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Pyroglutamate-3 amyloid-β (pGlu3 Aβ) is an N-terminally modified, toxic form of amyloid-β that is present in cerebral amyloid plaques and vascular deposits. Using the Fc-competent murine anti-pGlu3 Aβ monoclonal antibody (mAb), 07/2a, we present here a nonpharmacological approach using focused ultrasound (FUS) with intravenous (i.v.) injection of microbubbles (MB) to facilitate i.v. delivery of the 07/2a mAb across the blood brain barrier (BBB) in order to improve Aβ removal and restore memory in aged APP/PS1 mice, an Alzheimer’s disease (AD)-like model of amyloidogenesis.Compared to sham-treated controls, aged APP/PS1 mice treated with 07/2a immediately prior to FUS-mediated BBB disruption (mAb + FUS-BBBD combination treatment) showed significantly better spatial learning and memory in the Water T Maze. FUS-BBBD treatment alone improved contextual fear learning and memory in aged WT and APP/PS1 mice, respectively. APP/PS1 mice given the combination treatment had reduced Aβ42 and pGlu3 Aβ hippocampal plaque burden compared to PBS-treated APP/PS1 mice.Hippocampal synaptic puncta density and synaptosomal synaptic protein levels were also higher in APP/PS1 mice treated with 07/2a just prior to BBB disruption. Increased Iba-1+ microglia were observed in the hippocampi of AD mice treated with 07/2a with and without FUS-BBBD, and APP/PS1 mice that received hippocampal BBB disruption and 07/2a showed increased Ly6G+ monocytes in hippocampal CA3. FUS-induced BBB disruption did not increase the incidence of microhemorrhage in mice with or without 07/2a mAb treatment.Our findings suggest that FUS is useful tool that may enhance delivery of an anti-pGlu3 Aβ mAb for immunotherapy. FUS-mediated BBB disruption in combination with the 07/2a mAb also appears to facilitate monocyte infiltration in this AD model. Overall, these effects resulted in greater sparing of synapses and improved cognitive function without causing overt damage, suggesting the possibility of FUS as a noninvasive method to increase the therapeutic efficacy in AD patients.

Pyroglutamate-3 amyloid-β (pGlu3 Aβ) is an N-terminally modified, pathogenic form of amyloid-β that is present in cerebral amyloid plaques and vascular deposits. Here, for the first time, wee used focused ultrasound (FUS) with microbubbles to enhance the intravenous delivery of an Fc-competent anti-pGlu3 Aβ monoclonal antibody, 07/2a mAb, across the blood brain barrier (BBB) in an attempt to improve Aβ removal and memory in aged APP/PS1dE9 mice, an Alzheimer’s disease-like model of amyloidogenesis. First, we demonstrated that bilateral hippocampal FUS-BBBD led to a 5.5-fold increase of 07/2a mAb in the brains of sonicated aged APP/PS1dE9 mice compared to non-sonicated mice 72 hours following a single treatment. Then, we determined that three weekly treatments with 07/2a mAb alone improved spatial learning and memory in aged, plaque-rich APP/PS1dE9 mice, and that this improvement occurred faster and in a higher percentage of animals when combined with FUS-BBBD. APP/PS1dE9 mice given the combination treatment had reduced hippocampal plaque burden compared to PBS-treated APP/PS1dE9 mice. Furthermore, synaptic protein levels were higher in hippocampal synaptosomes from APP/PS1 mice given the combination treatment compared to sham controls, and there were more CA3 synaptic puncta labeled in the APP/PS1dE9 mice given the combination treatment compared to those given mAb alone. Plaque-associated microglia were present in the hippocampi of APP/PS1dE9 mice treated with 07/2a mAb with and without FUS-BBBD. However, we discovered that plaque-associated Ly6G+ monocytes were only present in the hippocampi of APP/PS1dE9 mice that were given FUS-BBBD alone or even more so, the combination treatment. Lastly, FUS- BBBD did not increase the incidence of microhemorrhage in mice with or without 07/2a mAb treatment. Our findings suggest that FUS is a useful tool to enhance delivery and efficacy of an anti-pGlu3 Aβ mAb for immunotherapy either via an additive effect or an independent mechanism. We revealed a potential novel mechanism wherein the combination of 07/2a mAb with FUS-BBBD led to greater monocyte infiltration and recruitment to plaques in this AD-like model. Overall, these effects resulted in greater plaque removal, sparing of synapses and improved cognitive function without causing overt damage, suggesting the possibility of FUS-BBBD as a noninvasive method to increase the therapeutic efficacy of drugs or biologics in AD patients.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P.��gingivalis (PgQC) and T.��forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ��-sheet surrounded by seven ��-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P.��gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.

Periodontitis is a severe yet underestimated oral disease. Since it is linked to several systemic diseases, such as diabetes, artheriosclerosis, and even Alzheimer’s disease, growing interest in treating periodontitis has emerged recently. The major cause of periodontitis is a shift in the oral microbiome. A keystone pathogen that is associated with this shift is Porphyromonas gingivalis. Hence, targeting P. gingivalis came into focus of drug discovery for the development of novel antiinfective compounds. Among others, glutaminyl cyclases (QCs) of oral pathogens might be promising drug targets. Here, we report the discovery and structure–activity relationship of a novel class of P. gingivalis QC inhibitors according to a tetrahydroimidazo[4,5-c]pyridine scaffold. Some compounds exhibited activity in the lower nanomolar range and thus were further characterized with regard to their selectivity and toxicity.

Objectives Human glutaminyl cyclases (QC and isoQC) play an important role in maintaining inflammatory conditions. Meanwhile a glutaminyl cyclase synthesized by Porphyromonas gingivalis (PgQC), a key pathogen in developing periodontitis and a potential link of periodontitis with rheumatoid arthritis (RA), was discovered. This study was aimed to determine the expression of QC, isoQC and PgQC in patients with chronic periodontitis (CP) and RA. Design Thirty volunteers were enrolled in a pilot study and divided into 3 groups (healthy, CP and RA individuals). Blood samples, biofilm and gingival crevicular fluid (GCF) were analysed for mRNA expression of QC, isoQC and P. gingivalis QC. Major bacteria being associated with periodontal disease were quantified in subgingival biofilm and protein levels for monocyte chemoattractant protein (MCP)-1, MCP-3 and interleukin (IL)-1β) were determined in the GCF. Expression of PgQC on the mRNA and protein levels was assessed in two P. gingivalis strains. Results PgQC is expressed in P. gingivalis strains and the protein seems to be located mainly in peri-plasmatic space. mRNA expression of QC was significantly increased in the peripheral blood from RA patients vs. healthy subjects and CP patients (p = 0.013 and p = 0.003, respectively). In GCF of RA patients, QC mRNA was detected more frequently than in healthy controls (p = 0.043). In these samples IL-1β levels were also elevated compared to GCF from periodontally healthy individuals (p = 0.003). PgQC was detected in eight out of the 13 P. gingivalis positive biofilm samples. Conclusion Activity of QC may play a supportive role in maintaining chronic periodontal inflammation and destruction in RA. PgQC is expressed in vivo but further research is needed to evaluate biological importance of this enzyme and if it constitutes a potential target in periodontal antimicrobial therapy.

The interplay of manganese and iron homeostasis and oxidative stress in Escherichia coli can give important insights into survival of bacteria in the phagosome and under differing iron or manganese bioavailabilities. Here, we characterized a mutant strain devoid of all know iron/manganese-uptake systems relevant for growth in defined medium. Based on these results an exit strategy enabling the cell to cope with iron depletion and use of manganese as an alternative for iron could be shown. Such a strategy would also explain why E. coli harbors some iron- or manganese-dependent iso-enzymes such as superoxide dismutases or ribonucleotide reductases. The benefits for gaining a means for survival would be bought with the cost of less efficient metabolism as indicated in our experiments by lower cell densities with manganese than with iron. In addition, this strain was extremely sensitive to the metalloid gallium but this gallium toxicity can be alleviated by low concentrations of manganese.

Bacterial contamination of touch surfaces poses a serious threat for public health. The use of bactericidal surface materials, such as copper and its alloys, might constitute a way to aid the use of antibiotics and disinfectants, thus minimizing the risk of emergence and spread of multiresistant germs. The survival of Escherichia coli on metallic copper surfaces has been studied previously; however, the mechanisms underlying bacterial inactivation on copper surfaces have not been elucidated. Data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems were slightly more sensitive than was the wild type. Preadaptation to copper enhanced survival rates upon copper surface exposure. This study constitutes a first step toward understanding the reasons for metallic copper surface-mediated killing of bacteria.

The Escherichia coli zupT (formerly ygiE) gene encodes a cytoplasmic membrane protein (ZupT) related to members of the eukaryotic ZIP family of divalent metal ion transporters. Previously, ZupT was shown to be responsible for uptake of zinc. In this study, we show that ZupT is a divalent metal cation transporter of broad substrate specificity. An E. coli strain with a disruption in all known iron uptake systems could grow in the presence of chelators only if zupT was expressed. Heterologous expression of Arabidopsis thaliana ZIP1 could also alleviate iron deficiency in this E. coli strain, as could expression of indigenous mntH or feoABC . Transport studies with intact cells showed that ZupT facilitates uptake of 55 Fe 2+ similarly to uptake of MntH or Feo. Other divalent cations were also taken up by ZupT, as shown using 57 Co 2+ . Expression of zupT rendered E. coli cells hypersensitive to Co 2+ and sensitive to Mn 2+ . ZupT did not appear to be metal regulated: expression of a Φ( zupT-lacZ ) operon fusion indicated that zupT is expressed constitutively at a low level.

The ZIP (ZRT-, IRT-like Protein) protein ZupT from Escherichia coli is a transporter with a broad substrate range. Phenotypic and transport analysis showed that ZupT, in addition to Zn(II), Fe(II) and Co(II) uptake, is also involved in transport of Mn(II) and Cd(II). Competition experiments with other substrate cations suggested that ZupT has a slight preference for Zn(II) and kinetic parameters for Zn(II) in comparison to Co(II) and Mn(II) transport support this observation. Metal uptake into cells by ZupT was optimum at near neutral pH and inhibited by ionophores. Bicarbonate or other ions did not influence metal-uptake via ZupT. Amino acid residues of ZupT contributing to substrate specificity were identified by site directed mutagenesis. ZupT with a H89A exchange lost Co(II) and Fe(II) transport activity, while the S117V mutant no longer transported Mn(II). ZupT with E152D was impaired in overall metal uptake but completely lost its ability to transport the substrates Zn(II) and Mn(II). These experimental findings expand our knowledge on the substrate specificity of ZupT and provide further insight into the function of ZupT as a bacterial member of the vastly distributed and important ZIP family.

Zinc is an essential micronutrient, and yet it can be toxic when present in excess. Zinc acquisition and distribution are dependent on tightly controlled transport of Zn(2+) ions. Schizosaccharomyces pombe represents a second eukaryotic model to study cellular metal homeostasis. In several ways its micronutrient metabolism is fundamentally different from Saccharomyces cerevisiae. We identified the first Zn(2+)-uptake system in S. pombe and named it SpZrt1. Knock-out strains for all three ZIP (Zrt, Irt-like protein) transporters in fission yeast were constructed. Only zrt1Delta cells were unable to grow at low Zn(2+) and showed reduced (65)Zn(2+) uptake. Elemental profiles revealed a strong decrease in zinc accumulation. Cd(2+) ions inhibited uptake but Fe(2+) or Mn(2+) did not. Both mRNA abundance and protein amount are tightly regulated. Zrt1 activity is rapidly shut down upon transfer of zinc-deficient cells to zinc-replete conditions. In cells lacking Zhf, a transporter mediating endoplasmic reticulum storage of zinc, this response is about 100-fold more sensitive. Thus, removal of excess of zinc from the cytosol is largely Zhf dependent. Moreover, cells deficient for both transporters are no longer able to adjust to changing external Zn(2+) concentrations. Optimal growth is restricted to a narrow range of Zn(2+) concentrations, illustrating the fine balance between micronutrient deficiency and toxicity.

Escherichia coli possesses multiple routes for iron uptake. Here we present EfeU (YcdN), a novel iron acquisition system of E. coli strain Nissle 1917. Laboratory strains of E. coli such as K12 lack a functional (efeU) ycdN gene caused by a frameshift mutation. EfeU, a member of the oxidase-dependent iron transporters (OFeT), is a homologue of the iron permease Ftr1p from yeast. The ycdN gene is part of the ycdNOB tricistronic operon which is expressed in response to iron deprivation in a Fur-dependent manner. Expression of efeU resulted in improved growth of an E. coli mutant lacking all known iron-uptake systems and mediated increased iron uptake into cells. Furthermore, the presence of other divalent metal cations did not impair growth of strains expressing efeU. The EfeU protein functioned as ferrous iron permease in proteoliposomes in vitro. Topology analysis indicated that EfeU is an integral cytoplasmic membrane protein exhibiting seven transmembrane helices. Two REXXE motifs within transmembrane helices of OFeT family members are implicated in iron translocation. Site-directed mutagenesis of each REGLE motif of EfeU diminished iron uptake in vivo and growth yield. In vitro the EfeU variant protein with an altered first REGLE motif was impaired in iron permeation, whereas activity of the EfeU variant with a mutation in the second motif was similar to the wild-type protein.

Escherichia coli excretes the catecholate siderophore enterobactin in response to iron deprivation. While the mechanisms underlying enterobactin biosynthesis and ferric enterobactin uptake and utilization are widely understood, nearly nothing is known about how enterobactin is exported from the cell. Mutant and high-performance liquid chromatography analyses demonstrated that the outer membrane channel tunnel protein TolC but none of the respective seven resistance nodulation cell division (RND) proteins CusA, AcrB, AcrD, AcrF, MdtF (YhiV), or the twin RND MdtBC (YegNO) was essential for enterobactin export across the outer membrane. Mutant E. coli strains with additional deletion of tolC or the major facilitator entS were growth deficient in iron-depleted medium. Strains with deletion of tolC or entS , but not with deletion of genes encoding RND transporters, excreted very little enterobactin into the growth medium. Enterobactin excretion in E. coli is thus probably a two-step process involving the major facilitator EntS and the outer membrane channel tunnel protein TolC. Quantitative reverse transcription-PCR analysis of gene-specific transcripts showed no significant changes in tolC expression upon iron depletion. However, iron starvation led to increased expression of the RND gene mdtF and a decrease in acrD .