Identification and Tetramer Structure of Hemin-Binding Protein SPD_0310 Linked to Iron Homeostasis and Virulence of Streptococcus pneumoniae.

Iron and iron-containing compounds are essential for bacterial virulence and host infection. Hemin is an important supplement compound for bacterial survival in an iron-deficient environment. Despite strong interest in hemin metabolism, the detailed mechanism of hemin transportation in Gram-positive bacteria is yet to be reported. The results of our study revealed that the homologous proteins of SPD_0310 were significantly conservative in Gram-positive bacteria (P < 0.001), and these proteins were identified as belonging to an uncharacterized protein family (UPF0371). The results of thermodynamic and kinetic studies have shown that SPD_0310 has a high hemin-binding affinity. Interestingly, we found that the crystal structure of SPD_0310 presented a homotetramer conformation, which is required for hemin binding. SPD_0310 can interact with many hemin-binding proteins (SPD_0090, SPD_1609, and GAPDH) located on the cell surface, which contributes to hemin transfer to the cytoplasm. It also has a high affinity with other iron transporters in the cytoplasm (SPD_0226 and SPD_0227), which facilitates iron redistribution in cells. More importantly, the knockout of the spd_0310 gene (Δspd_0310) resulted in a decrease in the iron content and protein expression levels of many bacterial adhesion factors. Moreover, the animal model showed that the Δspd_0310 strain has a lower virulence than the wild type. Based on the crystallographic and biochemical studies, we inferred that SPD_0310 is a hemin intermediate transporter which contributes to iron homeostasis and further affects the virulence of Streptococcus pneumoniae in the host. Our study provides not only an important theoretical basis for the in-depth elucidation of the hemin transport mechanism in bacteria but also an important candidate target for the development of novel antimicrobial agents based on metal transport systems. IMPORTANCE Iron is an essential element for bacterial virulence and infection of the host. The detailed hemin metabolism in Gram-positive bacteria has rarely been studied. SPD_0310 belongs to the UPF0371 family of proteins, and results of homology analysis and evolutionary tree analysis suggested that it was widely distributed and highly conserved in Gram-positive bacteria. However, the function of the UPF0371 family remains unknown. We successfully determined the crystal structure of apo-SPD_0310, which is a homotetramer. We found that cytoplasmic protein SPD_0310 with a special tetramer structure has a strong hemin-binding ability and interacts with many iron transporters, which facilitates hemin transfer from the extracellular space to the cytoplasm. The results of detailed functional analyses indicated that SPD_0310 may function as a hemin transporter similar to hemoglobin in animals and contributes to bacterial iron homeostasis and virulence. This study provides a novel target for the development of antimicrobial drugs against pathogenic Gram-positive bacteria.

Multiscale investigation on the effects of additional weight bearing in combination with low-magnitude high-frequency vibration on bone quality of growing female rats.

This study aimed to explore the effects of additional weight bearing in combination with low-magnitude high-frequency vibration (LMHFV; 45 Hz, 0.3 g) on bone quality. One hundred twenty rats were randomly divided into ten groups; namely, sedentary (SED), additional weight bearing in which the rat wears a backpack whose weight is x% of the body weight (WBx; x = 5, 12, 19, 26), basic vibration (V), and additional weight bearing in combination with LMHFV in which the rat wears a backpack whose weight is x% of the body weight (Vx; x = 5, 12, 19, 26). The experiment was conducted for 12 weeks, 7 days per week, and 15 min per day. A three-point bending mechanical test, micro computed tomography, and a nanoindentation test were used. Serum samples were analyzed chemically. Failure load in V19 rats was significantly lower than that in SED rats (P < 0.05). Vx (x = 5, 12, 19, 26) rats showed poor microarchitectures. The content of tartrate-resistant acid phosphatase 5b was significantly higher in Vx (x = 5, 12, 19, 26) rats than that in SED rats (P < 0.05). V26 rats demonstrated comparatively better nanomechanical properties of materials than the other vibrational groups. Additional weight bearing in combination with LMHFV negatively affected the macromechanical properties and microarchitecture of bone. Heavy additional weight bearing, such as 26% of body weight, in combination with LMHFV was able to improve the nanomechanical properties of growing bone material compared with LMHFV. A combined mechanical stimulation was used, which may provide useful information to understand the mechanism of this mechanical stimulation on bone.

Protective effect of Sheng-Nao-Kang decoction on focal cerebral ischemia-reperfusion injury in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Sheng-Nao-Kang decoction (SNK), a modified traditional Chinese medicine (TCM), has been used clinically for the treatment of acute and chronic cerebrovascular related diseases. To evaluate the protective effect of SNK on focal cerebral ischemia-reperfusion (I/R) injury in rats and investigate the underlying mechanisms. MATERIALS AND METHODS: Focal cerebral I/R injury in rats was induced by middle cerebral artery occlusion (MCAO) for 2h followed by reperfusion for 24h. Adult male Sprague-Dawley (SD) rats were randomly divided into six kinds of groups: Sham group; I/R group; SNK-treated groups at doses of 0.7 g/kg, 1.4 g/kg and 2.8 g/kg; and nimodipine (NMP)-treated group. The recoveries of neurological function in rats were estimated by neurological defect scoring and 2,3,5-triphenyltetrazolium chloride (TTC) staining after 24h reperfusion. Various biochemical indexes in serum were assayed by colorimetry, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), inducible nitric oxide synthase (iNOS) and total nitric oxide synthase (TNOS). Histological structures of the brain in rats were observed by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed to detect the caspase-3 protein content in rats. RESULTS: SNK administration significantly reduced the neurological defect scores and lessened the cerebral infarction volume. The treatment of SNK lowered MDA content, up-regulated SOD and GSH-Px levels, down-regulated iNOS and TNOS levels in serum. Furthermore, histological examination indicated that dense neuropil and largely surviving neurons were seen in SNK-treated rats. SNK administration restrained the expression of caspase-3 positive protein significantly. CONCLUSION: The results suggest that SNK demonstrates a strong and ameliorative effect on cerebral I/R damage in rats. The protective mechanisms of SNK are associated with its properties of anti-apoptosis and anti-oxidation as well as regulation of iNOS and TNOS.

New pregnane and steroidal glycosides from Tribulus terrestris L.

Three new steroidal saponins were isolated from the fruits of Tribulus terrestris L. Their structures were elucidated by spectroscopic and chemical analysis as 16beta-(4'-methyl-5'-O-beta-D-glucopyranosyl-pentanoxy)-5alpha-pregn-3beta-ol-12,20-dione-3-O-beta-D-glucopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 4)-beta-D-galactopyranoside (1), 2alpha,3beta-dihydroxy-5alpha-pregn-16-en-20-one 3-O-beta-D-glucopyranosyl-(1 --> 4)-beta-D-galactopyranoside (2) and 26-O-beta-D-glucopyranosyl-(25R)-5alpha-furostan-20(22)-en-2alpha,3beta,26-triol-3-O-beta-D-glucopyranosyl-(1 --> 4)-beta-D-galactopyranoside (3).