated and real SNP interaction data and GWA study results for carcass weight in a pig resource population; this resulted in detecting modules and key functional and biological pathways related to carcass weight.

Conclusions

We developed the WISH network method which is a novel 'systems genetics' approach to study genetic networks underlying complex trait variation. The WISH network method reduces data dimensionality and statistical complexity in associating genotypes with phenotypes in GWA studies and enables researchers to identify biologically relevant pathways and potential genetic biomarkers for any complex trait of interest.

     

Arterial branching in man and monkey

Vessel diameters and branching angles are measured from a large number of arterial bifurcations in the retina of a normal human subject and in that of a rhesus monkey. The results are compared with each other and with theoretical results on this subject.     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.     

Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization.     

Disulfide bond plasticity in epidermal growth factor

Epidermal growth factor (EGF) has a (1-3,2-4,5-6) disulfide-bonding pattern. This pattern is found in nearly all EGF-like domains, despite wide variation in sequences. Biological data from EGF and at least one EGF-like domain show that disulfide bond isomers have significant bioactivity and suggests that the EGF fold can accommodate alternate disulfide-bonding patterns. The disulfide bonds in murine EGF were altered to seven different patterns and structures were calculated incorporating all the restraints from the highest resolution restraint set available (Tejero et al., 1996). Results showed that besides the native (1-3,2-4,5-6), two other disulfide-bonding patterns: (1-2,3-4,5-6) and (1-3,2-5,4-6) satisfied the restraints as well as the native. The results for these two patterns were indistinguishable from the native on the basis of distance and dihedral violations, XPLOR energies, Procheck statistics, and RMSDs of the final set of structures. Two other disulfide bond patterns, (1-2,3-5, 4-6) and (1-4,2-3,5-6) were able to satisfy all the distance restraints but had one or more cysteine dihedral violations. For all seven isomers, the final calculated structures were highly similar to EGF with all-atom RMSD's in the 1. 5-2 A range. These results suggest that the EGF backbone fold has the unique property of accommodating several different disulfide-bonding patterns.