Vibrio vulnificus vulnibactin, but not metalloprotease VvpE, is essentially required for iron-uptake from human holotransferrin.

The roles of metalloprotease (VvpE) and catechol-siderophore (vulnibactin) in the uptake of iron from human transferrins by Vibrio vulnificus have been determined using different experimental conditions and methods. Therefore, in this study, we attempted to elucidate the roles of VvpE and vulnibactin using the same methods and experimental conditions, in an in vitro and a human ex vivo system, and in accordance with the molecular version of Koch's postulates. Neither vvpE mutation nor in trans vvpE complementation affected vulnibactin production, iron-assimilation from human holotransferrin (HT), and bacterial growth in a HT-containing deferrated Heart-Infusion medium (HT-DF-HI) or a HT-containing cirrhotic ascites (HT-CA). In contrast, the mutation of fur gene encoding Fur, a repressor regulating expression of the vulnibactin-mediated iron-uptake system, derepressed vulnibactin production, and facilitated iron-assimilation from HT and bacterial growth in HT-DF-HI or HT-CA. The mutation of vis gene encoding isochorismate synthase required for vulnibactin synthesis abolished vulnibactin production, iron-assimilation from HT and bacterial growth in HT-DF-HI or HT-CA. These results demonstrate that vulnibactin is essentially required for iron-assimilation from transferrin, and that VvpE has no direct effect on facilitating vulnibactin-mediated iron-assimilation from transferrin in vitro or in a human ex vivo system.

Proteases of a Bacillus subtilis clinical isolate facilitate swarming and siderophore-mediated iron uptake via proteolytic cleavage of transferrin.

We isolated a highly serine protease-producing Bacillus subtilis strain (PRY) from a clinical sample and identified it through biochemical testing and ribosomal DNA sequencing. The PRY strain exhibited a robust swarming behavior and was able to digest human transferrin efficiently, concomitantly with the production of catechol-siderophore in the exponential growth phase. The growth of PRY was in proportion to increased iron availability resulting from transferrin destruction. These results suggest that proteases of the B. subtilis PRY strain may play a significant role in the pathogenesis of human infections by facilitating siderophore-mediated iron uptake from transferrin and swarming motility.

Effect of the crp mutation on the utilization of transferrin-bound iron by Vibrio vulnificus.

Cyclic AMP-cAMP receptor protein (CRP) complex plays an essential role in the global regulation of Vibrio vulnificus virulence. We found that growth retardation of V. vulnificus caused by mutation of the crp gene encoding CRP was exacerbated under iron-limited conditions. Accordingly, we investigated the effect of crp mutation on the expression of the vulnibactin-mediated iron-uptake system and the ability of V. vulnificus to utilize transferrin-bound iron, and thus to grow in cirrhotic ascites, a human ex vivo system. The production of vulnibactin was suppressed, and the transcription of the vis and vuuA genes, which encode an enzyme required for vulnibactin synthesis and vulnibactin receptor protein, was also suppressed in the crp mutant. Moreover, the crp mutant could not utilize transferrin-bound iron, and its growth was severely suppressed both on transferrin-bound iron and in cirrhotic ascites. All the defects in the crp mutant were recovered by the in trans complementation of the wild-type crp gene. Putative CRP-binding sequences were found in the regulatory regions of the fur, vis and vuuA genes. These results indicate that crp mutation attenuates the ability to grow on transferrin-bound iron and in a human body fluid by down-regulating the vulnibactin-mediated iron-uptake system.

Assessment of a bioactive compound for its potential antiinflammatory property by tight junction permeability.

Lactobacillus probiotic strains are proving to be abundant sources of bioactive components, including antiinflammatory components. Lifree was made of fruits fermented by Lactobacillus paracasei, Lactobacillus reuterrii and Saccharomyces cerevisiae. This study was designed to test these compounds in cell assays measuring epithelial barrier function and proliferation in the first instance. Cell proliferation was measured in mouse fibroblasts cells (3T3NIH) and rat intestinal epithelial cells (IEC-6), and tight junction activity in the kidney epithelial cell line (MDCK). Tight junction permeability was assessed by measuring transepithelial electrical resistance (TER) across confluent monolayers, following the addition of Lifree with or without a challenge with EGTA. Lifree promoted tight junction formation and recovery following loss of TER from challenge with EGTA. On the other hand, Lifree did not stimulate cell growth in either 3T3NIH and IEC-6 cells. Lifree stimulates tight junction maintenance and formation, suggesting it may have potential antiinflammatory properties.

Staphylococcus aureus siderophore-mediated iron-acquisition system plays a dominant and essential role in the utilization of transferrin-bound iron.

Staphylococcus aureus is known to be capable of utilizing transferrin-bound iron, via both siderophore- and transferrin-binding protein (named IsdA)-mediated iron-acquisition systems. This study was designed in order to determine which iron-acquisition system plays the essential or dominant role with respect to the acquisition of iron from human transferrin, in the growth of S. aureus. Holotransferrin (HT) and partially iron-saturated transferrin (PT), but not apotransferrin (AT), were found to stimulate the growth of S. aureus. S. aureus consumed most of the transferrin-bound iron during the exponential growth phase. Extracellular proteases were not, however, involved in the liberation of iron from transferrin. Transferrin-binding to the washed whole cells via IsdA was not observed during the culture. The expression of IsdA was observed only in the deferrated media with AT, but not in the media supplemented with PT or HT. In contrast, siderophores were definitely produced in the deferrated media with PT and HT, as well as in the media supplemented with AT. The siderophores proved to have the ability to remove iron directly from transferrin, but the washed whole cells expressing IsdA did not. In the bioassay, the growth of S. aureus on transferrin-bound iron was stimulated by the siderophores alone. These results demonstrate that the siderophore-mediated iron-acquisition system plays a dominant and essential role in the uptake of iron from transferrin, whereas the IsdA-mediated iron-acquisition system may play only an ancillary role in the uptake of iron from transferrin.

Electro-acupuncture reverses nerve growth factor abundance in experimental polycystic ovaries in the rat.

Polycystic ovary syndrome (PCOS) remains one of the most common causes of anovulation in women of reproductive age. There is some evidence that nerve growth factor (NGF) is involved in the pathogenesis of PCOS. Therefore, seeking the pathogenesis of PCOS is important for controlling fertility. In traditional Oriental Medicine, acupuncture has been used for the function of ovaries. The present study was designed to determine whether electro-acupuncture (EA) could affect experimentally induced polycystic ovary (PCO) in the rat. The two acupoints Sp-6 and E-128 were stimulated to test for efficacy in the protein expression of NGF. Polycystic ovaries were induced by a single injection of estradiol valerate (4 mg i.m.). During the experimental period of 8 weeks, some of the rats were treated with EA twice weekly; this group was compared with a vehicle-treated control group and an estradiol-injected group not subjected to EA. At day 60, the protein expression of NGF was examined by immunohistochemistry in the ovaries, the adrenal glands and some parts of the brain. The estradiol treatment induced a clear PCO appearance, and was associated with a robust increase in NGF expression in the ovaries, the adrenal glands and the brain. EA treatment partly reversed the NGF abundance, particularly in the ovaries, but not in the brain. Our data show that EA affects the NGF involvement in ovarian dysfunction.

The effect of herbal medicine on nerve growth factor in estradiol valerate-induced polycystic ovaries in rats.

A type of polycystic ovary resembling some aspects of human polycystic ovarian syndrome (PCOS) can be induced in the rat with a single injection of long-acting estradiol valerate. Among several theories behind the development of polycystic ovaries (PCO), the involvement of the sympathetic nervous system draws much attention, and herbal medicine is known to relieve the abnormal symptoms of PCO. Two herbal formulas, Changbudodam-Tang (cang fu dao tan tang) and Yongdamsagan-Tang (long dan xie gan tang), were used in the present study. The administration of herbal medicine was done every other day for 60 days. The morphological changes of ovaries from herbal medicine treatment were compared to those from an oil-treated control group and an estradiol valerate-injected group. This study also examined the possible hypothesis of neurogenic participation in terms of nerve growth factor (NGF) in the pathology of ovarian dysfunction. The nerve growth factor was analyzed in the central nervous system and ovaries by immunohistochemistry. The main findings of the present study were: (1) PCO were fully developed in rats with a single intramuscular injection of estradiol valerate, (2) PCO resulted in the expression of NGF in the ovaries and the brain tissues, and (3) herbal medicine administration significantly decreased the elevated NGF staining in the ovaries without affecting the brain tissues significantly.