Staphylococcus aureus aggregation in the plasma fraction of silkworm hemolymph

Staphylococcus aureus formed bacterial aggregates in the plasma fraction of the hemolymph of silkworm, the larva of Bombyx mori, in a growth-dependent manner. The addition of arabinose or galactose inhibited the formation of S. aureus aggregates in the silkworm plasma. Formation of the bacterial aggregates depended on S. aureus genes required for the synthesis of bacterial surface polysaccharides-ypfP and ltaA, which are involved in lipoteichoic acid synthesis, and the tagO gene, which is involved in wall teichoic acid synthesis. These findings suggest that S. aureus forms bacterial aggregates in the silkworm plasma via bacterial surface teichoic acids

ｶｲｺ体液中に存在する細胞増殖抑制ならびに脂肪蓄積促進活性因子に関する研究

ｶｲｺ(Bombyx mori)の幼虫体液中にはｶｲｺ由来培養細胞の増殖を抑制する活性があることを見出した｡また､ｶｲｺ体液によって増殖を阻害された細胞中には脂肪滴の形成が認められた｡本研究ではこれらの細胞増殖抑制および脂肪蓄積促進活性の実体となるｶｲｺ体液因子の解明を行った｡ｶｲｺ体液を出発材料として､細胞増殖抑制活性を指標にﾀﾝﾊﾟｸ質の精製を行った｡6段階の精製過程の後､SDS-ﾎﾟﾘｱｸﾘﾙｱﾐﾄﾞｹﾞﾙ電気泳動において単一ﾊﾞﾝﾄﾞを示す画分を得た｡ｱﾐﾉ酸配列解析の結果､このﾀﾝﾊﾟｸ質はｶｲｺ Niemann-Pick disease type C2(BmNPC2)ﾀﾝﾊﾟｸ質であると同定された｡ﾘｺﾝﾋﾞﾅﾝﾄ BmNPC2 ﾀﾝﾊﾟｸ質は細胞増殖抑制活性を示したことから BmNPC2 ﾀﾝﾊﾟｸ質がｶｲｺ体液中の細胞増殖抑制活性の実体であることが示唆された｡さらに､ﾘｺﾝﾋﾞﾅﾝﾄ BmNPC2 ﾀﾝﾊﾟｸ質をｶｲｺ培養細胞に添加することによって､細胞内ﾄﾘｸﾞﾘｾﾘﾄﾞ量の増大が認められた｡以上の結果は､BmNPC2 ﾀﾝﾊﾟｸ質がｶｲｺにおいて細胞増殖と脂質代謝を制御する体液性因子であることを示唆している｡Silkworm hemolymph induced both the cessation of growth and an increase in neutral lipids storage in the silkworm-derived cell line, BmN4. In this study, we identified a responsible blood factor showing growth-inhibitory and lipid-accumulating activity. We subjected the silkworm hemolymph to successive column chromatographies and measured the growth-inhibitory activity of each fraction. The final purified fraction showed the highest specific activity and a single band of 15kDa protein on SDS-PAGE. The amino acid sequence revealed that the 15kDa protein was Bombyx mori Niemann-Pick disease type C2 (BmNPC2) protein. A recombinant BmNPC2 protein exhibited growth-inhibitory activity comparable with the final purified fraction, indicating that this protein is responsible for the activitiy of silkworm hemolymph. Moreover, the recombinant BmNPC2 protein induced an increase in triglyceride storage in BmN4 cells. These results indicate that BmNPC2 protein regulates both cell growth and lipid metabolism in silkworm

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

金沢大学医薬保健研究域薬学系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. © 2009 Blackwell Publishing Ltd

An Invertebrate Hyperglycemic Model for the Identification of Anti-Diabetic Drugs

The number of individuals diagnosed with type 2 diabetes mellitus, which is caused by insulin resistance and/or abnormal insulin secretion, is increasing worldwide, creating a strong demand for the development of more effective anti-diabetic drugs. However, animal-based screening for anti-diabetic compounds requires sacrifice of a large number of diabetic animals, which presents issues in terms of animal welfare. Here, we established a method for evaluating the anti-diabetic effects of compounds using an invertebrate animal, the silkworm, Bombyx mori. Sugar levels in silkworm hemolymph increased immediately after feeding silkworms a high glucose-containing diet, resulting in impaired growth. Human insulin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, decreased the hemolymph sugar levels of the hyperglycemic silkworms and restored growth. Treatment of the isolated fat body with human insulin in an in vitro culture system increased total sugar in the fat body and stimulated Akt phosphorylation. These responses were inhibited by wortmannin, an inhibitor of phosphoinositide 3 kinase. Moreover, AICAR stimulated AMPK phosphorylation in the silkworm fat body. Administration of aminoguanidine, a Maillard reaction inhibitor, repressed the accumulation of Maillard reaction products (advanced glycation end-products; AGEs) in the hyperglycemic silkworms and restored growth, suggesting that the growth defect of hyperglycemic silkworms is caused by AGE accumulation in the hemolymph. Furthermore, we identified galactose as a hypoglycemic compound in jiou, an herbal medicine for diabetes, by monitoring its hypoglycemic activity in hyperglycemic silkworms. These results suggest that the hyperglycemic silkworm model is useful for identifying anti-diabetic drugs that show therapeutic effects in mammals

A Novel Gene, fudoh, in the SCCmec Region Suppresses the Colony Spreading Ability and Virulence of Staphylococcus aureus

Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis

Intestinal Resident Yeast Candida glabrata Requires Cyb2p-Mediated Lactate Assimilation to Adapt in Mouse Intestine

The intestinal resident Candida glabrata opportunistically infects humans. However few genetic factors for adaptation in the intestine are identified in this fungus. Here we describe the C. glabrata CYB2 gene encoding lactate dehydrogenase as an adaptation factor for survival in the intestine. CYB2 was identified as a virulence factor by a silkworm infection study. To determine the function of CYB2, we analysed in vitro phenotypes of the mutant Δcyb2. The Δcyb2 mutant grew well in glucose medium under aerobic and anaerobic conditions, was not supersensitive to nitric oxide which has fungicidal-effect in phagocytes, and had normal levels of general virulence factors protease, lipase and adherence activities. A previous report suggested that Cyb2p is responsible for lactate assimilation. Additionally, it was speculated that lactate assimilation was required for Candida virulence because Candida must synthesize glucose via gluconeogenesis under glucose-limited conditions such as in the host. Indeed, the Δcyb2 mutant could not grow on lactate medium in which lactate is the sole carbon source in the absence of glucose, indicating that Cyb2p plays a role in lactate assimilation. We hypothesized that Cyb2p-mediated lactate assimilation is necessary for proliferation in the intestinal tract, as the intestine is rich in lactate produced by bacteria flora, but not glucose. The Δcyb2 mutant showed 100-fold decreased adaptation and few cells of Saccharomyces cerevisiae can adapt in mouse ceca. Interestingly, C. glabrata could assimilate lactate under hypoxic conditions, dependent on CYB2, but not yeast S. cerevisiae. Because accessible oxygen is limited in the intestine, the ability for lactate assimilation in hypoxic conditions may provide an advantage for a pathogenic yeast. From those results, we conclude that Cyb2p-mediated lactate assimilation is an intestinal adaptation factor of C. glabrata

Transcription and Translation Products of the Cytolysin Gene psm-mec on the Mobile Genetic Element SCCmec Regulate Staphylococcus aureus Virulence

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 PSMα 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 PSMα 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