Taxonomic study on Sinopoda Jäger, 1999 (Araneae, Sparassidae, Heteropodinae), with three new species from Korea.

The genus Sinopoda Jäger, 1999 is a group of huntsman spiders (Araneae: Sparassidae: Heteropodinae), and currently seven species have been reported in Korea. In this study, three new species are described from Korea, Sinopodabigibba sp. nov., Sinopodabogil sp. nov., and Sinopodapantherina sp. nov.; Sinopodajirisanensis Kim & Chae, 2013 is revalidated with neotype designation, and had been formerly synonymized with Sinopodaforcipata (Karsch, 1881). Additionally, all previous records of Sinopodastellatops Jäger & Ono, 2002 and S.forcipata from Korea are deemed misidentifications of S.jirisanensis and S.bogil sp. nov., respectively.

Novel bacterial lipoprotein structures conserved in low-GC content gram-positive bacteria are recognized by Toll-like receptor 2.

Bacterial lipoproteins/lipopeptides inducing host innate immune responses are sensed by mammalian Toll-like receptor 2 (TLR2). These bacterial lipoproteins are structurally divided into two groups, diacylated or triacylated lipoproteins, by the absence or presence of an amide-linked fatty acid. The presence of diacylated lipoproteins has been predicted in low-GC content gram-positive bacteria and mycoplasmas based on the absence of one modification enzyme in their genomes; however, we recently determined triacylated structures in low-GC gram-positive Staphylococcus aureus, raising questions about the actual lipoprotein structure in other low-GC content gram-positive bacteria. Here, through intensive MS analyses, we identified a novel and unique bacterial lipoprotein structure containing an N-acyl-S-monoacyl-glyceryl-cysteine (named the lyso structure) from low-GC gram-positive Enterococcus faecalis, Bacillus cereus, Streptococcus sanguinis, and Lactobacillus bulgaricus. Two of the purified native lyso-form lipoproteins induced proinflammatory cytokine production from mice macrophages in a TLR2-dependent and TLR1-independent manner but with a different dependence on TLR6. Additionally, two other new lipoprotein structures were identified. One is the "N-acetyl" lipoprotein structure containing N-acetyl-S-diacyl-glyceryl-cysteine, which was found in five gram-positive bacteria, including Bacillus subtilis. The N-acetyl lipoproteins induced the proinflammatory cytokines through the TLR2/6 heterodimer. The other was identified in a mycoplasma strain and is an unusual diacyl lipoprotein structure containing two amino acids before the lipid-modified cysteine residue. Taken together, our results suggest the existence of novel TLR2-stimulating lyso and N-acetyl forms of lipoproteins that are conserved in low-GC content gram-positive bacteria and provide clear evidence for the presence of yet to be identified key enzymes involved in the bacterial lipoprotein biosynthesis.

Purification and characterization of tenecin 4, a new anti-Gram-negative bacterial peptide, from the beetle Tenebrio molitor.

The biochemical characterization of novel antimicrobial peptides (AMPs) and the determination of ligand molecules that induce AMP production are essential for understanding the host innate immune response in insects. Here, we purified a new 14-kDa AMP, named tenecin 4, from the larval hemolymph of the beetle Tenebrio molitor. Tenecin 4 contains 14% glycine residues and has moderate similarities both to the C-terminal region of Drosophila attacin and to silk-moth gloverin proteins. Purified tenecin 4 showed bactericidal activity against Gram-negative Escherichia coli but not against Gram-positive Bacillus subtilis or the fungus Candida albicans. Tenecin 4 production was induced by Toll cascade-activating ligands, such as ß-1,3-glucan, lysine-type peptidoglycan and active Spätzle, and by the probable Imd pathway-activating ligand monomeric meso-diaminopimelic acid-type peptidoglycan. Taken together, these data show that tenecin 4 is a defense protein against Gram-negative pathogens and is induced by multiple ligands in Tenebrio larvae.

Biochemical characterization of evasion from peptidoglycan recognition by Staphylococcus aureus D-alanylated wall teichoic acid in insect innate immunity.

We recently reported that D-alanylation of Staphylococcus aureus wall teichoic acid (WTA) mitigates an induction of the Toll-mediated humoral response in Drosophila by interfering with peptidoglycan (PG) recognition by PG recognition protein-SA (PGRP-SA). Here, we investigated the mode of this interference by using an in vitro cell free system from larvae of the coleoptran insect Tenebrio molitor. WTA modification on PG had a potent inhibitory effect on PGRP-SA-mediated Toll proteolytic cascade activation, and the D-alanylation of WTA enhanced its inhibitory effect. Purified D-alanylated WTA released from PG lost its inhibitory action on both Toll cascade activation and PGRP-SA binding to insoluble PG. The inhibition of PGRP-SA binding to PG by D-alanylated WTA took place not only on polymeric PG but also on WTA-attached disaccharide units of monomeric PG. These results suggest that D-alanylation-mediated evasion requires the covalent bonding of D-alanylated WTA to PG, but not net-like cross-linking structure of PG.

Structural evidence of α-aminoacylated lipoproteins of Staphylococcus aureus.

Bacterial lipoproteins are known to be diacylated or triacylated and activate mammalian immune cells via Toll-like receptor 2/6 or 2/1 heterodimer. Because the genomes of low G+C content gram-positive bacteria, such as Staphylococcus aureus, do not contain Escherichia coli-type apolipoprotein N-acyltransferase, an enzyme converting diacylated lipoproteins into triacylated forms, it has been widely believed that native lipoproteins of S. aureus are diacylated. However, we recently demonstrated that one lipoprotein SitC purified from S. aureus RN4220 strain was triacylated. Almost simultaneously, another group reported that another lipoprotein SA2202 purified from S. aureus SA113 strain was diacylated. The determination of exact lipidated structures of S. aureus lipoproteins is thus crucial for elucidating the molecular basis of host-microorganism interactions. Toward this purpose, we intensively used MS-based analyses. Here, we demonstrate that SitC lipoprotein of S. aureus RN4220 strain has two lipoprotein lipase-labile O-esterified fatty acids and one lipoprotein lipase-resistant fatty acid. Further MS/MS analysis of the lipoprotein lipase digest revealed that the lipoprotein lipase-resistant fatty acid was acylated to α-amino group of the N-terminal cysteine residue of SitC. Triacylated forms of SitC with various length fatty acids were also confirmed in cell lysate of the RN4220 and Triton X-114 phase in three other S. aureus strains, including SA113 strain and one Staphylococcus epidermidis strain. Moreover, four other major lipoproteins including SA2202 in S. aureus strains were identified as N-acylated. These results strongly suggest that lipoproteins of S. aureus are mainly in the N-acylated triacyl form.