Effects of fermentation products of the commensal bacterium Clostridium ramosum on motility, intracellular pH, and flagellar synthesis of enterohemorrhagic Escherichia coli.

The flagellum and motility are crucial virulence factors for many pathogenic bacteria. In general, pathogens invade and translocate through motility and adhere to specific tissue via flagella. Therefore, the motility and flagella of pathogens are effectual targets for attenuation. Here, we show that the fermentation products of Clostridium ramosum, a commensal intestinal bacterium, decrease the intracellular pH of enterohemorrhagic Escherichia coli (EHEC) and influence its swimming motility. Quantifications of flagellar rotation in individual EHEC cells showed an increase in reversal frequency and a decrease in rotation rate in the presence of C. ramosum fermentation products. Furthermore, the C. ramosum fermentation products affected synthesis of flagellar filaments. The results were reproduced by a combination of organic acids under acidic conditions. Short-chain fatty acids produced by microbes in the gut flora are beneficial for the host, e.g. they prevent infection. Thus, C. ramosum could affect the physiologies of other enteric microbes and host tissues.

Detection of Clostridium perfringens in tsunami deposits after the Great East Japan Earthquake.

The Great East Japan Earthquake struck off the Tohoku and caused a tsunami in 2011. Most of the microbial characteristics of tsunami-affected soil remain unknown and no published study has shown how a tsunami affects the risk of infection by Clostridium perfringens living in soil. In 2011 and 2015, C. perfringens was assessed in deposits in soil from tsunami-damaged areas and undamaged areas of Miyagi. It was found that the number of C. perfringens was overwhelmingly greater in 2011 than in 2015 in the tsunami-damaged areas. According to real-time PCR, the prevalence C. perfringens organisms (%) was 103 fold greater in the damaged than in the undamaged areas.

Salmonella Typhimurium is Attracted to Egg Yolk and Repelled by Albumen.

Salmonella Typhimurium is the causative agent of non-typhoidal, foodborne salmonellosis. Contamination of hen eggs by the bacterium is a common source of S. Typhimurium infection. S. Typhimurium is peritrichous, and flagellum-dependent motility and chemotaxis are believed to facilitate egg contamination despite the presence of many antimicrobial egg components. We performed motility and chemotaxis assays to demonstrate that S. Typhimurium cells are attracted to egg yolks and are repelled by albumen. The bacterial flagellar motor shows bidirectional rotation, and counterclockwise-biased rotation allows cells to swim smoothly. A rotation assay for a single flagellum showed that, in comparison with thin albumen, the thick albumen more strongly affected the directional bias of the flagellar rotation, resulting in a remarkable suppression of the migration distance. Nevertheless, the S. Typhimurium cells retained positive chemotaxis toward the yolk in the presence of the albumens, suggesting that motility facilitates the growth of S. Typhimurium and survival in eggs.

Gastrointestinal colonisation and systemic spread of Candida albicans in mice treated with antibiotics and prednisolone.

Normally, Candida albicans is a commensal microbe that resides in the human oral cavity, gut and vagina. However, the fungus can cause mucosal and systemic infections in immunocompromised individuals. The mechanism by which local mucosal infections progress to systemic candidiasis is poorly understood. Here, a murine model of gastrointestinal (GI) candidiasis was developed by inoculation of the oral cavity, followed by treatment with tetracycline (TC) and prednisolone (PSL). Temporal progression from a local infection of the oral cavity to a systemic infection was then monitored. Histological analysis of tissues from mice treated with both TC and PSL revealed massive infiltration of the tongue and stomach by hyphae. PSL increased the fungal burden in the tongue, stomach and small intestine, and facilitated dissemination to the spleen, kidney and liver within 3 days post-infection. Treatment with both TC and PSL supressed interferon (IFN)-γ and interleukin (IL)-17 (cytokines that play key roles in host defence against fungal infection) levels in the tongue, which were induced by C. albicans infection. In addition, the mucosal layer of the small intestine of mice treated with both TC and PSL was almost destroyed by the fungal infection; this may be a critical event that allows passage of the fungus across the mucosa and into the systemic circulation. Thus, this mouse model is useful for studying mechanisms underlying progression of C. albicans from a local infection of the oral cavity to a systemic infection in immunocompromised individuals.

miR-663a regulates growth of colon cancer cells, after administration of antimicrobial peptides, by targeting CXCR4-p21 pathway.

BACKGROUND: Antimicrobial peptides (AMPs) play important roles in the innate immune system of all life forms and recently have been characterized as multifunctional peptides that have a variety of biological roles such as anticancer agents. However, detailed mechanism of antimicrobial peptides on cancer cells is still largely unknown. METHODS: miRNA array and real-time qPCR were performed to reveal the behavior of miRNA in colon cancer HCT116 cells during the growth suppression induced by the AMPs. Establishment of miR-663a over-expressing HCT116 cells was carried out for the evaluation of growth both in vitro and in vivo. To identify the molecular mechanisms, we used western blotting analysis. RESULTS: miR-663a is upregulated by administration of the human cathelicidin AMP, LL-37, and its analogue peptide, FF/CAP18, in the colon cancer cell line HCT116. Over-expression of miR-663a caused anti-proliferative effects both in vitro and in vivo. We also provide evidence supporting the view that these effects are attributed to suppression of the expression of the chemokine receptor CXCR4, resulting in the abrogation of phosphorylation of Akt and cell cycle arrest in G2/M via p21 activation. CONCLUSIONS: This study contributes to the understanding of the AMPs' mediated anti-cancer mechanisms in colon cancer cells and highlights the possibility of using AMPs and miRNAs towards developing future strategies for cancer therapy.

Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli on the L-alanine export.

The Escherichia coli alaE gene encodes the L-alanine exporter, AlaE, that catalyzes active export of L-alanine using proton electrochemical potential. The transporter comprises only 149 amino acid residues and four predicted transmembrane domains (TMs), which contain three charged amino acid residues. The AlaE-deficient L-alanine non-metabolizing cells (ΔalaE cells) appeared hypersusceptible to L-alanyl-L-alanine showing a minimum inhibitory concentration (MIC) of 2.5 µg/ml for the dipeptide due to a toxic accumulation of L-alanine. To elucidate the mechanism by which AlaE exports L-alanine, we replaced charged amino acid residues in the TMs, glutamic acid-30 (TM-I), arginine-45 (TM-II), and aspartic acid-84 (TM-III) with their respective charge-conserved amino acid or a net neutral cysteine. The ΔalaE cells producing R45K or R45C appeared hypersusceptible to the dipeptide, indicating that arginine-45 is essential for AlaE activity. MIC of the dipeptide in the ΔalaE cells expressing E30D and E30C was 156 µg/ml and >10,000 µg/ml, respectively, thereby suggesting that a negative charge at this position is not essential. The ΔalaE cells expressing D84E or D84C showed an MIC >10,000 and 78 µg/ml, respectively, implying that a negative charge is required at this position. These results were generally consistent with that of the L-alanine accumulation experiments in intact cells. We therefore concluded that charged amino acid residues (R45 and D84) in the AlaE transmembrane domain play a pivotal role in L-alanine export. Replacement of three cysteine residues at C22, C28 (both in TM-I), and C135 (C-terminal region) with alanine showed only a marginal effect on L-alanine export.

Gene expression analyses of the small intestine of pigs in the ex-evacuation zone of the Fukushima Daiichi Nuclear Power Plant.

BACKGROUND: After the accident at the Fukushima Daiichi Nuclear Power Plant, radioactive contaminants were released over a widespread area. Monitoring the biological effects of radiation exposure in animals in the ex-evacuation zone should be continued to understand the health effects of radiation exposure in humans. The present study aimed to clarify the effects of radiation by investigating whether there is any alteration in the morphology and gene expressions of immune molecules in the intestine of pigs and inobuta (wild boar and domestic pig hybrid) in the ex-evacuation zone in 2012. Gene expression analysis was performed in small intestine samples from pigs, which were collected from January to February 2012, in the ex-evacuation zone. Pigs lived freely in this zone, and their small intestine was considered to be affected by the dietary intake of radioactive contaminants. RESULTS: Several genes were selected by microarray analysis for further investigation using real-time polymerase chain reaction. IFN-γ, which is an important inflammatory cytokine, and TLR3, which is a pattern recognize receptor for innate immune system genes, were highly elevated in these pigs. The expressions of the genes of these proteins were associated with the radiation level in the muscles. We also examined the alteration of gene expressions in wild boars 5 years after the disaster. The expression of IFN-γ and TLR3 remained high, and that of Cyclin G1, which is important in the cell cycle, was elevated. CONCLUSIONS: We demonstrated that some changes in gene expression occurred in the small intestine of animals in the ex-evacuation zone after radiation. It is difficult to conclude that these alterations are caused by only artificial radionuclides from the Fukushima Daiichi Nuclear Power Plant. However, the animals in the ex-evacuation zone might have experienced some changes owing to radioactive materials, including contaminated soil, small animals, and insects. We need to continue monitoring the effects of long-term radiation exposure in living things.

Level of perception of technical terms regarding the effect of radiation on the human body by residents of Japan.

BACKGROUND: This study aimed to examine the level of perception of the technical terms related to the effect of radiation on the human body among residents of the six prefectures of Miyagi, Fukushima, Tokyo, Aichi, Hiroshima, and Nagasaki in Japan. Miyagi and Fukushima were selected as devastated area by Great East Japan Earthquake. Tokyo and Aichi were selected as control. Hiroshima and Nagasaki were selected as the A-bombed area. METHODS: A total of 1030 respondents, 172, 173, 171, 173, 171, and 170, respectively, were surveyed. Differences in the recognition level of technical terms related to the effect of radiation on the human body among residents of the six prefectures were assessed. RESULTS: The highest recognition levels were reported by the respondents from Fukushima (17 items). Those from Miyagi scored the second highest recognition levels (10 out of the 17 terms); the second highest recognition levels for the remaining seven terms were marked by the respondents of Tokyo. Respondents in the Tohoku region had a better recognition for the technical terminology relevant to the effect of radiation on the human body. CONCLUSIONS: Our findings indicate a need for continued, comprehensive risk communication pertaining to health hazards of radiation exposure in Tohoku region. Concerted efforts by central/local governments and other stakeholders are required to allay the anxiety/stress related to radiation exposure among the residents.

Secretion of d-alanine by Escherichia coli.

Escherichia coli has an l-alanine export system that protects the cells from toxic accumulation of intracellular l-alanine in the presence of l-alanyl-l-alanine (l-Ala-l-Ala). When a DadA-deficient strain was incubated with 6.0 mM l-Ala-l-Ala, we detected l-alanine and d-alanine using high-performance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with d-amino acid oxidase resulted in the disappearance of a signal corresponding to d-alanine. Additionally, the culture supernatant enabled a d-alanine auxotroph to grow without d-alanine supplementation, confirming that the signal detected by HPLC was authentic d-alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, alr or dadX, the extracellular level of d-alanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow from l-alanine to d-alanine enhanced d-alanine secretion. When high-density DadA-deficient cells preloaded with l-Ala-l-Ala were treated with 20 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), secretion of both l-alanine and d-alanine was enhanced ~twofold compared with that in cells without CCCP treatment. In contrast, the ATPase inhibitor dicyclohexylcarbodiimide did not exert such an effect on the l-alanine and d-alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the l-alanine exporter AlaE accumulated [3H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [3H]D-alanine was strongly inhibited by CCCP. These results indicate that E. coli has a transport system(s) that exports d-alanine and that this function is most likely modulated by proton electrochemical potential.

Establishment of cell lines derived from the genus Macaca through controlled expression of cell cycle regulators.

Nonhuman primates are useful animal models for the study of human diseases. However, the number of established cell lines from nonhuman primates is quite limited compared with the number established from other experimental animals. The establishment of nonhuman primate cell lines would allow drug testing on those cell lines before moving experiments into primates. In this study, we established nonhuman primate primary cell lines by introducing the genes for CDK4R24C, cyclin D1, and hTERT. These cell lines proliferated more rapidly than primary cells and bypassed cellular senescence. Karyotype analysis showed that the chromosome patterns were intact in the immortalized cell lines. Furthermore, we showed that the expression of introduced genes could be precisely controlled through the Tet-Off system with the addition of doxycycline. The present study shows that introduction of the CDK4R24C, cyclin D1, and hTERT genes are effective methods of establishing nonhuman primate cell lines.

Identification and characterization of common B cell epitope in bovine leukemia virus via high-throughput peptide screening system in infected cattle.

BACKGROUND: Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle. BLV is closely related to human T cell leukemia virus. B cell epitopes are important for the use of antibodies as therapeutic agents, the epitope-driven vaccine design, and immunological assays. A common B cell epitope for BLV has not yet been found due to individual differences in disease susceptibility. RESULTS: We used a peptide microarray with 156 synthetic 15-mer peptides covering the envelope glycoprotein gp51 and the Gag proteins p15, p24, and p12 to map B cell epitope and one B cell epitope, gp51p16, was recognized by all four cattle experimentally infected with BLV. A newly developed high-throughput peptide ELISA system revealed 590 (91.2%) of 647 cattle naturally infected with BLV, carrying 25 different bovine leukocyte antigen class II DRB3 (BoLA-DRB3) alleles, responded to a 20-mer gp51p16-C peptide containing a C-terminal cysteine and gp51p16. Alanine mutation and comparison of the sequences at 17 amino acid positions within gp51p16-C revealed that R7, R9, F10, V16, and Y18 were the common binding sites to BLV antibodies, and two of these sites were found to be highly conserved. Transient expression in the cells of five infectious molecular clones of BLV with a single alanine mutation at five common antibody binding sites had no effect syncytia formation of the gp51 protein. In addition, the mutant proteins, R7A and R9A had no effect on the expression of gp51 protein; the gp51 protein expressions of F10A, V16A and Y18A were lower than that of the wild type protein. CONCLUSIONS: This is the first report to identify a common B cell epitope in BLV by comprehensive screening of BLV-infected cattle with varied genetic backgrounds in BoLA-DRB3. Our results have important implications for disease control and diagnosis.

Development of a novel antimicrobial screening system targeting the pyoverdine-mediated iron acquisition system and xenobiotic efflux pumps.

The iron acquisition systems in Pseudomonas aeruginosa are inducible in response to low-iron conditions and important for growth of this organism under iron limitation. OprM is the essential outer membrane subunit of the MexAB-OprM xenobiotic efflux pump. We designed and constructed a new model antimicrobial screening system targeting both the iron-uptake system and xenobiotic efflux pumps. The oprM gene was placed immediately downstream of the ferri-pyoverdine receptor gene, fpvA, in the host lacking chromosomal oprM and the expression of oprM was monitored by an antibiotic susceptibility test under iron depleted and replete conditions. The recombinant cells showed wild-type susceptibility to pump substrate antibiotics, e.g., aztreonam, under iron limitation and became supersusceptible to them under iron repletion, suggesting that expression of oprM is under control of the iron acquisition system. Upon screening of a chemical library comprising 2952 compounds using this strain, a compound-ethyl 2-(1-acetylpiperidine-4-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate-was found to enhance the efficacy of aztreonam under iron limitation, suggesting that the compound inhibits either the iron acquisition system or the MexAB-OprM efflux pump. This compound was subsequently found to inhibit the growth of wild-type cells in the presence of sublethal amounts of aztreonam, regardless of the presence or absence of dipyridyl, an iron-chelator. The compound was eventually identified to block the function of the MexAB-OprM efflux pump, showing the validity of this new method.

Interaction between Leptospiral Lipopolysaccharide and Toll-like Receptor 2 in Pig Fibroblast Cell Line, and Inhibitory Effect of Antibody against Leptospiral Lipopolysaccharide on Interaction.

Leptospiral lipopolysaccharide (L-LPS) has shown potency in activating toll-like receptor 2 (TLR2) in pig fibroblasts (PEFs_NCC1), and causes the expression of proinflammatory cytokines. However, the stimulation by L-LPS was weak eliciting the function of TLR2 sufficiently in pig innate immunity responses during Leptospira infection. In this study, the immune response of pig embryonic fibroblast cell line (PEFs_SV40) was investigated and was found to be the high immune response, thus TLR2 is the predominate receptor of L-LPS in pig cells. Further, we found a strategy using the antibody against L-LPS, to prevent L-LPS interaction with TLR2 in pig cells which could impact on immune activation.

Ceragenin CSA-13 induces cell cycle arrest and antiproliferative effects in wild-type and p53 null mutant HCT116 colon cancer cells.

Antimicrobial peptides of the cathelicidin family play a central role in the host defense system. Our group has reported previously that cathelicidin-related or cathelicidin-modified antimicrobial peptides, such as FF/CAP-18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1 and colon cancer-derived cell line HCT116. Ceragenin CSA-13, which mimics the hydrophobic and cationic morphology of cathelicidin-related peptides, was developed to reduce synthetic costs and resolve stability issues in the presence of proteases. In this study, we evaluated the antiproliferative effect of CSA-13 on HCT116 cells. We evaluated the effects of CSA-13 in HCT116 cells by measuring cell growth, detecting apoptosis, analyzing the cell cycle, and examining mitochondrial membrane depolarization. Treatment with CSA-13 suppressed HCT116 cell proliferation in a dose-dependent manner, increasing the incidence of apoptosis detected by the binding of Annexin V. Furthermore, cell cycle analysis showed that the cell cycle of CSA-13-treated wild-type and p53 null mutant HCT116 cells was arrested at the G1/S phase, indicating that CSA-13 affects the cell cycle by a p53-independent pathway. Our study showed that CSA-13 exerts an antiproliferative effect in cancer cells similar to that of FF/CAP-18, suggesting that membrane-permeabilizing capability is the common underlying mechanism for anticancer and antimicrobial effects of CSA-13 and anitimicrobial peptides.

Frequent detection of 'azole' resistant Candida species among late presenting AIDS patients in northwest Ethiopia.

BACKGROUND: The chronic use of antifungal agents in the treatment of fungal infection in general and oropharyngeal candidiasis mainly in AIDS patient's leads to the selection of strain resistant to these therapies and a shift in the spectrum of Candida species. This study determines the species diversity and in vitro susceptibility of Candida isolates from late presenting AIDS patients in northwest Ethiopia. METHODS: Two hundred and twenty one HIV/AIDS patients were assessed with a standardized evaluation form at enrolment. Oral rinses were cultured on CHROMagar plates at 37°C for 48 hours and Candida species identification were made following standard microbiological techniques. In vitro drug susceptibility tests were made using broth microdilution method. RESULTS: The colonization rate of Candida species was found to be 82.3% (177/215). C. albicans was the predominant species isolated from 139 (81%) patients but there was a diversity of other species. C. glabrata was the most frequent non-albicans species isolated in 22.5% (40/177) of the patients followed by C. tropicalis 14.1% (27/177), C. krusei 5.6% (10) and other unidentifiable Candida species 4% (7/177). Recurrent episodes of oropharyngeal candidiasis and previous exposure to antifungal drugs were found to be predisposing factors for colonization by non-albicans species. Irrespective of the Candida species identified 12.2% (11/90), 7.7% (7/90) and 4.7% (4) of the isolates were resistant to fluconazole, ketoconazole and itraconazole, respectively. In contrast, resistance to micafungin, amphotericin B and 5-Fluorocytosine was infrequent. CONCLUSION: HIV/AIDS patients are orally colonized by single or multiple albicans and non- albicans Candida species that are frequently resistant to azoles and occasionally to amphotericin B, 5-Fluorocytosine and micafungin. These highlight the need for national surveillance for examining Candida epidemiology and resistance to antifungal drugs.

The inhibition effect of antiserum on the motility of Leptospira.

Leptospires are a group of bacteria with a unique ultrastructure and a fascinating swimming behavior that cause a number of emerging and re-emerging diseases worldwide called leptospirosis. The unusual form of motility is thought to play a critical role in the infection process. However, the inhibition mechanism of antiserum on the motility of Leptospira to attenuate the infection efficiency is unknown. In this study, effect of antiserum on motility was quantitatively investigated by swimming speed. Relatively low concentration of antiserum was found to inhibit leptospiral motility, suggesting that the basic immunization can affect the infection efficiency. Recovery of motility a few hours later after the addition of antiserum was observed. This raises a hypothesis that Leptospira carries surface molecules bound with antibodies toward the cell end to escape and recovers the motility.

Identification of hepatitis E virus in wild sika deer in Japan.

Hepatitis E virus (HEV) is a zoonotic agent mainly transmitted through the consumption of uncooked or undercooked meat products derived from infected animals. In Japan, domestic pigs and wild boars are the major animal reservoirs, and whether or not deer are an HEV reservoir remains controversial. We analyzed 395 serum and 199 liver samples from 405 sika deer (Cervus nippon) caught in the wild between 1997 and 2020 in 11 prefectures of Japan for markers of HEV infection. Overall, 17 deer had anti-HEV IgG (4.3%), while 1 (0.2%) had HEV RNA (genotype 3b), indicating the occurrence of ongoing HEV infection in wild deer in Japan. An analysis of the complete HEV genome (deJOI_14) recovered from a viremic deer in Oita Prefecture revealed only 88.8% identity with the first HEV strain in sika deer (JDEER-Hyo03L) in Japan, being closest (96.3%) to the HEV obtained from a hepatitis patient living in the same prefecture. Of note, the deJOI_14 strain was 8.7-9.0% different from the wild boar HEV strains obtained in the same habitat and the same year, suggesting that difference in infected HEV strains between boar and deer may be explained by the limited possibility of close contact with each other, although boars are a known source of HEV infection. Increased numbers of hepatitis E cases after consumption of raw or undercooked meat products of wild deer have been reported in Japan. These results suggest a low but nonnegligible zoonotic risk of HEV infection in wild deer in this country.

Inducible L-alanine exporter encoded by the novel gene ygaW (alaE) in Escherichia coli.

We previously isolated a mutant hypersensitive to L-alanyl-L-alanine from a non-L-alanine-metabolizing Escherichia coli strain and found that it lacked an inducible l-alanine export system. Consequently, this mutant showed a significant accumulation of intracellular L-alanine and a reduction in the L-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes, ygaW and ytfF, and two characterized genes, yddG and yeaS, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellular l-alanine level and enhancement of the L-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters of L-alanine. Since ygaW exhibited the most striking impact on both the intra- and the extracellular L-alanine levels among the four genes identified, we disrupted the ygaW gene in the non-L-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellular L-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only the ygaW gene conferred on the cells the ability to excrete alanine. In addition, expression of the ygaW gene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter for L-alanine in E. coli and proposed that the gene be redesignated alaE for alanine export.

Isolation of a mutant auxotrophic for L-alanine and identification of three major aminotransferases that synthesize L-alanine in Escherichia coli.

For Escherichia coli, it has been assumed that L-alanine is synthesized by alanine-valine transaminase (AvtA) in conjunction with an unknown alanine aminotransferase(s). We isolated alanine auxotrophs from a prototrophic double mutant deficient in AvtA and YfbQ, a novel alanine aminotransferase, by chemical mutagenesis. A shotgun cloning experiment identified two genes, uncharacterized yfdZ and serC, that complemented the alanine auxotrophy. When the yfdZ- or serC-mutation was introduced into the double mutant, one triple mutant (avtA yfbQ yfdZ) showed alanine auxotrophy, and another (avtA yfbQ serC), prototrophy. In addition, we found that four independent alanine auxotrophs possessed a point mutation in yfdZ but not in serC. We also found that yfdZ expression was induced in minimal medium. Furthermore, yfbQ-bearing plasmid conferred the ability to excrete alanine on the mutant lacking D-amino acid dehydrogenase-encoding gene, dadA. From these results, we concluded that E. coli synthesizes L-alanine by means of three aminotransferases, YfbQ, YfdZ, and AvtA.

Tertiary structure-related activity of tick defensin (persulcatusin) in the taiga tick, Ixodes persulcatus.

Defensins are small cysteine-rich cationic proteins found in both vertebrates and invertebrates constituting the front line of host innate immunity. To examine the importance of the tertiary structure of tick defensin in its antimicrobial activity, we synthesized two types of the peptides with tertiary structure or primary one on basis of the information of the sequence in the defensin originated from the taiga tick, Ixodes persulcatus. Chemically synthesized peptides were used to investigate the activity spectrum against Staphylococcus aureus, Borrelia garinii and flora-associated bacteria. Both synthetic peptides showed antimicrobial activity against S. aureus in short-time killing within 1 h, but they do not show the activity against B. garinii, Stenotrophomonas maltophila and Bacillus spp., which were frequently isolated from the midgut of I. persulcatus. The teriary structure brought more potent activity to S. aureus than primary one in short-time killing. We also examined its antimicrobial activity by evaluation of growth inhibition in the presence of the synthetic peptides. Minimum inhibitory concentration (MIC) was ranged from 1.2 to 5.0 µg/ml in tertiary peptide and from 10 to 40 µg/ml in primary peptide, when 10 strains of S. aureus were used. From the curve of cumulative inhibition rates, MIC50 (MIC which half of the strains showed) to S. aureus is about 1.2 µg/ml in the peptide with tertiary structure and about 10 µg/ml in the linear one. Corynebacterium renale is 10 times or more sensitive to tertiary peptide than primary one. In conclusion, the presence of 3 disulfide bridges, which stabilize the molecule and maintain the tertiary structure, is considered to have an effect on their antimicrobial activities against Gram-positive bacteria such as S. aureus.