Dissection of an ABC transporter LolCDE function analyzed by photo-crosslinking.

The envelope of Escherichia coli contains approximately 100 different species of lipoproteins, most of which are localized to the inner leaflet of the outer membrane. The localization of lipoprotein (Lol) system, consisting of five Lol proteins, is responsible for the trafficking of lipoproteins to the outer membrane. LolCDE binds to lipoproteins destined for the outer membrane and transfers them to the periplasmic chaperone LolA. Although the cryo-EM structures of E. coli LolCDE have been reported, the mechanisms by which outer membrane lipoproteins are transferred to LolA remain elusive. In this study, we investigated the interaction between LolCDE and lipoproteins using site-specific photo-crosslinking. We introduced a photo-crosslinkable amino acid into different locations across the four helices which form the central lipoprotein-binding cavity, and identified domains that crosslink with peptidoglycan-associated lipoprotein (Pal) in vivo. Using one of the derivatives containing the photo-crosslinkable amino acid, we developed an in vitro system to analyze the binding of lipoproteins to LolCDE. Our results indicate that compound 2, a LolCDE inhibitor, does not inhibit the binding of lipoproteins to LolCDE, but rather promotes the dissociation of bound lipoproteins from LolCDE.

An unusual case of fatal hypothermia involving topical diphenhydramine.

PURPOSE: Diphenhydramine is an antihistamine drug widely used to alleviate symptoms caused by allergies and the common cold. Diphenhydramine-involved fatalities have been reported in the past but usually involving overdose by ingestion. We report a peculiar case of fatal hypothermia during non-winter season involving topical diphenhydramine. METHODS: A 23-year-old male with no known preexisting medical conditions was found dead in the bathroom of his apartment with a small amount of running water on his back. Postmortem examinations and toxicological analysis on blood and urine were performed. RESULTS: Color difference was apparent between the right and left cardiac blood. Wischnewski spots were observed in the gastric mucosa. Histological examination revealed no obvious findings that could attribute to serious cardiovascular events. Drug screening by gas chromatograph-tandem mass spectrometry (GC/MS/MS) detected diphenhydramine in blood and urine. Further quantification revealed the postmortem concentrations to be 0.44 µg/mL in blood and 2500 µg/mL in urine. CONCLUSIONS: The cause of death was determined to be hypothermia. Diphenhydramine-induced drowsiness and possible intrinsic cardiac factor may have led to prolonged impaired consciousness, preventing his ability to escape from the running cold water leading to hypothermia and death.

Reversible autoinhibitory regulation of Escherichia coli metallopeptidase BepA for selective ß-barrel protein degradation.

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a ß-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

Structural Basis for the Function of the ß-Barrel Assembly-Enhancing Protease BepA.

The ß-barrel assembly machinery (BAM) complex mediates the assembly of ß-barrel membrane proteins in the outer membrane. BepA, formerly known as YfgC, interacts with the BAM complex and functions as a protease/chaperone for the enhancement of the assembly and/or degradation of ß-barrel membrane proteins. To elucidate the molecular mechanism underlying the dual functions of BepA, its full-length three-dimensional structure is needed. Here, we report the crystal structure of full-length BepA at 2.6-Å resolution. BepA possesses an N-terminal protease domain and a C-terminal tetratricopeptide repeat domain, which interact with each other. Domain cross-linking by structure-guided introduction of disulfide bonds did not affect the activities of BepA in vivo, suggesting that the function of this protein does not involve domain rearrangement. The full-length BepA structure is compatible with the previously proposed docking model of BAM complex and tetratricopeptide repeat domain of BepA.

No Association between the Polymorphism rs6943555 in the AUTS2 Gene and Personality Traits in Japanese University Students.

OBJECTIVE: The autism susceptibility candidate 2 (AUTS2) gene has been implicated in multiple neurological disorders. Several recent studies have revealed that the polymorphism rs6943555 in the AUTS2 gene is broadly associated with human mental function and behavior. Therefore, in the present study we investigated whether the polymorphism rs6943555 is associated with human personality traits in Japanese university students. In addition, our previous study reported that the AUTS2 rs6943555-rs9886351 haplotype is associated with alcohol dependence. As a preliminary analysis, we also examined whether the AUTS2 haplotypes are related to personality traits. METHODS: After written informed consent had been obtained from the participants, two AUTS2 polymorphisms were analyzed, and personality was assessed using the Temperament and Character Inventory (TCI) in 190 university students. In addition, in order to exclude the influence of the results for students with mental health problems, we gave the Patient Health Questionnaire-9 (PHQ-9) to all subjects. RESULTS: In all the subjects, there was a main effect of the polymorphism rs6943555 genotype on reward dependence (p=0.038) and cooperativeness (p=0.031), although the significance was lost on Bonferroni correction. Similarly, on analysis that excluded the subjects with PHQ-9 scores≥10, no significant association with any TCI dimension score among the rs6943555 genotypes was seen. There was no effect of the rs6943555-rs9886351 haplotypes on the TCI dimension scores. CONCLUSION: This study suggests that the polymorphism AUTS2 rs6943555 is not associated with personality traits. Further large-scale studies with more subjects using other self-report questionnaires are needed.

The TPR domain of BepA is required for productive interaction with substrate proteins and the ß-barrel assembly machinery complex.

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N-terminal protease domain and a C-terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone-like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the ß-barrel assembly machinery (BAM) driving integration of ß-barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site-directed in vivo photo-cross-linking was used to map the protein-protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.

Bacterial lipoproteins; biogenesis, sorting and quality control.

Bacterial lipoproteins are a subset of membrane proteins localized on either leaflet of the lipid bilayer. These proteins are anchored to membranes through their N-terminal lipid moiety attached to a conserved Cys. Since the protein moiety of most lipoproteins is hydrophilic, they are expected to play various roles in a hydrophilic environment outside the cytoplasmic membrane. Gram-negative bacteria such as Escherichia coli possess an outer membrane, to which most lipoproteins are sorted. The Lol pathway plays a central role in the sorting of lipoproteins to the outer membrane after lipoprotein precursors are processed to mature forms in the cytoplasmic membrane. Most lipoproteins are anchored to the inner leaflet of the outer membrane with their protein moiety in the periplasm. However, recent studies indicated that some lipoproteins further undergo topology change in the outer membrane, and play critical roles in the biogenesis and quality control of the outer membrane. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Association between AUTS2 haplotypes and alcohol dependence in a Japanese population.

OBJECTIVE: Recent genome-wide analysis has indicated that the autism susceptibility candidate 2 (AUTS2) gene is involved in the regulation of alcohol consumption. We hypothesised that AUTS2 might be associated with the development of alcohol dependence. Therefore, in this exploratory study, we compared the genotype and allele frequencies of the polymorphisms rs6943555 and rs9886351 in the AUTS2 gene between patients with alcohol dependence and healthy control subjects living in a Japanese provincial prefecture. We also examined whether or not the haplotypes consisting of these polymorphisms are related to alcohol dependence. METHODS: The subjects of this study consisted of 64 patients with alcohol dependence and 75 unrelated healthy people. The AUTS2 genotypes were determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. RESULTS: No significant differences in the genotype and allele frequencies of the polymorphisms AUTS2 rs6943555 and rs9886351 were found between alcohol dependence and control subjects. On the other hand, the frequencies of the AUTS2 haplotypes were significantly different between them, and the rs6943555 and rs9886351 A-A haplotype was associated with alcohol dependence (p=0.0187). CONCLUSION: This suggests that the rs6943555 and rs9886351 A-A haplotype might affect the vulnerability to alcohol dependence pathogenesis. Further studies are needed to confirm the reproducibility of the results of this study with increased numbers of subjects.

No Relationships between Psychosis and the Diazepam Binding Inhibitor rs2276596 (C/A) Polymorphism in Japanese: An Exploratory Study.

OBJECTIVE: Our recent study for the first time reported genotyping method of the diazepam binding inhibitor (DBI) rs2276596 polymorphism using a Polymerase Chain Reaction-Restriction Fragment Length Polymor- phism (PCR-RFLP), and revealed a significant relationships between this polymorphism and alcohol depend- ence. In this study, to facilitate elucidation of the pathogeneses of psychoses including schizophrenia and mood (affective) disorders, we investigated the relationship between the DBI rs2276596 polymorphism (C/A) and psychoses. METHOD: We analyzed the DBI genotypes using the PCR-RFLP method in healthy controls, and psychotics including schizophrenia and mood (affective) disorders (including recurrent depressive disorder and bipolar affective disorder) (ICD-10: F31, F33). RESULT: There was no significant difference in the rs2276596 genotype and allele frequencies of the DBI gene between these psychoses and healthy controls. CONCLUSION: The present data suggested that a mutated allele of the DBI was not one of the risk factors for schizophrenia and mood (affective) disorders, as for the rs2276596 polymorphism. [Original].

[Association between GSK3ß polymorphisms and the smoking habits in young Japanese].

Schizophrenia and bipolar disorder show high comorbidity with smoking dependence. Several previous studies reported that glycogen synthase kinase 3ß (GSK3ß), which is widely expressed in the brain including the dopamine projection areas such as the amygdala, nucleus accumbens and hippocampus, may play a role in neuropsychiatric disorders and dopamine- and serotonin-mediated behavior. In this study, we have analyzed the association of three single nucleotide polymorphisms (SNPs) within GSK3ß gene (rs3755557, rs334558, rs6438552) with the smoking habits and age at smoking initiation in a sample of 384 young adult Japanese, which included 172 smokers and 212 non-smokers. As a result, rs334558 was significantly associated with smoking habits in genotype frequency and allelic frequency (P < 0.05). Furthermore, higher haplotype 3 (T-T-T) and haplotype 5 (A-T-C) frequencies were observed in non-smokers than smokers (P < 0.05). Three functional polymorphisms examined in this study reportedly increase transcriptional activity when they have a high-activation allele such as the A allele of -1727A/T (rs3755557), the T allele of -50T/C (rs334558) or T allele of -157T/C (rs6438552). Thus, it was suggested in this study that changes in GSK3ß activity may have an impact on smoking habits.

Analysis of Association between Norepinephrine Transporter Gene Polymorphisms and Personality Traits of NEO-FFI in a Japanese Population.

OBJECTIVE: Norepinephrine is an important chemical messenger that is involved in mood and stress in humans, and is reabsorbed by the norepinephrine transporter (NET). According to Cloninger's theory, the noradrenergic system mediates the personality trait of reward dependence. Thus far, although association studies on NET gene polymorphisms and Cloninger's personality traits have been reported, they yielded inconsistent results. Therefore, in the present study we investigated whether or not the 1287G/A, -182T/C and -3081A/T polymorphisms of the NET gene (SLC6A2) are associated with reward dependence-related traits, as assessed by the five-factor model. METHODS: After written informed consent was obtained from participants, the three NET gene polymorphisms were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), and personality was assessed by the Neuroticism Extraversion Openness-Five Factor Inventory (NEO-FFI) in 270 Japanese university students. RESULTS: A significant relation was found between the -3081A/T functional promoter polymorphism and NEO-FFI scores: those with the T allele exhibited a lower extraversion (E) score than those without the T allele (Mann-Whitney U-test: z=-3.861, p<0.001). However, there was no correlation between the other NET gene polymorphisms and E score, and no association with other dimensions and these three polymorphisms. CONCLUSION: We conclude that the -3081A/T functional polymorphism in the NET gene may affect the extraversion of reward dependence-related traits, as measured by NEO-FFI. However, we used only the shortened version of NEO-PI-R in this study. Further investigations are necessary using the full version of self-rating personality questionnaires.

Activation of Toxin-Antitoxin System Toxins Suppresses Lethality Caused by the Loss of σE in Escherichia coli.

UNLABELLED: σ(E), an alternative σ factor that governs a major signaling pathway in envelope stress responses in Gram-negative bacteria, is essential for growth of Escherichia coli not only under stressful conditions, such as elevated temperature, but also under normal laboratory conditions. A mutational inactivation of the hicB gene has been reported to suppress the lethality caused by the loss of σ(E). hicB encodes the antitoxin of the HicA-HicB toxin-antitoxin (TA) system; overexpression of the HicA toxin, which exhibits mRNA interferase activity, causes cleavage of mRNAs and an arrest of cell growth, while simultaneous expression of HicB neutralizes the toxic effects of overproduced HicA. To date, however, how the loss of HicB rescues the cell lethality in the absence of σ(E) and, more specifically, whether HicA is involved in this process remain unknown. Here we showed that simultaneous disruption of hicA abolished suppression of the σ(E) essentiality in the absence of hicB, while ectopic expression of wild-type HicA, but not that of its mutant forms without mRNA interferase activity, restored the suppression. Furthermore, HicA and two other mRNA interferase toxins, HigB and YafQ, suppressed the σ(E) essentiality even in the presence of chromosomally encoded cognate antitoxins when these toxins were overexpressed individually. Interestingly, when the growth media were supplemented with low levels of antibiotics that are known to activate toxins, E. coli cells with no suppressor mutations grew independently of σ(E). Taken together, our results indicate that the activation of TA system toxins can suppress the σ(E) essentiality and affect the extracytoplasmic stress responses. IMPORTANCE: σ(E) is an alternative σ factor involved in extracytoplasmic stress responses. Unlike other alternative σ factors, σ(E) is indispensable for the survival of E. coli even under unstressed conditions, although the exact reason for its essentiality remains unknown. Toxin-antitoxin (TA) systems are widely distributed in prokaryotes and are composed of two adjacent genes, encoding a toxin that exerts harmful effects on the toxin-producing bacterium itself and an antitoxin that neutralizes the cognate toxin. Curiously, it is known that inactivation of an antitoxin rescues the σ(E) essentiality, suggesting a connection between TA systems and σ(E) function. We demonstrate here that toxin activation is necessary for this rescue and suggest the possible involvement of TA systems in extracytoplasmic stress responses.

Novel antibacterial targets and compounds revealed by a high-throughput cell wall reporter assay.

UNLABELLED: A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments with E. coli spheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy. IMPORTANCE: The prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targeting Escherichia coli outer membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.

Small-molecule inhibitors of gram-negative lipoprotein trafficking discovered by phenotypic screening.

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.

Psychological Evaluation of Animal-assisted Intervention (AAI) Programs Involving Visiting Dogs and Cats for Alcohol Dependents: A Pilot Study.

The purpose of this study was to develop an evaluation method for animal-assisted intervention (AAI) programs involving Mood Check List-Short form.2 (MCL-S.2) and the State-Trait Anxiety Inventory (STAI) for psychiatric daycare of Japanese alcohol. dependents. A total of 36 alcohol dependents completed the study and questionnaires assessing their state. A single session of AAI reduced both subjective and physiological measures of state anxiety (A-State); and this program induced a significant reduction in the anxiety after an AAI program session with the dogs and cats involved in the intervention (p = 0.001). The Wilcoxon t-test showed that there were also significant differences in the "anxiety", "pleasantness", and "relaxation". scores for MCL-S.2 among the alcohol dependents, before and after AAI; a significantly decreased "anxiety" score (p = 0.006), and increased "pleasantness" (p = 0.002) and "relaxation" (p=0.012) scores for MCL-S.2 after AAI. The results of this study indicated that alcohol dependents who experienced a group AAI session-program exhibited significant improvements in their feeling; decreased anxiety, and increased pleasantness and relaxation.

Haplotype analysis of GSK-3ß gene polymorphisms in bipolar disorder lithium responders and nonresponders.

The GSK-3ß gene, GSK3B, codes for an enzyme that is a target for the action of mood stabilizers, lithium and possibly valproic acid.In this study, the relationship between haplotypes consisting of single nucleotide polymorphisms (SNPs) of GSK3B -50T/C and -1727A/T and the effect of lithium was studied among Japanese bipolar disorder lithium nonresponders and responders.The distributions of the GSK3B haplotypes (-50T/C and -1727A/T) showed a trend for significant difference between the lithium nonresponders and responders (global P=0.07074). Haplotype 1 (T-A) was associated with a higher lithium response (haplotype-specific P=0.03477), whereas haplotype 2 (C-A) was associated with a lower lithium response (haplotype-specific P=0.03443).The pairwise D' and r values between the 2 SNPs in this study were 1.0 and 0.097, respectively. The 2 SNPs showed weak linkage disequilibrium with each other.

Relationship between alcohol dependence and the DBI rs2276596 (C/A) polymorphism in Japanese.

To facilitate elucidation of the pathogenesis of alcohol dependence, we investigated the relationship between a genetic variant of diazepam biding inhibitor (DBI) C/A polymorphism (rs2276596) and alcohol dependence. We determined the DBI genotypes using a novel method involving PCR-RFLP in healthy controls and alcoholics with a diagnosis of alcohol dependence by ICD-10 (F10.20). There was a significant difference in the rs2276596 polymorphism C/A allele frequency of the DBI gene (P < 0.0001) between alcoholics and healthy controls. The present data suggested that a mutant allele of the DBI was one of the risk factors for alcohol dependence as for the rs2276596 polymorphism.

Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.