Evolutionary transition in symbiotic syndromes enabled diversification of phytophagous insects on an imbalanced diet

Evolutionary adaptations for the exploitation of nutritionally challenging or toxic host plants represent a major force driving the diversification of phytophagous insects. Although symbiotic bacteria are known to have essential nutritional roles for insects, examples of radiations into novel ecological niches following the acquisition of specific symbionts remain scarce. Here we characterized the microbiota across bugs of the family Pyrrhocoridae and investigated whether the acquisition of vitamin-supplementing symbionts enabled the hosts to diversify into the nutritionally imbalanced and chemically well-defended seeds of Malvales plants as a food source. Our results indicate that vitamin-provisioning Actinobacteria (Coriobacterium and Gordonibacter), as well as Firmicutes (Clostridium) and Proteobacteria (Klebsiella) are widespread across Pyrrhocoridae, but absent from the sister family Largidae and other outgroup taxa. Despite the consistent association with a specific microbiota, the Pyrrhocoridae phylogeny is neither congruent with a dendrogram based on the hosts'' microbial community profiles nor phylogenies of individual symbiont strains, indicating frequent horizontal exchange of symbiotic partners. Phylogenetic dating analyses based on the fossil record reveal an origin of the Pyrrhocoridae core microbiota in the late Cretaceous (81.2–86.5 million years ago), following the transition from crypt-associated beta-proteobacterial symbionts to an anaerobic community localized in the M3 region of the midgut. The change in symbiotic syndromes (that is, symbiont identity and localization) and the acquisition of the pyrrhocorid core microbiota followed the evolution of their preferred host plants (Malvales), suggesting that the symbionts facilitated their hosts'' adaptation to this imbalanced nutritional resource and enabled the subsequent diversification in a competition-poor ecological niche.     

Novel clade of alphaproteobacterial endosymbionts associated with stinkbugs and other arthropods

Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genus Nysius (Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiont Schneideria nysicola within a pair of bacteriomes in the abdomen. Our survey of Nysius species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those of Wolbachia. Diagnostic PCR survey of 343 individuals representing 24 populations of four Nysius species revealed overall detection rates of the alphaproteobacteria at 77.6% in Nysius plebeius, 87.7% in Nysius sp. 1, 81.0% in Nysius sp. 2, and 100% in Nysius expressus. Further survey of diverse stinkbugs representing 24 families, 191 species, and 582 individuals detected the alphaproteobacteria from an additional 12 species representing six families. Molecular phylogenetic analysis showed that the alphaproteobacteria from the stinkbugs form a distinct and coherent monophyletic group in the order Rickettsiales together with several uncharacterized endosymbionts from fleas and ticks. The alphaproteobacterial symbiont clade was allied to bacterial clades such as the endosymbionts of acanthamoebae, the endosymbionts of cnidarians, and Midichloria spp., the mitochondrion-associated endosymbionts of ticks. In situ hybridization and electron microscopy identified small filamentous bacterial cells in various tissues of N. plebeius, including the bacteriome and ovary. The concentrated localization of the symbiont cells at the anterior pole of oocytes indicated its vertical transmission route through host insect generations. The designation "Candidatus Lariskella arthropodarum" is proposed for the endosymbiont clade.     

Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.     

Elevated extracellular calcium stimulates secretion of bone morphogenetic protein 2 by a macrophage cell line

It has been suggested that macrophages and multinucleated giant cells are responsible for phagocytosis of resorbable calcium phosphate (CaP) compounds implanted in bone defects. However, function of macrophages around the CaP, if continuously exposed to various concentration of extracellular calcium ions ([Ca(2+)](o)), is still unknown. The present study showed that when resorbable octacalcium phosphate was implanted in mouse calvaria, macrophage-like cells were observed around the implant during bone formation. Then, experiments were designed to investigate whether secretion of bone morphogenetic protein 2 (BMP-2) is enhanced by [Ca(2+)](o) in a macrophage cell line (J774A.1) in vitro. The mRNA expression and the secretion of BMP-2 in J774A.1 cells were significantly increased when incubated in the medium with [Ca(2+)](o) up to 14mM. The promotion of mRNA expression was maintained even when incubated with a small amount of minute CaP crystals. The present results suggest that [Ca(2+)](o) above physiological concentration may stimulate macrophages to induce osteogenic cytokine, such as BMP-2, for bone formation by osteoblast.     

Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells

Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 Å. The protein is unusually elongated and mainly comprises long β-strands aligned with its long axis. It is similar to aerolysin-type β-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small β-sheet sandwiched by short α-helices, is probably the target-binding module. Two other domains are both β-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming β-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The β-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.     

Synthesis and pharmacological evaluation of 1,1,3-substituted urea derivatives as potent TNF-α production inhibitors

A three substituted urea derivative, SA13353 (compound 1a), exhibited potent inhibitory activity against lipopolysaccharide (LPS)-induced TNF-α production. We focused on the 1,1-substituted moiety (R¹ and R²) of SA13353 and investigated substituent effects of this moiety on LPS-induced TNF-α production by oral administration in rats. The synthesis of the urea derivatives was performed rapidly in a one-pot manner using a manual synthesizer. Several compounds containing hydrophobic substituents at this moiety showed more potent inhibitory activities than SA13353.