In Staphylococcus aureus, the Particulate State of the Cell Envelope Is Required for the Efficient Induction of Host Defense Responses.

Upon microbial infection, host immune cells recognize bacterial cell envelope components through cognate receptors. Although bacterial cell envelope components function as innate immune molecules, the role of the physical state of the bacterial cell envelope (i.e., particulate versus soluble) in host immune activation has not been clearly defined. Here, using two different forms of the staphylococcal cell envelope of Staphylococcus aureus RN4220 and USA300 LAC strains, we provide biochemical and immunological evidence that the particulate state is required for the effective activation of host innate immune responses. In a murine model of peritoneal infection, the particulate form of the staphylococcal cell envelope (PCE) induced the production of chemokine (C-X-C motif) ligand 1 (CXCL1) and CC chemokine ligand 2 (CCL2), the chemotactic cytokines for neutrophils and monocytes, respectively, resulting in a strong influx of the phagocytes into the peritoneal cavity. In contrast, compared with PCE, the soluble form of cell envelope (SCE), which was derived from PCE by treatment with cell wall-hydrolyzing enzymes, showed minimal activity. PCE also induced the secretion of calprotectin (myeloid-related protein 8/14 [MRP8/14] complex), a phagocyte-derived antimicrobial protein, into the peritoneal cavity at a much higher level than did SCE. The injected PCE particles were phagocytosed by the infiltrated neutrophils and monocytes and then delivered to mediastinal draining lymph nodes. More importantly, intraperitoneally (i.p.) injected PCE efficiently protected mice from S. aureus infection, which was abolished by the depletion of either monocytes/macrophages or neutrophils. This study demonstrated that the physical state of bacterial cells is a critical factor for efficient host immune activation and the protection of hosts from staphylococcal infections.

Adsorption of Staphylococcus viruses S13' and S24-1 on Staphylococcus aureus strains with different glycosidic linkage patterns of wall teichoic acids.

The group of phages belonging to the family Podoviridae, genus P68virus, including Staphylococcus viruses S13' and S24-1, are important because of their benefits in phage therapy against Staphylococcus aureus infections. The O-glycosidic linkage patterns of wall teichoic acids (WTAs) in S. aureus cell walls seem to be important for adsorption of this phage group. In this study, the adsorption of Staphylococcus viruses S13' and S24-1 to S. aureus was examined using strains with modified WTA glycosidic linkage patterns. We found that the ß-O-N-acetylglucosamine of WTAs was essential for S13' adsorption, while N-acetylglucosamine, regardless of the α- and ß-O-glycosidic linkages of the WTAs, was essential for S24-1 adsorption. Next, examining the binding activities of their receptor-binding proteins (RBPs) to cell walls with different WTA glycosidic patterns, the ß-O-N-acetylglucosamine of the WTAs was essential for S13' RBP binding, while N-acetylglucosamine, regardless of the α- and ß-O-glycosidic linkages of the WTAs, was essential for S24-1 RBP binding. Therefore, the results of the RBP binding assays were consistent with those of the phage adsorption assays. Bioinformatic analysis suggested that the RBPs of Staphylococcus viruses S13' and S24-1 were structurally similar to the RBPs of phage phi11 of thefamily Siphoviridae. Phylogenetic analysis of the RBPs indicated that two phylogenetic subclusters in the family Podoviridae were related to the glycosidic linkage patterns required for phage adsorption, possibly mediated by RBPs. We hope that this study will encourage the future development of therapeutic phages.

Polyester synthesis genes associated with stress resistance are involved in an insect-bacterium symbiosis.

Many bacteria accumulate granules of polyhydroxyalkanoate (PHA) within their cells, which confer resistance to nutritional depletion and other environmental stresses. Here, we report an unexpected involvement of the bacterial endocellular storage polymer, PHA, in an insect-bacterium symbiotic association. The bean bug Riptortus pedestris harbors a beneficial and specific gut symbiont of the ß-proteobacterial genus Burkholderia, which is orally acquired by host nymphs from the environment every generation and easily cultivable and genetically manipulatable. Biochemical and cytological comparisons between symbiotic and cultured Burkholderia detected more PHA granules consisting of poly-3-hydroxybutyrate and associated phasin (PhaP) protein in the symbiotic Burkholderia. Among major PHA synthesis genes, phaB and phaC were disrupted by homologous recombination together with the phaP gene, whereby ΔphaB, ΔphaC, and ΔphaP mutants were generated. Both in culture and in symbiosis, accumulation of PHA granules was strongly suppressed in ΔphaB and ΔphaC, but only moderately in ΔphaP. In symbiosis, the host insects infected with ΔphaB and ΔphaC exhibited significantly lower symbiont densities and smaller body sizes. These deficient phenotypes associated with ΔphaB and ΔphaC were restored by complementation of the mutants with plasmids encoding a functional phaB/phaC gene. Retention analysis of the plasmids revealed positive selection acting on the functional phaB/phaC in symbiosis. These results indicate that the PHA synthesis genes of the Burkholderia symbiont are required for normal symbiotic association with the Riptortus host. In vitro culturing analyses confirmed vulnerability of the PHA gene mutants to environmental stresses, suggesting that PHA may play a role in resisting stress under symbiotic conditions.

Surface Glycopolymers Are Crucial for In Vitro Anti-Wall Teichoic Acid IgG-Mediated Complement Activation and Opsonophagocytosis of Staphylococcus aureus.

The cell envelopes of many Gram-positive bacteria contain wall teichoic acids (WTAs). Staphylococcus aureus WTAs are composed of ribitol phosphate (RboP) or glycerol phosphate (GroP) backbones substituted with D-alanine and N-acetyl-D-glucosamine (GlcNAc) or N-acetyl-D-galactosamine (GalNAc). Two WTA glycosyltransferases, TarM and TarS, are responsible for modifying the RboP WTA with α-GlcNAc and ß-GlcNAc, respectively. We recently reported that purified human serum anti-WTA IgG specifically recognizes ß-GlcNAc of the staphylococcal RboP WTA and then facilitates complement C3 deposition and opsonophagocytosis of S. aureus laboratory strains. This prompted us to examine whether anti-WTA IgG can induce C3 deposition on a diverse set of clinical S. aureus isolates. To this end, we compared anti-WTA IgG-mediated C3 deposition and opsonophagocytosis abilities using 13 different staphylococcal strains. Of note, the majority of S. aureus strains tested was recognized by anti-WTA IgG, resulting in C3 deposition and opsonophagocytosis. A minority of strains was not recognized by anti-WTA IgG, which correlated with either extensive capsule production or an alteration in the WTA glycosylation pattern. Our results demonstrate that the presence of WTAs with TarS-mediated glycosylation with ß-GlcNAc in clinically isolated S. aureus strains is an important factor for induction of anti-WTA IgG-mediated C3 deposition and opsonophagocytosis.

Human SAP is a novel peptidoglycan recognition protein that induces complement-independent phagocytosis of Staphylococcus aureus.

The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer--a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an FcγR-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain-like receptors.

Glycoepitopes of staphylococcal wall teichoic acid govern complement-mediated opsonophagocytosis via human serum antibody and mannose-binding lectin.

Serum antibodies and mannose-binding lectin (MBL) are important host defense factors for host adaptive and innate immunity, respectively. Antibodies and MBL also initiate the classical and lectin complement pathways, respectively, leading to opsonophagocytosis. We have shown previously that Staphylococcus aureus wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with α- or ß-O-N-acetyl-d-glucosamine (GlcNAc) and d-alanine, is recognized by MBL and serum anti-WTA IgG. However, the exact antigenic determinants to which anti-WTA antibodies or MBL bind have not been determined. To answer this question, several S. aureus mutants, such as α-GlcNAc glycosyltransferase-deficient S. aureus ΔtarM, ß-GlcNAc glycosyltransferase-deficient ΔtarS, and ΔtarMS double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant S. aureus strain. Here, we describe the unexpected finding that ß-GlcNAc WTA-deficient ΔtarS mutant cells (which have intact α-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas α-GlcNAc WTA-deficient ΔtarM mutant cells (which have intact ß-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in S. aureus cells was lost in the ΔtarMS double mutant but not in either single mutant. When we determined the serum concentrations of the anti-α- or anti-ß-GlcNAc-specific WTA IgGs, anti-ß-GlcNAc WTA-IgG was dominant in pooled human IgG fractions and in the intact sera of healthy adults and infants. These data demonstrate the importance of the WTA sugar conformation for human innate and adaptive immunity against S. aureus infection.

Inhibitory receptor paired Ig-like receptor B is exploited by Staphylococcus aureus for virulence.

The innate immune system has developed to acquire a wide variety of pattern-recognition receptors (PRRs) to identify potential pathogens, whereas pathogens have also developed to escape host innate immune responses. ITIM-bearing receptors are attractive targets for pathogens to attenuate immune responses against them; however, the in vivo role of the inhibitory PRRs in host-bacteria interactions remains unknown. We demonstrate in this article that Staphylococcus aureus, a major Gram-positive bacteria, exploits inhibitory PRR paired Ig-like receptor (PIR)-B on macrophages to suppress ERK1/2 and inflammasome activation, and subsequent IL-6 and IL-1ß secretion. Consequently, Pirb(-/-) mice infected with S. aureus showed enhanced inflammation and more effective bacterial clearance, resulting in resistance to the sepsis. Screening of S. aureus mutants identified lipoteichoic acid (LTA) as an essential bacterial cell wall component required for binding to PIR-B and modulating inflammatory responses. In vivo, however, an LTA-deficient S. aureus mutant was highly virulent and poorly recognized by macrophages in both wild-type and Pirb(-/-) mice, demonstrating that LTA recognition by PRRs other than PIR-B mediates effective bacterial elimination. These results provide direct evidence that bacteria exploit the inhibitory receptor for virulence, and host immune system counterbalances the infection.

Specific serum Ig recognizing staphylococcal wall teichoic acid induces complement-mediated opsonophagocytosis against Staphylococcus aureus.

Wall teichoic acid (WTA) of Staphylococcus aureus is a major cell envelope-associated glycopolymer that is a key molecule in promoting colonization during S. aureus infection. The complement system plays a key role in the opsonization and clearance of pathogens. We recently reported that S. aureus WTA functions as a ligand of human serum mannose-binding lectin (MBL), a recognition molecule of the lectin complement pathway. Intriguingly, serum MBL in adults does not bind to WTA because of an inhibitory effect of serum anti-WTA-IgG. In this study, serum anti-WTA-IgG was purified to homogeneity using a purified S. aureus WTA-coupled affinity column to examine the biological function of human anti-WTA-IgG. The purified anti-WTA-IgG contained the IgG2 subclass as a major component and specifically induced C4 and C3 deposition on the S. aureus surface in the anti-WTA-IgG-depleted serum, but not in C1q-deficient serum. Furthermore, the anti-WTA-IgG-dependent C3 deposition induced phagocytosis of S. aureus cells by human polymorphonuclear leukocytes. These results demonstrate that serum anti-WTA-IgG is a real trigger for the induction of classical complement-dependent opsonophagocytosis against S. aureus. Our results also support the fact that a lack of the lectin complement pathway in MBL-deficient adults is compensated by Ag-specific, Ab-mediated adaptive immunity.

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.

Environment-mediated accumulation of diacyl lipoproteins over their triacyl counterparts in Staphylococcus aureus.

Bacterial lipoproteins are believed to exist in only one specific lipid-modified structure, such as the diacyl form or the triacyl form, in each bacterium. In the case of Staphylococcus aureus, recent extensive matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis revealed that S. aureus lipoproteins exist in the α-aminoacylated triacyl form. Here, we discovered conditions that induce the accumulation of diacyl lipoproteins that lack α-aminoacylation in S. aureus. The accumulation of diacyl lipoproteins required a combination of conditions, including acidic pH and a post-logarithmic-growth phase. High temperatures and high salt concentrations additively accelerated the accumulation of the diacyl lipoprotein form. Following a post-logarithmic-growth phase where S. aureus MW2 cells were grown at pH 6, SitC lipoprotein was found almost exclusively in its diacyl structure rather than in its triacyl structure. This is the first report showing that the environment mediates lipid-modified structural alterations of bacterial lipoproteins.

93-kDa twin-domain serine protease inhibitor (Serpin) has a regulatory function on the beetle Toll proteolytic signaling cascade.

Serpins are protease inhibitors that play essential roles in the down-regulation of extracellular proteolytic cascades. The core serpin domain is highly conserved, and typical serpins are encoded with a molecular size of 35-50 kDa. Here, we describe a novel 93-kDa protein that contains two complete, tandemly arrayed serpin domains. This twin serpin, SPN93, was isolated from the larval hemolymph of the large beetle Tenebrio molitor. The N-terminal serpin domain of SPN93 forms a covalent complex with the Spätzle-processing enzyme, a terminal serine protease of the Toll signaling cascade, whereas the C-terminal serpin domain of SPN93 forms complexes with a modular serine protease and the Spätzle-processing enzyme-activating enzyme, which are two different enzymes of the cascade. Consequently, SPN93 inhibited ß-1,3-glucan-mediated Toll proteolytic cascade activation in an in vitro system. Site-specific proteolysis of SPN93 at the N-terminal serpin domain was observed after activation of the Toll proteolytic cascade in vivo, and down-regulation of SPN93 by RNAi sensitized ß-1,3-glucan-mediated larval death. Therefore, SPN93 is the first serpin that contains twin tandemly arrayed and functionally active serpin domains that have a regulatory role in the larval Toll proteolytic signaling cascade.

Inhibitory role for D-alanylation of wall teichoic acid in activation of insect Toll pathway by peptidoglycan of Staphylococcus aureus.

Pathogenic bacteria mitigate host immunity to establish infections, but the mechanism of this bacterial action has not been fully elucidated. To search for cell wall components that modulate innate immune responses in host organisms, we examined Staphylococcus aureus mutants, which were deficient in components of the cell wall, for pathogenicity in Drosophila. A mutation of dltA, which is responsible for the D-alanylation of teichoic acids, brought about an increase in the survival rate of adult flies that had received a septic infection with the bacteria. The growth of dltA-deficient S. aureus in adult flies was less efficient than that of the parental strain. The level of mRNA of Toll pathway-dependent antimicrobial peptides was higher in flies infected with the dltA mutant than that observed after the infection with the parental strain. The defective phenotype associated with the mutation of dltA, reduced pathogenicity and growth, was not evident in flies lacking the Toll pathway. Finally, a fraction of peptidoglycan prepared from the dltA mutant induced the expression of mRNA of a Toll-dependent antimicrobial peptide in flies and was bound by peptidoglycan recognition protein-SA in vitro more effectively than that obtained from the parental strain, and this difference was lost after the removal of wall teichoic acid from peptidoglycan. Taken together, we conclude that D-alanylated wall teichoic acid of S. aureus mitigates a Toll-mediated humoral response in Drosophila interfering with the recognition of peptidoglycan by a pattern recognition receptor.

Heterogeneous IgE reactivities to Staphylococcus pseudintermedius strains in dogs with atopic dermatitis, and the identification of DM13-domain-containing protein as a bacterial IgE-reactive molecule.

Staphylococcus pseudintermedius is one of the major pathogens causing canine skin infection. In canine atopic dermatitis (AD), heterogeneous strains of S. pseudintermedius reside on the affected skin site. Because an increase in specific IgE to this bacterium has been reported, S. pseudintermedius is likely to exacerbate the severity of canine AD. In this study, the IgE reactivities to various S. pseudintermedius strains and the IgE-reactive molecules of S. pseudintermedius were investigated. First, examining the IgE reactivities to eight strains of S. pseudintermedius using 141 sera of AD dogs, strain variation of S. pseudintermedius showed 10-63% of the IgE reactivities. This is different from the expected result based on the concept of Staphylococcus aureus clonality in AD patients. Moreover, according to the western blot analysis, there were more than four proteins reactive to IgE. Subsequently, the analysis of the common IgE-reactive protein at ∼15 kDa confirmed that the DM13-domain-containing protein was reactive in AD dogs, which is not coincident with any S. aureus IgE-reactive molecules. Considering these, S. pseudintermedius is likely to exacerbate AD severity in dogs, slightly different from the case of S. aureus in human AD.

Diversity of innate immune recognition mechanism for bacterial polymeric meso-diaminopimelic acid-type peptidoglycan in insects.

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal ß-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.

Human serum mannose-binding lectin senses wall teichoic acid Glycopolymer of Staphylococcus aureus, which is restricted in infancy.

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.

The role of phagocytosis in IL-8 production by human monocytes in response to lipoproteins on Staphylococcus aureus.

Cytokine responses to microbes are triggered by pattern recognition receptors, such as Toll-like receptors (TLRs), which sense pathogen-associated molecular patterns. Cell wall-associated triacylated lipoproteins in Staphylococcus aureus are known to be native TLR2 ligands that mediate host inflammatory responses against S. aureus. However, the mechanism by which these lipidated lipoproteins, which are buried under the thick S. aureus cell wall, work to stimulate TLR2 remains unclear. Heat-killed wild type S. aureus cells activated human monocytic THP-1 cells to produce proinflammatory cytokines, including interleukin (IL)-8, whereas the lipoprotein lipidation-deficient lgt mutant induced less than an eighth of the amount of IL-8 induced by the wild type. IL-8 induction in response to heat-killed S. aureus cells in THP-1 cells was not inhibited by a blocking antibody against cell surface TLR2, suggesting that intracellular TLR2 might be involved in the induction of IL-8 by S. aureus lipoprotein. The relationship between phagocytosis and IL-8 production in THP-1 cells was analyzed on a single-cell level by flow cytometry using fluorescein-labeled S. aureus cells and phycoerythrin-labeled anti-IL-8 antibody. Production of intracellular IL-8 was correlated with phagocytosis of S. aureus cells in THP-1 cells and in human peripheral blood mononuclear cells. Opsonization of S. aureus cells enhanced both the phagocytosis of S. aureus cells and the production of intracellular IL-8 in THP-1 cells. These results suggest that lipidated lipoproteins on S. aureus cells stimulate human monocytes after phagocytosis.

Identification of lipoteichoic acid as a ligand for draper in the phagocytosis of Staphylococcus aureus by Drosophila hemocytes.

Phagocytosis is central to cellular immunity against bacterial infections. As in mammals, both opsonin-dependent and -independent mechanisms of phagocytosis seemingly exist in Drosophila. Although candidate Drosophila receptors for phagocytosis have been reported, how they recognize bacteria, either directly or indirectly, remains to be elucidated. We searched for the Staphylococcus aureus genes required for phagocytosis by Drosophila hemocytes in a screening of mutant strains with defects in the structure of the cell wall. The genes identified included ltaS, which encodes an enzyme responsible for the synthesis of lipoteichoic acid. ltaS-dependent phagocytosis of S. aureus required the receptor Draper but not Eater or Nimrod C1, and Draper-lacking flies showed reduced resistance to a septic infection of S. aureus without a change in a humoral immune response. Finally, lipoteichoic acid bound to the extracellular region of Draper. We propose that lipoteichoic acid serves as a ligand for Draper in the phagocytosis of S. aureus by Drosophila hemocytes and that the phagocytic elimination of invading bacteria is required for flies to survive the infection.

Rapid exchange of bound ADP on the Staphylococcus aureus replication initiation protein DnaA.

In Escherichia coli, regulatory inactivation of the replication initiator DnaA occurs after initiation as a result of hydrolysis of bound ATP to ADP, but it has been unknown how DnaA is controlled to coordinate cell growth and chromosomal replication in gram-positive bacteria such as Staphylococcus aureus. This study examined the roles of ATP binding and its hydrolysis in the regulation of the S. aureus DnaA activity. In vitro, S. aureus DnaA melted S. aureus oriC in the presence of ATP but not ADP by a mechanism independent of ATP hydrolysis. Unlike E. coli DnaA, binding of ADP to S. aureus DnaA was unstable. As a result, at physiological concentrations of ATP, ADP bound to S. aureus DnaA was rapidly exchanged for ATP, thereby regenerating the ability of DnaA to form the open complex in vitro. Therefore, we examined whether formation of ADP-DnaA participates in suppression of replication initiation in vivo. Induction of the R318H mutant of the AAA+ sensor 2 protein, which has decreased intrinsic ATPase activity, caused over-initiation of chromosome replication in S. aureus, suggesting that formation of ADP-DnaA suppresses the initiation step in S. aureus. Together with the biochemical features of S. aureus DnaA, the weak ability to convert ATP-DnaA into ADP-DnaA and the instability of ADP-DnaA, these results suggest that there may be unidentified system(s) for reducing the cellular ratio of ATP-DnaA to ADP-DnaA in S. aureus and thereby delaying the re-initiation of DNA replication.

Three pairs of protease-serpin complexes cooperatively regulate the insect innate immune responses.

Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and beta-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.

Auxiliary role for D-alanylated wall teichoic acid in Toll-like receptor 2-mediated survival of Staphylococcus aureus in macrophages.

We previously reported that Staphylococcus aureus avoids killing within macrophages by exploiting the action of Toll-like receptor 2 (TLR2), which leads to the c-Jun N-terminal kinase (JNK)-mediated inhibition of superoxide production. To search for bacterial components responsible for this event, a series of S. aureus mutants, in which the synthesis of the cell wall was interrupted, were screened for the level of JNK activation in macrophages. In addition to a mutant lacking the lipoproteins that have been suggested to act as a TLR2 ligand, two mutant strains were found to activate the phosphorylation of JNK to a lesser extent than the parental strain, and this defect was recovered by acquisition of the corresponding wild-type genes. Macrophages that had phagocytosed the mutant strains produced more superoxide than those engulfing the parental strain, and the mutant bacteria were more efficiently killed in macrophages than the parent. The genes mutated, dltA and tagO, encoded proteins involved in the synthesis of D-alanylated wall teichoic acid. Unlike a cell wall fraction rich in lipoproteins, D-alanine-bound wall teichoic acid purified from the parent strain by itself did not activate JNK phosphorylation in macrophages. These results suggest that the d-alanylated wall teichoic acid of S. aureus modulates the cell wall milieu for lipoproteins so that they effectively serve as a ligand for TLR2.