Microbiology and Immunology

Based on the results of phenotypic characterization, cellular lipid and fatty acid analysis, phylogenetic analysis of 16S rDNA nucleotide sequences and rRNA‐DNA hybridization, Burkholderia pickettii, Burkholderia solanacearum and Alcaligenes eutrophus are transferred to the new genus Ralstonia, and Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb, nov., and R. eutropha (Davis 1969) comb. nov. are proposed. The type species of the new genus is R. pickettii. Type strain of R. pickettii is ATCC 27511^T, of R. solanacearum is ATCC 10696^T, and of R. eutropha is ATCC 17697^T.

The nucleic acid fraction from cells of 6 species of bacterium and 2 kinds of vertebrate, calf and salmon, was extracted and purified by the same procedures as described previously. When the spleen cells from BALB/c mice were incubated with the nucleic acid fraction from either of the bacteria, natural killer (NK) activity of the cells was remarkably elevated and the cells produced factors to activate macrophages and to inhibit viral growth. It was shown that the factor to activate macrophages was interferon (IFN)‐gamma and that to inhibit viral growth was IFN‐alpha/beta. On the other hand, the nucleic acid fraction from either of the vertebrate cells did not show such activities. Pretreatment of the bacterial nucleic acid fraction with DNase, but not with RNase, abrogated completely the biological activities. The activities of the bacterial nucleic acid were not influenced by the presence of polymyxin B, an inhibitor of lipopolysaccharide (LPS), and the spleen cells from not only BALB/c mice but also LPS‐insensitive C3H/HeJ mice were activated, indicating that the activities of the fraction were not ascribed to LPS contaminated possibly into the fraction, but to DNA itself. Intralesional injection with the bacterial DNA fraction caused regression of mouse IMC tumors, but the injection with the vertebrate DNA fraction did not. These findings prompted us to examine the biological activities of DNA samples from a variety of animals and plants, which were provided from other laboratories or purchased from manufacturers. All of the DNA samples from cells of 5 kinds of bacterium, 2 of virus and 4 of invertebrate augmented NK activity and induced IFN, more or less, in mouse spleen calls, while the DNA from 10 kinds of vertebrate, including 3 of fish and 5 of mammal, showed no such activities. The DNA from 2 species of plants, were also inactive. Possible mechanisms to explain the different biological activities of DNA from different cell sources were discussed based on our previous finding that the particular palindromic sequences with a G‐C motif (s) are required for induction of IFNs and activation of NK cells with synthetic 30‐mer oligonucleotides.

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. In this study, we isolated 36 polyphenols and 4 terpenoids from medicinal plants, and investigated their antibacterial activity against H. pylori in vitro. All hydrolyzable tannins tested demonstrated promising antibacterial activity against H. pylori. Monomeric hydrolyzable tannins revealed especially strong activity. Other compounds demonstrated minimal antibacterial activity with a few exceptions. A monomeric hydrolyzable tannin, Tellimagrandin I demonstrated time‐ and dose‐dependent bactericidal activity against H. pylori in vitro. On the other hand, hydrolyzable tannins did not affect the viability of MKN‐28 cells derived from human gastric epithelium. Hydrolyzable tannins, therefore, have potential as new and safe therapeutic regimens against H. pylori infection. Furthermore, we investigated effects of hydrolyzable tannins on lipid bilayer membranes. All the hydrolyzable tannins tested demonstrated dose‐dependent membrane‐damaging activity. However, it remains to be elucidated whether their membrane‐damaging activity directly contributes to their antibacterial action.

Interleukin (IL)‐17 is a proinflammatory cytokine which induces differentiation and migration of neutrophils through induction of cytokines and chemokines including granulocyte‐colony stimulating factor and CXCL8/IL‐8. IL‐17‐producing CD4⁺ T cells (Th17) have pivotal role in pathogenesis of autoimmune diseases. IL‐17 is also involved in protective immunity against various infections. IL‐17 has important role in induction of neutrophil‐mediated protective immune response against extracellular bacterial or fungal pathogens such as Klebsiella pneumoniae and Candida albicans. Importance of IL‐17 in protection against intracellular pathogens including Mycobacterium has also been reported. Interestingly, not only CD4⁺ T cells but atypical CD4^–CD8^– T cells expressing T cell receptor (TCR) γδ produce IL‐17, and IL‐17 producing cells participate in both innate and acquired immune response to infections. Furthermore, neutrophil induction may not be the only mechanism of IL‐17‐mediated protective immunity. IL‐17 seems to participate in host defense through regulation of cell‐mediated immunity or induction of antimicrobial peptides such as β‐defensins. In this review, we summarize recent progress on the role of IL‐17 in immune response against infections, and discuss possible application of IL‐17 in prevention and treatment of infectious diseases.

Antimicrobial peptides contribute to innate host defense against a number of bacteria and fungal pathogens. Some of antimicrobial synthetic peptides were systemically administered in vivo; however, effective protection has so far not been obtained because the effective dose of peptides in vivo seems to be very high, often close to the toxic level against the host. Alternatively, peptides administered in vivo may be degraded by certain proteases present in serum. In this study, D‐amino acids were substituted for the L‐amino acids of antimicrobial peptides to circumvent these problems. Initially a peptide (L‐peptide) rich in five arginine residues and consisting of an 11‐amino acid peptide (residues 32–42) of human granulysin was synthesized. Subsequently, the L‐amino acids of the 11‐amino acid peptide were replaced partially (D‐peptide) or wholly (AD‐peptide) with D‐amino acids. Activity and stability to proteolysis, in particular, in the serum of antimicrobial peptides with D‐amino acid substitutions were examined. Peptides with D‐amino acid substitutions were found to lyse bacteria as efficiently as their all‐L‐amino acid parent, L‐peptide. In addition, the peptide composed of L‐amino acids was susceptible to trypsin, whereas peptides containing D‐amino acid substitutions were highly stable to trypsin treatment. Similarly, the peptide consisting of L‐amino acids alone was also susceptible to fetal calf serum (FCS), however, protease inhibitors restored the lowered antimicrobial activity of the FCS‐incubated peptide. Thus, D‐amino acid substitutions can make antimicrobial peptides resistant to proteolysis, suggesting that the antimicrobial peptides consisting of D‐amino acids are potential candidates for clinical therapeutic use.

Corilagin and tellimagrandin I are polyphenols isolated from the extract of Arctostaphylos uvaursi and Rosa canina L. (rose red), respectively. We have reported that corilagin and tellimagrandin I remarkably reduced the minimum inhibitory concentration (MIC) of β‐lactams in methicillin‐resistant Staphylococcus aureus (MRSA). In this study, we investigated the effect of corilagin and tellimagrandin I on the penicillin binding protein 2′ (2a) (PBP2′ (PBP2a)) which mainly confers the resistance to β‐lactam antibiotics in MRSA. These compounds when added to the culture medium were found to decrease production of the PBP2′ (PBP2a) slightly. Using BOCILLIN FL, a fluorescent‐labeled benzyl penicillin, we found that PBP2′ (PBP2a) in MRSA cells that were grown in medium containing corilagin or tellimagrandin I almost completely lost the ability to bind BOCILLIN FL. The binding activity of PBP2 and PBP3 were also reduced to some extent by these compounds. These results indicate that inactivation of PBPs, especially of PBP2′ (PBP2a), by corilagin or tellimagrandin I is the major reason for the remarkable reduction in the resistance level of β‐lactams in MRSA. Corilagin or tellimagrandin I suppressed the activity of β‐lactamase to some extent.

The effect of γ‐toxin, a thermolysin‐like metalloprotease of Clostridium perfringens, on the inactive ε‐prototoxin produced by the same organism was examined. When the purified ε‐prototoxin was incubated with the purified γ‐toxin at 37 C for 2 hr, the 32.5‐kDa ε‐prototoxin was processed into a 30.5‐kDa polypeptide, as determined by SDS‐polyacrylamide gel electrophoresis. A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD₅₀) of the prototoxin with and without γ‐toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. The lethal activity of the prototoxin activated by γ‐toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD₅₀ of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. The ε‐toxin gene was cloned and sequenced. Determination of the N‐terminal amino acid sequence of each activated ε‐prototoxin revealed that γ‐toxin cleaved between the 10th and 11th amino acid residues from the N‐terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues. The molecular weight of each activated ε‐prototoxin was also determined by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The C‐terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C‐terminus of the prototoxin, suggesting that removal of not only N‐terminal but also C‐terminal peptide is responsible for activation of the prototoxin.

The fecal microbiota of 10 hospitalized elderly subjects and 14 healthy adults were analyzed by terminal‐restriction fragment length polymorphism (T‐RFLP) analysis using HhaI, MspI, HaeIII, and AluI, as well as fecal polyamine (PA) concentration. The T‐RFLP profiles of the fecal microbiota of the subjects were roughly divided into 2 clusters—I (9 out of 11 were derived from hospitalized elderly subjects) and II (12 out of 13 were derived from healthy adults). The average concentration of putrescine in Cluster II was 5.8 times higher than that of putrescine in Cluster I (P=0.0015). Using a phylogenetic assignment database for T‐RFLP analysis of human colonic microbiota, the terminal‐restriction fragments (T‐RFs) characteristically detected in the case of subjects with high fecal PA concentration were predicted to be derived from bacterial species and phylotypes belonging to Clostridium subcluster XIVa, particularly including Clostridium xylanolyticum, Clostridium saccharolyticum, the uncultured human intestinal bacterium clone JW1H4 (a relative of Desulfotomaculum guttoideum), Roseburia intestinalis, the uncultured bacterium clone 41F10 (a relative of Eubacterium ramulus), Roseburia cecicola, Ruminococcus obeum and its relatives. From these results, we concluded that fecal microbiota may be linked with fecal PA concentration and that some bacterial species belonging to Clostridium subcluster XIVa may play a major role in the control of intestinal PA concentration in humans.

Certain Bifidobacterium strains have been shown to inhibit inflammatory responses in intestinal epithelial cells. However, the precise mechanisms of these effects, including the chemical nature of the active compounds, remain to be elucidated. Here partial characterization of the anti‐inflammatory properties of Bifidobacterium strains isolated from feces of healthy infants is reported. It was found that conditioned media (CM) of all strains studied are capable of attenuating tumor necrosis factor‐α (TNF‐α) and lipopolysaccharide‐ (LPS) induced inflammatory responses in the HT‐29 cell line. In contrast, neither killed bifidobacterial cells, nor cell‐free extracts showed such activities. Further investigations resulted in attribution of this activity to heat‐stable, non‐lipophilic compound(s) resistant to protease and nuclease treatments and of molecular weight less than 3  kDa. The anti‐inflammatory effects were dose‐ and time‐dependent and associated with inhibition of IκB phosphorylation and nuclear factor‐κ light chain enhancer of activated B cells (NF‐κB)‐dependent promoter activation. The combined treatments of cells with CMs and either LPS or TNF‐α, but not with CMs alone, resulted in upregulation of transforming growth factor‐β1, IκBζ, and p21^CIP mRNAs. Our data suggest certain species‐specificities of the anti‐inflammatory properties of bifidobacteria. This observation should prompt additional validation studies using larger set of strains and employing the tools of comparative genomics.