Yersinia virulence depends on mimicry of host Rho-family nucleotide dissociation inhibitors

Yersinia spp. cause gastroenteritis and the plague, representing historically devastating pathogens that are currently an important biodefense and antibiotic resistance concern. A critical virulence determinant is the Yersinia protein kinase A, or YpkA, a multidomain protein that disrupts the eukaryotic actin cytoskeleton. Here we solve the crystal structure of a YpkA-Rac1 complex and find that YpkA possesses a Rac1 binding domain that mimics host guanidine nucleotide dissociation inhibitors (GDIs) of the Rho GTPases. YpkA inhibits nucleotide exchange in Rac1 and RhoA, and mutations that disrupt the YpkA-GTPase interface abolish this activity in vitro and impair in vivo YpkA-induced cytoskeletal disruption. In cell culture experiments, the kinase and the GDI domains of YpkA act synergistically to promote cytoskeletal disruption, and a Y. pseudotuberculosis mutant lacking YpkA GDI activity shows attenuated virulence in a mouse infection assay. We conclude that virulence in Yersinia depends strongly upon mimicry of host GDI proteins by YpkA.     

Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms

The influence of salicylic acid (SA) on elicitation of defense mechanisms in Arabidopsis (Arabidopsis thaliana) seeds and seedlings was assessed by physiological measurements combined with global expression profiling (proteomics). Parallel experiments were carried out using the NahG transgenic plants expressing the bacterial gene encoding SA hydroxylase, which cannot accumulate the active form of this plant defense elicitor. SA markedly improved germination under salt stress. Proteomic analyses disclosed a specific accumulation of protein spots regulated by SA as inferred by silver-nitrate staining of two-dimensional gels, detection of carbonylated (oxidized) proteins, and neosynthesized proteins with [35S]-methionine. The combined results revealed several processes potentially affected by SA. This molecule enhanced the reinduction of the late maturation program during early stages of germination, thereby allowing the germinating seeds to reinforce their capacity to mount adaptive responses in environmental water stress. Other processes affected by SA concerned the quality of protein translation, the priming of seed metabolism, the synthesis of antioxidant enzymes, and the mobilization of seed storage proteins. All the observed effects are likely to improve seed vigor. Another aspect revealed by this study concerned the oxidative stress entailed by SA in germinating seeds, as inferred from a characterization of the carbonylated (oxidized) proteome. Finally, the proteomic data revealed a close interplay between abscisic signaling and SA elicitation of seed vigor.     

Amyloid by Design: Intrinsic Regulation of Microbial Amyloid Assembly

The term amyloid has historically been used to describe fibrillar aggregates formed as the result of protein misfolding and that are associated with a range of diseases broadly termed amyloidoses. The discovery of “functional amyloids” expanded the amyloid umbrella to encompass aggregates structurally similar to disease-associated amyloids but that engage in a variety of biologically useful tasks without incurring toxicity. The mechanisms by which functional amyloid systems ensure nontoxic assembly has provided insights into potential therapeutic strategies for treating amyloidoses. Some of the most-studied functional amyloids are ones produced by bacteria. Curli amyloids are extracellular fibers made by enteric bacteria that function to encase and protect bacterial communities during biofilm formation. Here we review recent studies highlighting microbial functional amyloid assembly systems that are tailored to enable the assembly of non-toxic amyloid aggregates.     

Specialized morphology corresponds to a generalist diet: linking form and function in smashing mantis shrimp crustaceans

Many animals are considered to be specialists because they have feeding structures that are fine-tuned for consuming specific prey. For example, “smasher” mantis shrimp have highly specialized predatory appendages that generate forceful strikes to break apart hard-shelled prey. Anecdotal observations suggest, however, that the diet of smashers may include soft-bodied prey as well. Our goal was to examine the diet breadth of the smasher mantis shrimp, Neogonodactylus bredini, to determine whether it has a narrow diet of hard-shelled prey. We combined studies of prey abundance, feeding behavior, and stable isotope analyses of diet in both seagrass and coral rubble to determine if N. bredini’s diet was consistent across different habitat types. The abundances of hard-shelled and soft-bodied prey varied between habitats. In feeding experiments, N. bredini consumed both prey types. N. bredini consumed a range of different prey in the field as well and, unexpectedly, the stable isotope analysis demonstrated that soft-bodied prey comprised a large proportion (29–53 %) of the diet in both habitats. Using a Bayesian mixing model framework (MixSIAR), we found that this result held even when we used uninformative, or generalist, priors and informative priors reflecting a specialist diet on hard-shelled prey and prey abundances in the field. Thus, contrary to expectation, the specialized feeding morphology of N. bredini corresponds to a broad diet of both hard-shelled and soft-bodied prey. Using multiple lines of study to describe the natural diets of other presumed specialists may demonstrate that specialized morphology often broadens rather than narrows diet breadth.     

Epigenetic regulation of vitamin D hydroxylase expression and activity in normal and malignant human prostate cells

It was previously suggested that the 25-Vitamin-D₃-1-hydroxylase (CYP27B1) is downregulated during human prostate tumor pathogenesis while the catabolic 25-Vitamin-D₃-24-hydroxylase (CYP24) expression is increased. The latter could lead to resistance against the antimitotic, prodifferentiating activity of 1,25-dihydroxycholecalciferol. Our hypothesis was that regulation of Vitamin D hydroxylase expression during prostate tumor progression might be under epigenetic control. We demonstrate by real time RT-PCR that PNT-2 human normal prostate cells indeed possess CYP27B1, but are practically devoid of CYP24 mRNA, whereas DU-145 cancer cells have constitutive expression of CYP24, and very low levels of CYP27B1 mRNA. Treatment of PNT-2 cells with the methylation inhibitor 5-aza-2′-deoxycytidine together with the deacetylation inhibitor trichostatin A resulted in elevation of both CYP27B1 and CYP24 mRNA expression demonstrating that even in normal human prostate cells expression of Vitamin D hydroxylases may be under epigenetic control. In the DU-145 malignant cell line trichostatin A together with 5-aza-2′-deoxycytidine increased CYP27B1 mRNA expression to a smaller extent than in normal cells, however this resulted in a highly significant increase in 1-hydroxylation capacity. This demonstrates for the first time that synthesis of 1,25-dihydroxycholecalciferol in human prostate tumors could be reinitiated by epigenetic regulators.     

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as “genomic storm” in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous “danger” signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.     

Radial construction of an arterial wall

Some of the most serious diseases involve altered size and structure of the arterial wall. Elucidating how arterial walls are built could aid understanding of these diseases, but little is known about how concentric layers of muscle cells and the outer adventitial layer are assembled and patterned around endothelial tubes. Using histochemical, clonal, and genetic analysis in mice, here we show that the pulmonary artery wall is constructed radially, from the inside out, by two separate but coordinated processes. One is sequential induction of successive cell layers from surrounding mesenchyme. The other is controlled invasion of outer layers by inner layer cells through developmentally regulated cell reorientation and radial migration. We propose that a radial signal gradient controls these processes and provide?evidence that PDGF-B and at least one other signal contribute. Modulation of such radial signaling pathways may underlie vessel-specific differences and pathological changes in arterial wall size and structure. VIDEO ABSTRACT:     

Finding rigid bodies in protein structures: Application to flexible fitting into cryoEM maps

We present RIBFIND, a method for detecting flexibility in protein structures via the clustering of secondary structural elements (SSEs) into rigid bodies. To test the usefulness of the method in refining atomic structures within cryoEM density we incorporated it into our flexible fitting protocol (Flex-EM). Our benchmark includes 13 pairs of protein structures in two conformations each, one of which is represented by a corresponding cryoEM map. Refining the structures in simulated and experimental maps at the 5–15 Å resolution range using rigid bodies identified by RIBFIND shows a significant improvement over using individual SSEs as rigid bodies. For the 15 Å resolution simulated maps, using RIBFIND-based rigid bodies improves the initial fits by 40.64% on average, as compared to 26.52% when using individual SSEs. Furthermore, for some test cases we show that at the sub-nanometer resolution range the fits can be further improved by applying a two-stage refinement protocol (using RIBFIND-based refinement followed by an SSE-based refinement). The method is stand-alone and could serve as a general interactive tool for guiding flexible fitting into EM maps.