高度バンコマイシン耐性黄色ブドウ球菌(VRSA)感染症に対する治療法の確立

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 関水 和久, 東京大学教授 堅田 利明, 東京大学教授 三浦 正幸, 東京大学教授 一條 秀憲, 東京大学准教授 八代田 英樹University of Tokyo(東京大学

Negative Regulation of Quorum-Sensing Systems in Pseudomonas aeruginosa by ATP-Dependent Lon Protease▿

Lon protease, a member of the ATP-dependent protease family, regulates numerous cellular systems by degrading specific substrates. Here, we demonstrate that Lon is involved in the regulation of quorum-sensing (QS) signaling systems in Pseudomonas aeruginosa, an opportunistic human pathogen. The organism has two acyl-homoserine lactone (HSL)-mediated QS systems, LasR/LasI and RhlR/RhlI. Many reports have demonstrated that these two systems are regulated and interconnected by global regulators. We found that lon-disrupted cells overproduce pyocyanin, the biosynthesis of which depends on the RhlR/RhlI system, and show increased levels of a transcriptional regulator, RhlR. The QS systems are organized hierarchically: the RhlR/RhlI system is subordinate to LasR/LasI. To elucidate the mechanism by which Lon negatively regulates RhlR/RhlI, we examined the effect of lon disruption on the LasR/LasI system. We found that Lon represses the expression of LasR/LasI by degrading LasI, an HSL synthase, leading to negative regulation of the RhlR/RhlI system. RhlR/RhlI was also shown to be regulated by Lon independently of LasR/LasI via regulation of RhlI, an HSL synthase. In view of these findings, it is suggested that Lon protease is a powerful negative regulator of both HSL-mediated QS systems in P. aeruginosa

Cell-Surface Phenol Soluble Modulins Regulate Staphylococcus aureus Colony Spreading.

Staphylococcus aureus produces phenol-soluble modulins (PSMs), which are amphipathic small peptides with lytic activity against mammalian cells. We previously reported that PSMα1-4 stimulate S. aureus colony spreading, the phenomenon of S. aureus colony expansion on the surface of soft agar plates, whereas δ-toxin (Hld, PSMγ) inhibits colony-spreading activity. In this study, we revealed the underlying mechanism of the opposing effects of PSMα1-4 and δ-toxin in S. aureus colony spreading. PSMα1-4 and δ-toxin are abundant on the S. aureus cell surface, and account for 18% and 8.5% of the total amount of PSMα1-4 and δ-toxin, respectively, in S. aureus overnight cultures. Knockout of PSMα1-4 did not affect the amount of cell surface δ-toxin. In contrast, knockout of δ-toxin increased the amount of cell surface PSMα1-4, and decreased the amount of culture supernatant PSMα1-4. The δ-toxin inhibited PSMα3 and PSMα2 binding to the S. aureus cell surface in vitro. A double knockout strain of PSMα1-4 and δ-toxin exhibited decreased colony spreading compared with the parent strain. Expression of cell surface PSMα1-4, but not culture supernatant PSMα1-4, restored the colony-spreading activity of the PSMα1-4/δ-toxin double knockout strain. Expression of δ-toxin on the cell surface or in the culture supernatant did not restore the colony-spreading activity of the PSMα1-4/δ-toxin double knockout strain. These findings suggest that cell surface PSMα1-4 promote S. aureus colony spreading, whereas δ-toxin suppresses colony-spreading activity by inhibiting PSMα1-4 binding to the S. aureus cell surface

Summary of the cell surface PSMα1–4 and the colony spreading in <i>S</i>. <i>aureus</i> gene knockout strains.

<p>PSMα1–4 and δ-toxin are presented as orange and blue dots, respectively. Knockout of δ-toxin increases the amount of cell surface PSMα1–4. In contrast, knockout of PSMα1–4 does not affect the amount of cell surface δ-toxin. The amount of cell surface PSMα1–4 and the colony-spreading activity in the wild-type strain, the δ-toxin knockout strain, the PSMα1–4 knockout strain, and the PSMα1-4/δ-toxin knockout strain is summarized in the lower part of this figure. The amount of cell surface PSMα1–4 is a determinant of colony-spreading activity.</p

Effect of PSMα1–4 knockout on δ-toxin distribution.

<p><i>S</i>. <i>aureus</i> Newman strain (parent) and PSMα1–4 knockout strain (Δ<i>psmα</i>) were cultured for 19 h. The amount of δ-toxin on the cell surface or in the culture supernatant was measured. Vertical axis represents the amount of δ-toxin per 1 ml bacterial culture. Data are means±standard errors from four independent experiments. Asterisk indicates Student’s t-test p value less than 0.05 between parent and Δ<i>psmα</i>.</p

Competitive binding assay of PSMs against <i>S</i>. <i>aureus</i> cell surface.

<p><b>A.</b> Inhibitory activity of δ-toxin against PSMα2 binding to the <i>S</i>. <i>aureus</i> cell surface of the PSMα1-4/δ-toxin knockout strain was measured. Binding assay of PSMα2 (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of δ-toxin (0, 10, 20, and 30 nmol) and the amount of PSMα2 bound to the cell surface was measured (left graph). In the competition assay, the binding of δ-toxin to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (PSMα2 and δ-toxin) is presented (right graph). In all graphs, horizontal axis represents the amount of PSM added to <i>S</i>. <i>aureus</i> cells and vertical axis represents the amount of PSM bound to <i>S</i>. <i>aureus</i> cells (3 x 10⁸ CFU). B. Inhibitory activity of PSMα2 against δ-toxin binding to the <i>S</i>. <i>aureus</i> cell surface was measured. Binding assay of δ-toxin (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of PSMα2 (0, 10, 20, and 30 nmol) and the amount of δ-toxin bound to the cell surface was measured (left graph). In the competition assay, binding of PSMα2 to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (δ-toxin and PSMα2) is presented (right graph). C. Inhibitory activity of δ-toxin against PSMα3 binding to the <i>S</i>. <i>aureus</i> cell surface was measured. Binding assay of PSMα3 (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of δ-toxin (0, 10, 20, and 30 nmol) and the amount of PSMα3 bound to the cell surface was measured (left graph). In the competition assay, binding of δ-toxin to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (PSMα3 and δ-toxin) is presented (right graph). D. Inhibitory activity of PSMα3 against δ-toxin binding to <i>S</i>. <i>aureus</i> cell surface was measured. Binding assay of δ-toxin (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of PSMα3 (0, 10, 20, and 30 nmol) and the amount of δ-toxin bound to the cell surface was measured (left graph). In the competition assay, binding of PSMα3 to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (δ-toxin and PSMα3) is presented (right graph).</p

Bacterial strains and plasmids used in the study.

<p>Bacterial strains and plasmids used in the study.</p

Effect of δ-toxin knockout on the PSMαs distribution in SA564 and FRP3757 strains.

<p>A. Schematic representation of genomic region of the <i>hld-</i>wild-type strain (WT) and the two different δ-toxin knockout strains (Δ<i>hld1</i>, Δ<i>hld2</i>) of SA564 and FRP3757 strains. The <i>agr</i> locus in the chromosome of <i>S</i>. <i>aureus</i> SA564 or FRP3757 (USA300) was replaced with the <i>agr</i> locus having wild-type <i>hld</i> gene or mutated <i>hld</i> genes, which carries antibiotic resistance markers. The Hld amino acid sequences are presented in the parentheses. B. SA564 <i>hld</i>-wild-type strain (WT) or the δ-toxin knockout strains (Δ<i>hld1</i>, Δ<i>hld2</i>) were cultured for 19 h. The amount of PSMα1–4 on the cell surface (left graph) or in the culture supernatant (right graph) was measured. Data are the means±standard errors from triplicate experiments. Asterisks indicate Student’s t-test p value less than 0.05 between WT and Δ<i>hld1</i> or between WT and Δ<i>hld2</i>. <i>C</i>. FRP3757 <i>hld</i>-wild-type strain (WT) or the δ-toxin knockout strains (Δ<i>hld1</i>, Δ<i>hld2</i>) were cultured for 19 h. The amount of PSMα1–4 on the cell surface (left graph) or in the culture supernatant (right graph) was measured. Data are the means±standard errors from triplicate experiments. Asterisks indicate Student’s t-test p value less than 0.05 between WT and Δ<i>hld1</i> or between WT and Δ<i>hld2</i>.</p

Amount of PSMα1–4 and δ-toxin on the <i>S</i>. <i>aureus</i> cell surface and in the culture supernatant.

<p>A. <i>S</i>. <i>aureus</i> Newman strain was cultured for 19 h. Cells were washed with 6 M guanidine HCl and PSMs on the cell surface were obtained. PSMs on the cell surface (from 1.33 ml bacterial culture) and in the culture supernatant (from 0.267 ml bacterial culture) were analyzed by HPLC. Dotted line indicates the respective PSMs. B. The amount of PSMs on the cell surface or in the culture supernatant was measured. Vertical axis represents the amount of each PSM per 1 ml bacterial culture. Data are means±standard errors from three independent experiments.</p

Correlation analysis between the amount of cell surface PSMs and the colony-spreading activity in <i>S</i>. <i>aureus</i> clinical isolates.

<p>HA-MRSA isolates (n = 40), CA-MRSA isolates (n = 14), and Newman strain were cultured for 19 h. The total amount of PSMα1, PSMα2, and PSMα3 (PSMα1–3) (<i>A</i>) or the amount of δ-toxin (<i>B</i>) in each strain was measured by HPLCs and the mean value from three independent experiments was plotted on the horizontal axis as the relative value against that of Newman strain. The colony-spreading activity of each strain, which was reported in our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164523#pone.0164523.ref018" target="_blank">18</a>], was plotted on the vertical axis. A linear approximation and correlation coefficient are presented in the graph.</p