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Kurokawa, Kenji,Lee, Hanna,Roh, Kyung-Baeg,Asanuma, Miwako,Kim, Young Sook,Nakayama, Hiroshi,Shiratsuchi, Akiko,Choi, Youngnim,Takeuchi, Osamu,Kang, Hee Jung,Dohmae, Naoshi,Nakanishi, Yoshinobu,Akira, American Society for Biochemistry and Molecular Bi 2009 The Journal of biological chemistry Vol.284 No.13
<P>Some synthetic lipopeptides, in addition to native lipoproteins derived from both Gram-negative bacteria and mycoplasmas, are known to activate TLR2 (Toll-like receptor 2). However, the native lipoproteins inherent to Gram-positive bacteria, which function as TLR2 ligands, have not been characterized. Here, we have purified a native lipoprotein to homogeneity from Staphylococcus aureus to study as a native TLR2 ligand. The purified 33-kDa lipoprotein was capable of stimulating TLR2 and was identified as a triacylated SitC lipoprotein, which belongs to a family of ATP binding cluster (ABC) transporter substrate-binding proteins. Analyses of the SitC-mediated production of cytokine using mouse peritoneal macrophages revealed that the SitC protein (3 nm) induced the production of tumor necrosis factor-alpha and interleukin-6. Moreover, analysis of knock-out mice showed that SitC required TLR2 and MyD88, but not TLR1 or TLR6, for the induction of cytokines. In addition to the S. aureus SitC lipoprotein, we purified two other native ABC transporter substrate-binding lipoproteins from Bacillus subtilis and Micrococcus luteus, which were both shown to stimulate TLR2. These results demonstrate that S. aureus SitC lipoprotein is triacylated and that the ABC transporter substrate-binding lipoproteins of Gram-positive bacteria function as native ligands for TLR2.</P>
Kurokawa, Kenji,Jung, Dong-Jun,An, Jang-Hyun,Fuchs, Katharina,Jeon, Yu-Jin,Kim, Na-Hyang,Li, Xuehua,Tateishi, Koichiro,Park, Ji Ae,Xia, Guoqing,Matsushita, Misao,Takahashi, Kazue,Park, Hee-Ju,Peschel, American Society for Biochemistry and Molecular Bi 2013 The Journal of biological chemistry Vol.288 No.43
<P>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 <I>Staphylococcus aureus</I> wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with α- or β-<I>O-N</I>-acetyl-<SMALL>d</SMALL>-glucosamine (GlcNAc) and <SMALL>d</SMALL>-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 <I>S. aureus</I> mutants, such as α-GlcNAc glycosyltransferase-deficient <I>S. aureus</I> Δ<I>tarM</I>, β-GlcNAc glycosyltransferase-deficient Δ<I>tarS</I>, and Δ<I>tarMS</I> double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant <I>S. aureus</I> strain. Here, we describe the unexpected finding that β-GlcNAc WTA-deficient Δ<I>tarS</I> mutant cells (which have intact α-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas α-GlcNAc WTA-deficient Δ<I>tarM</I> mutant cells (which have intact β-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in <I>S. aureus</I> cells was lost in the Δ<I>tarMS</I> double mutant but not in either single mutant. When we determined the serum concentrations of the anti-α- or anti-β-GlcNAc-specific WTA IgGs, anti-β-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 <I>S. aureus</I> infection.</P>
Kurokawa, Kenji,Hamamoto, Hiroshi,Matsuo, Miki,Nishida, Satoshi,Yamane, Noriko,Lee, Bok Luel,Murakami, Kazuhisa,Maki, Hideki,Sekimizu, Kazuhisa American Society for Microbiology 2009 Antimicrobial Agents and Chemotherapy Vol.53 No.9
<B>ABSTRACT</B><P>The availability of a silkworm larva infection model to evaluate the therapeutic effectiveness of antibiotics was examined. The 50% effective doses (ED50) of d-cycloserine against the <I>Staphylococcus aureus ddlA</I> mutant-mediated killing of larvae were remarkably lower than those against the parental strain-mediated killing of larvae. Changes in MICs and ED50 of other antibiotics were negligible, suggesting that these alterations are d-cycloserine selective. Therefore, this model is useful for selecting desired compounds based on their therapeutic effectiveness during antibiotic development.</P>
Kurokawa, Kenji,Ryu, Kyoung-Hwa,Ichikawa, Rie,Masuda, Akiko,Kim, Min-Su,Lee, Hanna,Chae, Jun-Ho,Shimizu, Takashi,Saitoh, Tatsuya,Kuwano, Koichi,Akira, Shizuo,Dohmae, Naoshi,Nakayama, Hiroshi,Lee, Bok American Society for Biochemistry and Molecular Bi 2012 The Journal of biological chemistry Vol.287 No.16
Structural evidence of <b>α</b>‐aminoacylated lipoproteins of <i>Staphylococcus aureus</i>
Asanuma, Miwako,Kurokawa, Kenji,Ichikawa, Rie,Ryu, Kyoung‐,Hwa,Chae, Jun‐,Ho,Dohmae, Naoshi,Lee, Bok Luel,Nakayama, Hiroshi Blackwell Publishing Ltd 2011 FEBS JOURNAL Vol.278 No.5
<P>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 <I>Staphylococcus aureus</I>, do not contain <I>Escherichia coli</I>‐type apolipoprotein <I>N</I>‐acyltransferase, an enzyme converting diacylated lipoproteins into triacylated forms, it has been widely believed that native lipoproteins of <I>S. aureus</I> are diacylated. However, we recently demonstrated that one lipoprotein SitC purified from <I>S. aureus</I> RN4220 strain was triacylated. Almost simultaneously, another group reported that another lipoprotein SA2202 purified from <I>S. aureus</I> SA113 strain was diacylated. The determination of exact lipidated structures of <I>S. aureus</I> 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 <I>S. aureus</I> RN4220 strain has two lipoprotein lipase‐labile <I>O</I>‐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 <I>S. aureus</I> strains, including SA113 strain and one <I>Staphylococcus epidermidis</I> strain. Moreover, four other major lipoproteins including SA2202 in <I>S. aureus</I> strains were identified as <I>N</I>‐acylated. These results strongly suggest that lipoproteins of <I>S. aureus</I> are mainly in the <I>N</I>‐acylated triacyl form.</P>
Inhibitory receptor paired Ig-like receptor B is exploited by Staphylococcus aureus for virulence.
Nakayama, Masafumi,Kurokawa, Kenji,Nakamura, Kyohei,Lee, Bok Luel,Sekimizu, Kazuhisa,Kubagawa, Hiromi,Hiramatsu, Keiichi,Yagita, Hideo,Okumura, Ko,Takai, Toshiyuki,Underhill, David M,Aderem, Alan,Ogas American Association of Immunologists 2012 Journal of Immunology Vol. No.
<P>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-1β 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.</P>
Oku, Yusuke,Kurokawa, Kenji,Matsuo, Miki,Yamada, Sakuo,Lee, Bok-Luel,Sekimizu, Kazuhisa American Society for Microbiology 2009 Journal of Bacteriology Vol.191 No.1
<B>ABSTRACT</B><P>Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium <I>Staphylococcus aureus</I>. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient <I>S. aureus</I> Δ<I>ltaS</I> mutant could grow at 30°C but not at 37°C. Even at the permissive temperature, Δ<I>ltaS</I> mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of Δ<I>ltaS</I> mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the Δ<I>ltaS</I> mutant at 37°C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the Δ<I>ltaS</I> mutation was found to be synthetically lethal with the Δ<I>tagO</I> mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30°C, suggesting that LTA and WTA compensate for one another in an essential function.</P>
Shuttle Vectors Derived from pN315 for Study of Essential Genes in <i>Staphylococcus aureus</i>
Matsuo, Miki,Kurokawa, Kenji,Lee, Bok-Luel,Sekimizu, Kazuhisa Pharmaceutical Society of Japan 2010 BIOLOGICAL & PHARMACEUTICAL BULLETIN Vol.33 No.2
<P>Using the <I>par</I> to <I>rep</I> region of the 24653 bp plasmid pN315, which is present in <I>Staphylococcus aureus</I> strain N315, we constructed three vectors that can be shuttled between <I>Escherichia coli</I> and <I>S. aureus</I> and maintained stably in <I>S. aureus</I>. Due to plasmid incompatibility, the resident plasmid in <I>S. aureus</I> cells can be replaced <I>via</I> transformation with an entering plasmid, which carries a different drug resistance gene. To evaluate the applicability of this plasmid-based approach for identifying genes essential for <I>S. aureus</I> cell growth, the chromosomal <I>mraY</I> gene, which is involved in peptidoglycan biosynthesis, was deleted in cells harboring a resident plasmid with an intact <I>mraY</I> gene. The resultant disruptant was then transformed with an empty vector. Cells with a chromosomal <I>mraY</I> deletion but lacking the plasmid supplying <I>mraY</I> could not be recovered, suggesting that <I>mraY</I> is indispensable for staphylococcal cell growth or viability. In contrast, other two genes were shown to be dispensable by this system. Thus, the pN315-based plasmids appear to be useful for studying genes essential for <I>S. aureus</I> cell growth.</P>