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DOI： 10.1371/journal.pone.0270166

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DOI： 10.1128/JB.00515-20

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DOI： 10.1038/s41598-021-82212-5

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© 2020 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

DOI： 10.1111/gtc.12737

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DOI： 10.1016/j.biochi.2019.08.008

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DOI： 10.5582/ddt.2017.01052

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DOI： 10.5582/ddt.2017.01053

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

Staphylococcus aureus is a predominant pathogen in keratitis, and the rate of methicillin-resistant S. aureus (MRSA) is increasing. In our previous study, genotypes of MRSA isolates from keratitis cases were classified into ST5 or ST764 lineage by multi-locus sequence typing. In this study, we examined the virulence properties of these MRSA keratitis isolates and its virulence determinants. There was no difference in the prevalence of virulence genes, such as adhesion and toxins, between ST5 and ST764 isolates. All ST5 isolates carried the intact psm-mec gene, which suppresses exotoxin production and colony spreading, but promotes biofilm formation. In contrast, all ST764 isolates had one point mutation in the psm-mec gene. Biofilm production in ST5 isolates was significantly higher than that in ST764 isolates, whereas colony spreading, hemolytic activity, and production of alpha-phenol-soluble modulins were higher in ST764 than in ST5 isolates. The toxicity of ST764 supernatants to corneal epithelial cells was higher than that of ST5 supernatants. These results suggest that the point mutation in the psm-mec gene contributes to the difference in virulence properties between ST5 and ST764 isolates in MRSA keratitis.

DOI： 10.1089/mdr.2015.0315

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Staphylococcus aureus produces phenol-soluble modulins (PSMs), which are amphipathic small peptides with lytic activity against mammalian cells. We previously reported that PSM alpha 1-4 stimulate S. aureus colony spreading, the phenomenon of S. aureus colony expansion on the surface of soft agar plates, whereas delta-toxin (Hld, PSM gamma) inhibits colony-spreading activity. In this study, we revealed the underlying mechanism of the opposing effects of PSM alpha 1-4 and delta-toxin in S. aureus colony spreading. PSM alpha 1-4 and delta-toxin are abundant on the S. aureus cell surface, and account for 18% and 8.5% of the total amount of PSM alpha 1-4 and delta-toxin, respectively, in S. aureus overnight cultures. Knockout of PSM alpha 1-4 did not affect the amount of cell surface delta-toxin. In contrast, knockout of d-toxin increased the amount of cell surface PSM alpha 1-4, and decreased the amount of culture supernatant PSM alpha 1-4. The d-toxin inhibited PSM alpha 3 and PSM alpha 2 binding to the S. aureus cell surface in vitro. A double knockout strain of PSM alpha 1-4 and delta-toxin exhibited decreased colony spreading compared with the parent strain. Expression of cell surface PSM alpha 1-4, but not culture supernatant PSM alpha 1-4, restored the colony-spreading activity of the PSM alpha 1-4/delta-toxin double knockout strain. Expression of delta-toxin on the cell surface or in the culture supernatant did not restore the colony-spreading activity of the PSMa1-4/delta-toxin double knockout strain. These findings suggest that cell surface PSM alpha 1-4 promote S. aureus colony spreading, whereas delta-toxin suppresses colony-spreading activity by inhibiting PSM alpha 1-4 binding to the S. aureus cell surface.

DOI： 10.1371/journal.pone.0164523

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We identified SA1684 as a Staphylococcus aureus virulence gene using a silkworm infection model. The SA1684 gene product carried the DUF402 domain, which is found in RNA-binding proteins, and had amino acid sequence similarity with a nucleoside diphosphatase, Streptomyces coelicolor SC4828 protein. The SA1684-deletion mutant exhibited drastically decreased virulence, in which the LD50 against silkworms was more than 10 times that of the parent strain. The SA1684-deletion mutant also exhibited decreased exotoxin production and colony-spreading ability. Purified SA1684 protein had Mn2+ -or Co2+-dependent hydrolyzing activity against nucleoside diphosphates. Alanine substitutions of Tyr-88, Asp-106, and Asp-123/Glu-124, which are conserved between SA1684 and SC4828, diminished the nucleoside diphosphatase activity. Introduction of the wild-type SA1684 gene restored the hemolysin production of the SA1684-deletion mutant, whereas none of the alanine-substituted SA1684 mutant genes restored the hemolysin production. RNA sequence analysis revealed that SA1684 is required for the expression of the virulence regulatory genes agr, sarZ, and sarX, as well as metabolic genes involved in glycolysis and fermentation pathways. These findings suggest that the novel nucleoside diphosphatase SA1684 links metabolic-pathways and virulence gene expression and plays an important role in S. aureus virulence.

DOI： 10.1074/jbc.M116.721845

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Invertebrate animal species that can withstand temperatures as high as 37 degrees C, the human body temperature, are limited. In the present study, we utilized the two-spotted cricket, Gryllus bimaculatus, which lives in tropical and subtropical regions, as an animal model of human pathogenic bacterial infection. Injection of Pseudomonas aeruginosa or Staphylococcus aureus into the hemolymph killed crickets. Injected P. aeruginosa or S. aureus proliferated in the hemolymph until the cricket died. The ability of these pathogenic bacteria to kill the crickets was blocked by the administration of antibiotics. S. aureus gene-knockout mutants of virulence factors, including cvfA, agr and srtA, exhibited decreased killing ability compared with the parent strain. The dose at which 50% of crickets were killed by P. aeruginosa or S. aureus was not decreased at 37 degrees C compared with that at 27 degrees C. Injection of Listeria monocytogenes, which upregulates toxin expression at 37 degrees C, killed crickets, and the dose at which 50% of crickets were killed was decreased at 37 degrees C compared with that at 27 degrees C. These findings suggest that the two-spotted cricket is a useful model animal for evaluating the virulence properties of various human pathogenic bacteria at variable temperature including 37 degrees C.

DOI： 10.1093/femsle/fnw163

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Honeybee royal jelly is reported to have body-enlarging effects in holometabolous insects such as the honeybee, fly and silkmoth, but its effect in non-holometabolous insect species has not yet been examined. The present study confirmed the body-enlarging effect in silkmoths fed an artificial diet instead of mulberry leaves used in the previous literature. Administration of honeybee royal jelly to silkmoth from early larval stage increased the size of female pupae and adult moths, but not larvae (at the late larval stage) or male pupae. We further examined the body-enlarging effect of royal jelly in a non-holometabolous species, the two-spotted cricket Gryllus bimaculatus, which belongs to the evolutionarily primitive group Polyneoptera. Administration of royal jelly to G. bimaculatus from its early nymph stage enlarged both males and females at the mid-nymph and adult stages. In the cricket, the body parts were uniformly enlarged in both males and females; whereas the enlarged female silkmoths had swollen abdomens. Administration of royal jelly increased the number, but not the size, of eggs loaded in the abdomen of silkmoth females. In addition, fat body cells were enlarged by royal jelly in the silkmoth, but not in the cricket. These findings suggest that the body-enlarging effect of royal jelly is common in non-holometabolous species, G. bimaculatus, but it acts in a different manner than in holometabolous species.

DOI： 10.1242/bio.019190

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DOI： 10.5582/ddt.2016.01020

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The relationship between body size and vocalization parameters has been studied in many animal species. In insect species, however, the effect of body size on song frequency has remained unclear. Here we analyzed the effect of body size on the frequency spectra of mating songs produced by the two-spotted cricket, Gryllus bimaculatus. We recorded the calling songs and courtship songs of male crickets of different body sizes. The calling songs contained a frequency component that peaked at 5.7 kHz. On the other hand, courtship songs contained two frequency components that peaked at 5.8 and 14.7 kHz. The dominant frequency of each component in both the calling and courtship songs was constant regardless of body size. The size of the harp and mirror regions in the cricket forewings, which are the acoustic sources of the songs, correlated positively with body size. These findings suggest that the frequency contents of both the calling and courtship songs of the cricket are unaffected by whole body, harp, or mirror size.

DOI： 10.1371/journal.pone.0146999

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Phenol-soluble modulins (PSMs) are Staphylococcus aureus cytolytic toxins that lyse erythrocytes and neutrophils and have important functions in the S. aureus infectious process. The molecular mechanisms of PSM secretion, however, are not well understood. Here we report that knockout of the multidrug-resistance ABC transporter AbcA, which contributes to S. aureus resistance against antibiotics and chemicals, diminished the secreted amount of PSM, leading to the accumulation of PSM in the intracellular fraction. The amount of PSM in the culture supernatants of the abcA knockout mutants was restored by introduction of the wild-type abcA gene, whereas it was not completely restored by introduction of mutant abcA genes encoding AbcA mutant proteins carrying amino acid substitutions in the adenosine triphosphate binding motifs. The abcA knockout mutant exhibited attenuated virulence in a mouse systemic infection model. These findings suggest that the multidrug resistance transporter AbcA secretes PSMs and contributes to S. aureus virulence.

DOI： 10.1093/infdis/jiv376

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DOI： 10.1016/j.biochi.2015.10.027

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DOI： 10.1038/srep17092

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DOI： 10.1111/febs.13302

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DOI： 10.1371/journal.pone.0130486

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DOI： 10.5582/ddt.2015.01026

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DOI： 10.5582/ddt.2015.01000

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Over-expression of alpha-phenol-soluble modulins (PSMs) results in high virulence of community-associated methicillin-resistant Staphylococcus aureus (MRSA). The psm-mec gene, located in the mobile genetic element SCCmec-II, suppresses PSMs production. Fifty-two patients with MRSA bacteraemia were enrolled. MRSA isolates were evaluated with regard to the psm-mec gene sequence, bacterial virulence, and the minimum inhibitory concentration (MIC) of vancomycin and teicoplanin. Fifty-one MRSA isolates were classified as SCCmec-II, and 10 had one point mutation in the psm-mec promoter. We compared clinical characteristics and outcomes between mutant MRSA and wild-type MRSA. Production of PSM3 in mutant MRSA was significantly increased, but biofilm formation was suppressed. Wild-type MRSA caused more catheter-related bloodstream infections (30/41 vs. 3/10, p0.0028), whereas mutant MRSA formed more deep abscesses (4/10 vs. 3/41, p0.035). Bacteraemia caused by mutant MRSA was associated with reduced 30-day mortality (1/10 vs. 13/41, p0.25), although this difference was not significant. The MIC90 of teicoplanin was higher for wild-type MRSA (1.5mg/L vs. 1mg/L), but the MIC of vancomycin was not different between the two groups. The 30-day mortality of MRSA with a high MIC of teicoplanin (1.5mg/L) was higher than that of strains with a lower MIC (0.75mg/L) (6/10 vs. 6/33, p0.017). Mutation of the psm-mec promoter contributes to virulence of SCCmec-II MRSA, and the product of psm-mec may determine the clinical characteristics of bacteraemia caused by SCCmec-II MRSA, but it does not affect mortality.

DOI： 10.1111/1469-0691.12575

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Staphylococcal species acquire antibiotic resistance by incorporating the mobile-genetic element SCCmec. We previously found that SCCmec-encoded psm-mec RNA suppresses exotoxin production as a regulatory RNA, and the psm-mec translation product increases biofilm formation in Staphylococcus aureus. Here, we examined whether the regulatory role of psm-mec on host bacterial virulence properties is conserved among other staphylococcal species, S. epidermidis and S. haemolyticus, both of which are important causes of nosocomial infections. In S. epidermidis, introduction of psm-mec decreased the production of cytolytic toxins called phenol-soluble modulins (PSMs) and increased biofilm formation. Introduction of psm-mec with a stop-codon mutation that did not express PSM-mec protein but did express psm-mec RNA also decreased PSM production, but did not increase biofilm formation. Thus, the psm-mec RNA inhibits PSM production, whereas the PSM-mec protein increases biofilm formation in S. epidermidis. In S. haemolyticus, introduction of psm-mec decreased PSM production, but did not affect biofilm formation. The mutated psm-mec with a stop-codon also caused the same effect. Thus, the psm-mec RNA also inhibits PSM production in S. haemolyticus. These findings suggest that the inhibitory role of psm-mec RNA on exotoxin production is conserved among staphylococcal species, although the stimulating effect of the psm-mec gene on biofilm formation is not conserved.

DOI： 10.1371/journal.pone.0100260

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Staphylococcus aureus forms giant colonies on soft-agar surfaces, which is called colony-spreading. In the present study, we searched for host factors that influence S. aureus colony-spreading activity. The addition of calf serum, porcine serum, or silkworm hemolymph to soft-agar medium stimulated S. aureus colony-spreading activity. Gel filtration column chromatography of calf serum produced a high molecular weight fraction and a low molecular weight fraction, both of which exhibited colony-spreading stimulatory activity. In the low molecular weight fraction, we identified the stimulatory factor as bovine serum albumin. The stimulatory fraction in the high molecular weight fraction was identified as high-density lipoprotein (HDL) particles. Delipidation of HDL abolished the stimulatory activity of HDL. Phosphatidylcholine, which is the major lipid component in HDL particles, stimulated the colony-spreading activity. Other phosphatidylcholine-containing lipoprotein particles, low-density lipoprotein and very low-density lipoprotein, also showed colony-spreading stimulatory activity. These findings suggest that S. aureus colony-spreading activity is stimulated by albumin and lipoprotein particles in mammalian serum.

DOI： 10.1371/journal.pone.0097670

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Background: Primed immune responses contribute to vertebrate host defense. Results: Silkworms acquire resistance to a pathogen by a preinjection of its heat-killed cells or its cell surface peptidoglycans. The amount of antimicrobial peptides is increased at the second round of infection. Conclusion: Invertebrates acquire infection resistance by peptidoglycan recognition and antimicrobial peptide increase. Significance: Molecular mechanisms of invertebrate primed immunity were revealed.
A heightened immune response, in which immune responses are primed by repeated exposure to a pathogen, is an important characteristic of vertebrate adaptive immunity. In the present study, we examined whether invertebrate animals also exhibit a primed immune response. The LD50 of Gram-negative enterohemorrhagic Escherichia coli O157:H7 Sakai in silkworms was increased 100-fold by pre-injection of heat-killed Sakai cells. Silkworms pre-injected with heat-killed cells of a Gram-positive bacterium, Staphylococcus aureus, did not have resistance to Sakai. Silkworms preinjected with enterohemorrhagic E. coli peptidoglycans, cell surface components of bacteria, were resistant to Sakai infection. Silkworms preinjected with S. aureus peptidoglycans, however, were not resistant to Sakai. Silkworms preinjected with heat-killed Sakai cells showed persistent resistance to Sakai infection even after pupation. Repeated injection of heat-killed Sakai cells into the silkworms induced earlier and greater production of antimicrobial peptides than a single injection of heat-killed Sakai cells. These findings suggest that silkworm recognition of Gram-negative peptidoglycans leads to a primed immune reaction and increased resistance to a second round of bacterial infection.

DOI： 10.1074/jbc.M113.525139

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Background: Production of 3-phosphorylated RNA by CvfA affects S. aureus virulence gene expression. Results: Disrupting pnpA-encoding exonuclease suppressed the cvfA-deleted mutant phenotype. Purified PNPase did not degrade 3-phosphorylated RNA. Conclusion: CvfA-produced 3-phosphorylated RNA inhibits PNPase-induced RNA degradation, resulting in hemolysin production by S. aureus. Significance: Altering the nucleotide structure at the RNA 3 terminus regulates S. aureus virulence.
We previously identified CvfA (SA1129) as a Staphylococcus aureus virulence factor using a silkworm infection model. S. aureus cvfA-deleted mutants exhibit decreased expression of the agr locus encoding a positive regulator of hemolysin genes and decreased hemolysin production. CvfA protein hydrolyzes a 2,3-cyclic phosphodiester bond at the RNA 3 terminus, producing RNA with a 3-phosphate (3-phosphorylated RNA, RNA with a 3-phosphate). Here, we report that the cvfA-deleted mutant phenotype (decreased agr expression and hemolysin production) was suppressed by disrupting pnpA-encoding polynucleotide phosphorylase (PNPase) with 3- to 5-exonuclease activity. The suppression was blocked by introducing a pnpA-encoding PNPase with exonuclease activity but not by a pnpA-encoding mutant PNPase without exonuclease activity. Therefore, loss of PNPase exonuclease activity suppressed the cvfA-deleted mutant phenotype. Purified PNPase efficiently degraded RNA with 2,3-cyclic phosphate at the 3 terminus (2,3-cyclic RNA), but it inefficiently degraded 3-phosphorylated RNA. These findings indicate that 3-phosphorylated RNA production from 2,3-cyclic RNA by CvfA prevents RNA degradation by PNPase and contributes to the expression of agr and hemolysin genes. We speculate that in the cvfA-deleted mutant, 2,3-cyclic RNA is not converted to the 3-phosphorylated form and is efficiently degraded by PNPase, resulting in the loss of RNA essential for expressing agr and hemolysin genes, whereas in the cvfA/pnpA double-disrupted mutant, 2,3-cyclic RNA is not degraded by PNPase, leading to hemolysin production. These findings suggest that CvfA and PNPase competitively regulate RNA degradation essential for S. aureus virulence.

DOI： 10.1074/jbc.M114.554329

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We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease the saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS.

DOI： 10.1074/jbc.M113.495051

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DOI： 10.1371/journal.ppat.1003269

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DOI： 10.1111/j.1574-6968.2012.02599.x

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DOI： 10.1074/jbc.M112.357848

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DOI： 10.1111/j.1574-6968.2011.02439.x

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Silkworm hemolymph inhibits hemolysin production by Staphylococcus aureus. We purified a factor in the silkworm hemolymph responsible for this inhibitory activity. The final fraction with the greatest specific activity contained 220- and 74-kDa proteins. Determination of the N-terminal amino acid sequence revealed that the 220- and 74-kDa proteins were apolipophorin I and apolipophorin II, respectively, indicating that the factor was apolipophorin (ApoLp). The purified ApoLp fraction showed decreased expression of S. aureus hla encoding alpha-hemolysin, hlb encoding beta-hemolysin, saeRS, and RNAIII, which activate the expression of these hemolysin genes. Injection of an anti-ApoLp antibody into the hemolymph increased the sensitivity of silkworms to the lethal effect of S. aureus. Hog gastric mucin, a mammalian homologue of ApoLp, decreased the expression of S. aureus hla and hlb. These findings suggest that ApoLp in the silkworm hemolymph inhibits S. aureus virulence and contributes to defense against S. aureus infection and that its activity is conserved in mammalian mucin.

DOI： 10.1074/jbc.M111.278416

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Staphylococcus aureus spreads on soft agar surfaces and forms giant colonies. Here, we examined the inhibitory role of extracellular DNA on the colony spreading activity. The double-deletion mutation of nuc1 and nuc2, which encode secretory nucleases, increased extracellular DNA and showed a decreased ability to form giant colonies. The addition of DNase I or micrococcal nuclease to the soft agar restored the ability of the nuc1-nuc2 double mutant to form giant colonies. In addition, the promoter activities of nuc1 and nuc2 in the wild-type strain were elevated in the peripheral region of the giant colony. These findings suggest that the digestion of extracellular DNA by secretory nucleases is required for the colony spreading activity of S. aureus. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.micpath.2011.04.007

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Staphylococcus aureus spreads on soft agar surfaces, which is called "colony spreading". Here, we report that the colony spreading in S. aureus was promoted by the addition of glucose to soft agar plates. Disruption of ccpA and hprK, which are involved in catabolite repression, decreased the colony spreading ability promoted by glucose. Deletion of the agr locus, a virulence regulatory element whose expression is activated by glucose in a ccpA-dependent manner, abolished the colony spreading promoted by glucose. Disruption of clpP and arlRS, which contributes to agr expression, also decreased glucose-promoted colony spreading. These findings suggest that S. aureus colony spreading requires the expression of agr, which is positively regulated by environmental carbon sources, and that virulence gene expression and colony spreading induced by agr are simultaneously activated in the S. aureus infectious process. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.micpath.2011.04.003

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The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSM alpha production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSM alpha production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.

DOI： 10.1371/journal.ppat.1001267

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DOI： 10.5582/ddt.2011.v5.2.66

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DOI： 10.1016/j.micpath.2010.03.010

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DOI： 10.1016/j.str.2010.02.007

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DOI： 10.1016/j.micpath.2008.10.010

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DOI： 10.1371/journal.pone.0003921

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DOI： 10.1074/jbc.M705309200

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DOI： 10.1128/IAI.01552-06

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DOI： 10.1128/JB.01635-06

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DOI： 10.1016/S0076-6879(06)22011-1

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DOI： 10.1111/j.1365-2958.2006.05480.x

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DOI： 10.1093/jb/mvj171

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DOI： 10.1002/bem.20215

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DOI： 10.1016/j.bbrc.2005.08.144

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DOI： 10.1111/j.1365-2958.2005.04596.x

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DOI： 10.1111/j.1365-2443.2005.00849.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The injection of bacteria (Staphylococcus aureus, Stenotrophomonas maltophilia) or true fungi (Candida albicans, Candida tropicalis) that are pathogenic to humans into the silkworm hemolymph leads to death of the larvae within 2 days. Antibiotics used for clinical purposes have therapeutic effects on silkworms infected with these pathogens. The 50% effective doses obtained by injection into the silkworm hemolymph are consistent with those reported for mice. Injection of vancomycin and kanamycin into the silkworm hemolymph was effective, but oral administration was not. Chloramphenicol, which is effective by oral administration, appeared in the silkworm hemolymph soon after injection into the midgut, whereas vancomycin did not. Isolated midgut membranes were impermeable to vancomycin. Thus, the ineffectiveness of oral administration of vancomycin to silkworms is due to a lack of intestinal absorption.

DOI： 10.1128/AAC.48.3.774-779.2004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

DnaA protein activity, the initiator of chromosomal DNA replication in bacteria, is regulated by acidic phospholipids such as phosphatidylglycerol ( PG) or cardiolipin ( CL) via facilitation of the exchange reaction of bound adenine nucleotide. Total lipid isolated from exponentially growing Staphylococcus aureus cells facilitated the release of ATP bound to S. aureus DnaA protein, whereas that from stationary phase cells was inert. Fractionation of total lipid from stationary phase cells revealed that the basic phospholipid, lysylphosphatidylglycerol (LPG), inhibited PG- or CL-facilitated release of ATP from DnaA protein. There was an increase in LPG concentration during the stationary phase. A fraction of the total lipid from stationary phase cells of an integrational deletion mprF mutant, in which LPG was lost, facilitated the release of ATP from DnaA protein. A zwitterionic phospholipid, phosphatidylethanolamine, also inhibited PG- facilitated ATP release. These results indicate that interaction of DnaA protein with acidic phospholipids might be regulated by changes in the phospholipid composition of the cell membrane at different growth stages. In addition, the mprF mutant exhibited an increased amount of origin per cell in vivo, suggesting that LPG is involved in regulating the cell cycle event(s).

DOI： 10.1074/jbc.M212202200

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/sj.cr.7290159

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The murB gene encodes UDP-N-acetylenolpyruvylglucosamine reductase and functions in bacterial peptidoglycan biosynthesis. A plasmid carrying the murB gene restored the temperature-sensitive growth of six Staphylococcus aureus mutants, in which peptidoglycan biosynthesis stopped at a restrictive temperature. Specific activity of UDP-N-acetylenolpyruvylglucosamine reductase in extracts from the mutants was lower than that from wild-type cells. Nucleotide sequence determination revealed that each mutant had a single amino acid substitution in the mur-B gene and five of six mutations were located within domain 3, where the proposed substrate binding site is located. These results suggest that the murB gene is essential for growth of S. aureus and that domain 3 is important for the MurB activity. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1097(03)00260-X

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Authorship：Lead author  

DOI： 10.1016/S0378-1097(02)00603-1

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1006/mpat.2002.0494

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DOI： 10.1046/j.1440-169x.2001.00579.x

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DOI： 10.1016/S0140-6736(00)04403-2

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DOI： 10.1007/s004380100564

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：UNIV BASQUE COUNTRY PRESS  

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) mRNA in 1- and 2-cell stage Xenopus embryos induces cell autonomous dissociation at the late blastula stage and developmental arrest at the early gastrula stage. The induction of cell dissociation took place "punctually" at the late blastula stage in the SAMDC-overexpressing cells, irrespective of the stage of the microinjection of SAMDC mRNA. When we examined the cells undergoing the dissociation, we found that they were TUNEL-positive and contained fragmented nuclei with condensed chromatin and fragmented DNA. Furthermore, by injecting Xenopus Bcl-2 mRNA together with SAMDC mRNA, we showed that SAMDC-overexpressing embryos are rescued completely by Bcl-2 and become tadpoles. These results indicate that cell dissociation induced by SAMDC overexpression is due to apoptotic cell death. Since the level of S-adenosylmethionine (SAM) is greatly reduced in SAMDC-overexpressing embryos and this induces inhibition of protein synthesis accompanied by the inhibition of DNA and RNA syntheses, we conclude that deficiency in SAM induced by SAMDC overexpression activates the maternal program of apoptosis in Xenopus embryos at the late blastula stage, but not before. We propose that this mechanism serves as a surveillance mechanism to check and eliminate cells physiologically damaged during the cleavage stage.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos. (C) 2000 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S0305-0491(00)00193-0

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Total pages：135  

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