Aspects for development of novel antibacterial medicines using a vitamin D3 decomposition product in Helicobacter pylori infection.

A previous study by our group demonstrated that a vitamin D3 decomposition product (VDP1) acts as the selective bactericidal substance on Helicobacter pylori. VDP1 is an indene compound modified with a carbonyl and an alkyl. The alkyl of VDP1 turned out to be a mandatory structure to exert effective bactericidal action on H. pylori. Meanwhile, it still remains to be clarified as to how influence the alteration of the carbonyl in VDP1 has on the anti-H. pylori activity. In this study, we synthesized novel VDP1 derivatives that replaced the carbonyl of VDP1 by various functional groups and investigated the antibacterial action of the VDP1 derivatives on H. pylori. VDP1 derivatives retaining either a hydroxy (VD3-1) or an acetic ester (VD3-3) exhibited more effective bactericidal action to H. pylori than VDP1. The replacement of the carbonyl of VDP1 by either an allyl acetate (VD3-2) or an acrylic acid (VD3-5) provided almost no change to the anti-H. pylori activity. Apart from this, an isomer of VDP1 (VD3-4) slightly improved anti-H. pylori activity of VDP1. Meanwhile, the replacement of the carbonyl of VDP1 by a methyl acrylate (VD3-6) attenuated the anti-H. pylori activity. As with VDP1, its derivatives also were suggested to exert the anti-H. pylori action through the interaction with myristic acid side chains of dimyristoyl-phosphatidylethanolamine, a characteristic membrane lipid constituent of this pathogen. These results indicate that it is capable of developing specific antibacterial medicines for H. pylori targeting the biomembranal dimyristoyl-phosphatidylethanolamine using VDP1 as the fundamental structure.

The Chronic and Acute Toxicity of Traditional Medicines Containing Terminalia chebula.

Objectives: Terminalia chebula, the main ingredient of Altan Arur 5, has been used for many years in traditional medicine. This medicine is more effective than other drugs and is used to treat chronic gastritis and gastrointestinal disorders such as peptic ulcers and esophageal reflux. Other ingredients of Altan Arur 5 are Punica granatum (pomegranate), tulip seeds, black balm, and excreta of Trogopterus xanthipes. The main ingredients of T. chebula are antibacterial and analgesic in traditional medicine. Despite having been used for many years and although many studies have been conducted on the beneficial effects of this medicine and its ingredients, the toxicity of Altan Arur 5 has not yet been elucidated. Therefore, we aimed to study the toxicity of Altan Arur 5 to ensure that it is safe to use. Methods: Acute and chronic toxicity of Altan Arur 5 were assessed in 10 Kunming mice and 8 Sprague-Dawley rats, respectively, in different doses. In the acute toxicity study, Altan Arur 5 was orally administered to Kunming mice in doses of 12 g/kg, 24 g/kg, and 48 g/kg for 14 days. In the chronic toxicity study, it was orally administered to Sprague-Dawley rats in doses of 1.25 g/kg, 2.5 g/kg, and 5 g/kg for 12 weeks. Results: No significant differences were observed in the relative organ weights for mice treated with Altan Arur 5 compared with those in the control group. Furthermore, no macro- or microstructural changes were noted in the organs of any group. Conclusion: Our toxicity testing revealed that the traditional medicine Altan Arur 5 has no toxic effects in vivo.

A novel role of catalase in cholesterol uptake of Helicobacter pylori.

Helicobacter pylori infection is known to be a significant risk factor for the development of gastric cancers in humans. This pathogen exhibits unique biological characteristics in membrane lipid composition. Specifically, H. pylori incorporates exogenous cholesterol into biomembranes and uses cholesterol as the membrane lipid constituents. A previous study by our group demonstrated that phosphatidylethanolamine of H. pylori functions as the cholesterol-binding lipid. It is, however, unclear whether H. pylori is equipped with protein molecules involved in the cholesterol uptake. We, therefore, examined H. pylori proteins that tightly bind to cholesterol. As a consequence, H. pylori catalase (KatA) turned out to be a candidate of the cholesterol uptake-associated protein. In addition, an H. pylori mutant strain that expresses KatA protein lacking catalase activity was significantly lower in total cholesterol contents than the wild-type H. pylori strain. The putative amino acid sequence of KatA found out to contain a number of the cholesterol recognition/interaction amino acid consensus sequence domains (CRAC and CARC domains). These results suggest that H. pylori KatA with normal folding conformation acts as the cholesterol-binding or -storage protein.

A short review, effect of dimethyl-ß-cyclodextrin on the interaction between Helicobacter pylori and steroidal compounds.

The 2,6-di-O-methyl-ß-cyclodextrin (dMßCD) is an amphiphilic annular compound consisting of seven dimethyl-glucose molecules. This compound is well known as a solubilizer of lipophilic compounds. Especially, dMßCD extracts cholesterol from the plasma membrane of mammalian cells and releases the cholesterol to the aqueous solution. The experimental use of dMßCD, therefore, serves to investigate the role of cholesterol in the mammalian cell membrane. It is, however, unclear as to how dMßCD extracts cholesterol incorporated into the glycerophospholipid biomembrane. Meanwhile, dMßCD acts as a beneficial compound for Helicobacter pylori and is used as the standard component for supporting the growth of this bacterium in the serum-free culture. However, the detailed mechanism of dMßCD for supporting the growth of H. pylori is still to be clarified. H. pylori is a Gram-negative microaerophilic bacillus recognized as a pathogen concerned with gastrointestinal diseases in human. Previous studies by our group have successfully obtained the H. pylori strains culturable without dMßCD and demonstrated the distinct effects of dMßCD on the interaction between H. pylori and exogenous steroidal compounds. For instance, dMßCD promotes and inhibits the absorption of cholesterol and several steroidal compounds respectively into the biomembranes of H. pylori. In this study we summarized behaviors of dMßCD toward steroidal compounds relevant to H. pylori.

Unique responses of Helicobacter pylori to exogenous hydrophobic compounds.

Helicobacter pylori is a pathogen responsible for peptic ulcers and gastric cancers in human. One of the unique biological features of this bacterium is a membrane lipid composition significantly differed from that of typical Gram-negative bacteria. Due to its unique lipid composition, the responses of H. pylori to various exogenous lipophilic compounds significantly differ from the responses of typical Gram-negative bacteria to the same lipophilic compounds. For instance, some steroidal compounds are incorporated into the biomembranes of H. pylori through the intermediation of the myristoyl-phosphatidylethanolamine (PE). In addition, H. pylori shows high susceptibility to bacteriolytic action of lipids such as 3-carbonyl steroids, vitamin D, and indene compounds. These lipids are also considered to interact with myristoyl-PE of H. pylori membranes, and to ultimately confer the bactericidal action to this bacterium. In this study we summarize the lipids concerned with H. pylori and suggest the possibility of the development of chemotherapeutic medicines that act on the membrane lipid component of H. pylori.

Antibacterial effect of indene on Helicobacter pylori correlates with specific interaction between its compound and dimyristoyl-phosphatidylethanolamine.

Recent studies by our group have suggested that the vitamin D3 decomposition product VDP1 [(1R,3aR,7aR)-1-[(1R)-1,5-dimethylhexyl]octahydro-7a-methyl-4H-inden-4-one] confers the potent bactericidal action to Helicobacter pylori by targeting the membranal dimyristoyl-phosphatidylethanolamine (di-14:0 PE). In this study we synthesized a new VDP1 derivative to advance further investigation as for the correlative relationship between VDP1 structure and anti-H. pylori activity or PE vesicle collapse induction activity. The derivative VD3-7 [(1R,7aR)-4-fluoro-7a-methyl-1-((R)-6-methylheptan-2-yl)octahydro-1H-indene] retained a fluorine atom in place of the oxygen atom of VDP1. The fluorination of the carbonyl portion of VDP1 forfeited the effective anti-H. pylori activity. We, therefore, prepared Coomassie brilliant blue (CBB)-containing unilamellar vesicles consisting of various PE molecular species, and examined the vesicle collapse induction activity of either VDP1 or VD3-7 by detecting the CBB eluted from the PE unilamellar vesicles. VDP1 strongly induced CBB elution from the unilamellar vesicles of rectus-PE retaining the same two fatty acid side-chains shorter than carbon numbers 14, indicating that VDP1 specifically disrupted the vesicular conformation of those PE unilamellar vesicles. Meanwhile, VD3-7 had no influence on the structural stability of any PE unilamellar vesicles. This study obtained additional evidence that VDP1 acts as a bactericidal agent on H. pylori by targeting the membranal di-14:0 PE.

Erratum: Author Correction: Induction of Mucosal Humoral Immunity by Subcutaneous Injection of an Oil-emulsion Vaccine against Salmonella Enterica subsp. enterica serovar Enteritidis in Chickens.

[This corrects the article DOI: 10.14252/foodsafetyfscj.2018003.].

Indene Compounds Synthetically Derived from Vitamin D Have Selective Antibacterial Action on Helicobacter pylori.

Helicobacter pylori infects the human stomach and is closely linked with the development of gastric cancer. When detected, this pathogen can be eradicated from the human stomach using wide-spectrum antibiotics. However, year by year, H. pylori strains resistant to the antibacterial action of antibiotics have been increasing. The development of new antibacterial substances effective against drug-resistant H. pylori is urgently required. Our group has recently identified extremely selective bactericidal effects against H. pylori in (1R,3aR,7aR)-1-[(1R)-1,5-dimethylhexyl]octahydro-7a-methyl-4H-inden-4-one (VDP1) (otherwise known as Grundmann's ketone), an indene compound derived from the decomposition of vitamin D3 and proposed the antibacterial mechanism whereby VDP1 induces the bacteriolysis by interacting at least with PtdEtn (dimyristoyl-phosphatidylethanolamine [di-14:0 PtdEtn]) retaining two 14:0 fatty acids of the membrane lipid constituents. In this study, we synthesized new indene compounds ((1R,3aR,7aR)-1-((2R,E)-5,6-dimethylhept-3-en-2-yl)-7a-methyloctahydro-4H-inden-4-one [VD2-1], (1R,3aR,7aR)-1-((S)-1-hydroxypropan-2-yl)-7a-methyloctahydro-1H-inden-4-ol [VD2-2], and (1R,3aR,7aR)-7a-methyl-1-((R)-6-methylheptan-2-yl)octahydro-1H-inden-4-ol [VD3-1]) using either vitamin D2 or vitamin D3 as materials. VD2-1 and VD3-1 selectively disrupted the di-14:0 PtdEtn vesicles without destructing the vesicles of PtdEtn (dipalmitoyl-phosphatidylethanolamine) retaining two 16:0 fatty acids. In contrast, VD2-2, an indene compound lacking an alkyl group, had no influence on the structural stability of both PtdEtn vesicles. In addition, VD2-1 and VD3-1 exerted extremely selective bactericidal action against H. pylori without affecting the viability of commonplace bacteria. Meanwhile, VD2-2 almost forfeited the bactericidal effects on H. pylori. These results suggest that the alkyl group of the indene compounds has a crucial conformation to interact with di-14:0 PtdEtn of H. pylori membrane lipid constituents whereby the bacteriolysis is ultimately induced.

Induction of Mucosal Humoral Immunity by Subcutaneous Injection of an Oil-emulsion Vaccine against Salmonella enterica subsp. enterica serovar Enteritidis in Chickens.

Salmonella enterica subsp. enterica serovar Enteritidis (SE) is one of the major causes of food poisoning. Much effort has been made to develop a vaccine for the prevention of SE colonization and infection in poultry. However, the effect of inactivated whole-cell SE vaccines on the bacterial attachment has not been clarified. This study investigated the immune responses to a killed whole-cell SE vaccine in chickens and the effect of vaccination on the bacterial attachment of SE to cultured Vero cells. A 1 ml dose of 108-109 CFU viable SE bacterial cells was orally administered to chickens at 4 weeks or 10 months post vaccination. The number (CFU) of SE in 1 g of cecal droppings was counted on day 6 after administration. The SE CFUs were significantly lower (p < 0.05) in the vaccinated chickens, not only at 4 weeks but also at 10 months after vaccination, than in the unvaccinated control chickens. Anti-SE IgG and anti-SE IgA were detected using enzyme-linked immunosorbent assay (ELISA) in serum and intestinal and oviduct fluid samples from vaccinated chickens. Adhesion of heat-killed SE cells to Vero cells was reduced by pre-treatment of the bacteria by the vaccinated chicken-derived intestinal fluid, indicating the potential of the vaccine-induced antibody to prevent SE adhesion to epithelial cell surfaces.

Phosphatidylethanolamine, but not Phosphatidylglycerol-Cardiolipin, Isolated from Two Species of Helicobacter Binds Cholesterol over Cholesteryl Ester.

This study demonstrated that the cells of Helicobacter felis and Helicobacter cinaedi spontaneously absorb cholesterol added to the medium. A recent study by our group has revealed that phosphatidylethanolamine (PtdEtn) of Helicobacter pylori contains myristic acid as the most predominant saturated fatty acid and that the PtdEtn of this bacterium binds cholesterol more selectively than cholesteryl ester. We, therefore, isolated the PtdEtn from the two Helicobacter species to analyze the hydrophobic interaction between cholesterol and its glycerophospholipid. PtdEtn of the Helicobacter bacteria interacted more selectively with cholesterol than with cholesteryl ester, and the degree of the selective binding of cholesterol was higher in the PtdEtn than in the phosphatidylglycerol-cardiolipin of the same bacteria. These results suggest the possibility that the cells of H. felis and H. cinaedi may contain abundant PtdEtn with myristic acid. On this basis, we analyzed the PtdEtn molecular species of the Helicobacter bacteria and demonstrated that the PtdEtn containing myristic acid accounts for more than 35% in the total PtdEtn. These results suggest that the myristoyl PtdEtn takes part in the absorption of cholesterol in H. felis and H. cinaedi.

Identification and characterization of a vitamin D3 decomposition product bactericidal against Helicobacter pylori.

This study demonstrated that the vitamin D3 decomposition product VDP1 exerts an antibacterial action against Helicobacter pylori but not against other bacteria. Treatment with VDP1 induced a collapse of cell membrane structures of H. pylori and ultimately lysed the bacterial cells. A unique dimyristoyl phosphatidylethanolamine in the membrane lipid compositions contributed to the interaction of VDP1 with H. pylori cells. In separate experiments, VDP1 had no influence on the viability of the human cancer cell lines MKN45 and T47D and lacked any vitamin D3-like hormonal action against the latter. In both (1)H and (13)C NMR analyses, the spectra patterns of VDP1 corresponded with those of Grundmann's ketone. These results suggest that VDP1 (or Grundmann's ketone-type indene compound) may become a fundamental structure for the development of new antibacterial substances with selective bactericidal action against H. pylori.

Antibacterial activity of a novel synthetic progesterone species carrying a linoleic acid molecule against Helicobacter pylori and the hormonal effect of its steroid on a murine macrophage-like cell line.

Helicobacter pylori, a pathogen responsible for gastric and duodenal diseases, absorbs various steroid compounds into the cell membrane even though some are toxic to this bacterium. An earlier study by our group has demonstrated that progesterone is bactericidal to H. pylori. In this study, we newly synthesized a steroid compound, 17α-hydroxyprogesterone linoleic acid ester (17hPL), to examine antibacterial activity against H. pylori. As expected, 17hPL acted as a bactericidal agent to H. pylori and had no effect on the survival of other common bacterial species. This steroidal substance interacted with phosphatidylethanolamine (PE) on the outer membrane of H. pylori to induce the release of PE from the bacterial cell membrane and to ultimately lyse the bacterial cells. One of the hormonal effects of progesterone is the inhibition of nitric oxide (NO) production from mouse macrophages activated by lipopolysaccharide (LPS). We therefore examined the inhibition effect of 17hPL on the NO production of RAW 264.7 cells, a murine macrophage-like cell line, stimulated with LPS and demonstrated that 17hPL is relatively weaker in its capability to inhibit NO production in LPS-activated cells than progesterone. These results suggest the possibility that 17hPL could be an oral medicine for selectively treating patients infected with H. pylori.

Detoxification of 7-dehydrocholesterol fatal to Helicobacter pylori is a novel role of cholesterol glucosylation.

The glucosylation of free cholesterol (FC) by Helicobacter pylori cells has various biological significances for the survival of this bacterium. H. pylori cells with glucosylated FC are capable of evading host immune systems, such as phagocytosis by macrophages and activation of antigen-specific T cells, and surviving in the gastric mucosal tissues for long periods. An additional role of cholesterol glucosylation in the survival of H. pylori which is distinct from the role of escaping the host immune system, however, has yet to be identified. This study demonstrated that 7-dehydrocholesterol (7dFC), an FC precursor, is a toxic compound fatal to H. pylori cells, but the cell membrane of H. pylori is capable of absorbing this toxic sterol via glucosylation. In contrast to the case with 7dFC, no toxicity to H. pylori cells was detected from the glucosylated 7dFC. In addition, cgt gene mutant H. pylori cells that cannot glucosylate cholesterols had higher susceptibility to the toxic action of 7dFC than wild-type H. pylori cells. These results indicate that the cgt gene product of H. pylori serves to detoxify the sterol fatal to this bacterium and to permit this toxic sterol as a cell membrane lipid component. In summary, this study defined a novel role of cholesterol glucosylation in H. pylori.

Phosphatidylethanolamine of Helicobacter pylori functions as a steroid-binding lipid in the assimilation of free cholesterol and 3ß-hydroxl steroids into the bacterial cell membrane.

One of the unique features of Helicobacter pylori is its ability to assimilate free-cholesterol (FC) into its membranes. Via FC assimilation, H. pylori strengthens the membrane lipid barrier and/or evades the host immune system. No previous studies, however, have investigated the FC uptake mechanisms of the H. pylori cell. Phosphatidylethanolamine (PE) is the most prevalent lipid component of bacteria, including H. pylori, but the function of PE remains unclear. We were therefore interested in H. pylori PE (HpPE) and investigated the interaction of its PE with cholesterols. The PE isolated from H. pylori underwent a unique molecular interaction with FC, cholesterol ester (CE), and 2,6-di-O-methyl-ß-cyclodextrin (dMßCD), a sterol solubilizer. HpPE interacted not only with the FC molecule, but also with the FC-dMßCD inclusion complex. In contrast, Escherichia coli PE (EcPE), prepared as a reference PE, seemed to bind only FC, and only via a hydrophobic interaction, without binding dMßCD. HpPE was clearly more potent than EcPE in binding FC. Intriguingly, HpPE had a negligible affinity for CE, while EcPE had a high affinity for CE, comparable to its affinity for FC. Further, HpPE interacted with 3ß-OH steroids, pregnenolone and dehydroepiandrosterone, in the absence of dMßCD. Gas chromatogram-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analyses revealed that the fatty acid compositions of HpPE were quite distinct from those of EcPE, and the C(14:0) fatty acid in the HpPE molecule was found to be significant in binding FC selectively. These results indicate that PE is a key candidate of nonesterified steroid-binding lipids in H. pylori.

Steroid hormones as bactericidal agents to Helicobacter pylori.

Helicobacter pylori is a unique bacterial species that assimilates various steroids as membrane lipid components. Our group has recently found, however, that certain steroids may impair the viability of H. pylori. In this study, we go on to reveal that estradiol, androstenedione, and progesterone (PS) all have the potential to inhibit the growth of H. pylori. Of these three steroid hormones, progesterone demonstrated the most effective anti-H. pylori action. 17α-hydroxyprogesterone caproate (17αPSCE), a synthetic progesterone derivative, had a much stronger anti-H. pylori action than progesterone, whereas 17α-hydroxyprogesterone, a natural progesterone derivative, completely failed to inhibit the growth of the organism. Progesterone and 17αPSCE were both found to kill H. pylori through their bacteriolytic action. Among five bacterial species investigated, H. pylori was the only species susceptible to the bactericidal action of progesterone and 17αPSCE. The other four species, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epiderimidis, all resisted this action. Progesterone and free-cholesterol (FC) obstructed each other's effects against the H. pylori cell. Taken in sum, these results suggest that progesterone and FC may bind to the identical region on the H. pylori cell surface. We expect these findings to contribute to the development of a novel anti-H. pylori steroidal agent.

[Latest advances in non-pylori Helicobacter species].

The genus Helicobacter presently comprises 32 validly named species. Helicobacter equorum, Helicobacter baculiformis and Helicobacter suis were recognized as new species in 2007 and 2008. Non-pylori Helicobacter species are occasionally isolated from human clinical specimens, though their natural hosts are mammals other than human and birds. It remains unclear whether they are associated with human diseases. 'Helicobacter heilmannii' causes human chronic gastritis. Several reports suggest that Helicobacter hepaticus and Helicobacter bilis are associated with human hepatic and biliary disorders, respectively. Non-pylori Helicobacter species are occasionally isolated from clinical specimens of immunocompromised hosts. These cases suggest that they are important opportunistic pathogens.

Steroids mediate resistance to the bactericidal effect of phosphatidylcholines against Helicobacter pylori.

Helicobacter pylori assimilates various steroids as membrane lipid components, but it can also survive in the absence of steroids. It thus remains to be clarified as to why the organism relies on steroid physiologically. In this study, we have found that phosphatidylcholine carrying a linoleic acid molecule or arachidonic acid molecule has the potential to kill steroid-free H. pylori. The bactericidal action of phosphatidylcholines against H. pylori was due to the lytic activity of the phosphatidylcholines themselves and not due to the lytic activity of the unsaturated fatty acids or lyso-phosphatidylcholine resulting from the hydrolysis of the phosphatidylcholines. In contrast to the steroid-free H. pylori, the organism that absorbed and glucosylated free cholesterol was unaffected by the bactericidal action of the phosphatidylcholines. Similarly, H. pylori that absorbed estrone without glucosylating it also resisted the bactericidal action of the phosphatidylcholines. The steroids absorbed by H. pylori existed in both the outer and inner membranes, while the glucosyl-steroids produced via the steroid absorption were localized in the outer membrane rather than in the inner membrane. These results indicate that H. pylori absorbs the steroids to reinforce the membrane lipid barrier and thereby expresses resistance to the bacteriolytic action of hydrophobic compounds such as phosphatidylcholine.

Anabolic utilization of steroid hormones in Helicobacter pylori.

In this study, we have demonstrated that Helicobacter pylori absorbs a steroid prehormone (pregnenolone) and two androgens (dehydroepiandrosterone and epiandrosterone), glucosylates these steroids, and utilizes glucosyl-steroid hormone compounds as the membrane lipid components. The only common structure among the steroid prehormone and the two androgens is a 3beta-OH in the steroid framework. Our results indicate that the 3beta-OH in the steroid hormones is a crucial conformation required for steroid glucosylation by H. pylori. In addition, we found that H. pylori absorbs and holds estrogens possessing 3-OH (estrone and estradiol) into the membrane. The effective absorption of estrogen into the membrane appeared to be controlled by the number of hydroxyl groups modifying the steroid framework. In contrast, H. pylori induced neither membrane absorption nor glucosylation of the other steroid hormones possessing 3=O (progesterone, androstenedione and testosterone) or 3alpha-OH (androsterone). These results indicate that H. pylori selectively absorbs 3beta-OH and 3-OH steroid hormones, and utilizes only 3beta-OH steroid hormones as the materials for glucosylation.

Conversion of flavodoxin from holoenzyme to apoprotein during growth phase changes in Helicobacter pylori.

The catabolic pathway for flavodoxin has yet to be clarified for any bacterial species. In this study, we found that the flavin mononucleotide in the flavodoxin of Helicobacter pylori is degraded to riboflavin via the phosphomonoesterase activity of class C acid phosphatase. The result is a conversion of holoflavodoxin to apoflavodoxin.

Alteration in the composition of cholesteryl glucosides and other lipids in Helicobacter pylori undergoing morphological change from spiral to coccoid form.

In this analysis of membrane lipid compositions in Helicobacter pylori, the membrane lipid profiles drastically changed during coccoid formation: cholesteryl-6-O-tetradecanoyl-alpha-D-glucopyranoside levels increased, cholesteryl-alpha-D-glucopyranoside and phosphatidyl ethanolamine decreased, and a coordinated increase in cardiolipin and decrease in phosphatidyl glycerol were observed. Cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside was hardly detected in the spiral forms in the logarithmic phase, but subsequently increased throughout the coccoid conversion. These results suggest that environmental stresses induce the expression of certain regulatory systems for lipid metabolism in H. pylori, and that the resulting alterations in lipid composition play an important role in inducing the coccoid conversion.