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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>
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>
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>
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>
Shiratsuchi, Akiko,Shimizu, Kaori,Watanabe, Ikuko,Hashimoto, Yumi,Kurokawa, Kenji,Razanajatovo, Iony M.,Park, Keun H.,Park, Hae K.,Lee, Bok L.,Sekimizu, Kazuhisa,Nakanishi, Yoshinobu Blackwell Publishing Ltd 2010 Immunology Vol.129 No.2
<P>Summary</P><P>We previously reported that <I>Staphylococcus aureus</I> 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 <I>S. aureus</I> 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, <I>dltA</I> and <I>tagO</I>, encoded proteins involved in the synthesis of <SMALL>D</SMALL>-alanylated wall teichoic acid. Unlike a cell wall fraction rich in lipoproteins, <SMALL>D</SMALL>-alanine-bound wall teichoic acid purified from the parent strain by itself did not activate JNK phosphorylation in macrophages. These results suggest that the <SMALL>D</SMALL>-alanylated wall teichoic acid of <I>S. aureus</I> modulates the cell wall milieu for lipoproteins so that they effectively serve as a ligand for TLR2.</P>