Potential Mechanisms of Mucin-Enhanced Acinetobacter baumannii Virulence in the Mouse Model of Intraperitoneal Infection.

Porcine mucin has been commonly used to enhance the infectivity of bacterial pathogens, including Acinetobacter baumannii, in animal models, but the mechanisms for enhancement by mucin remain relatively unknown. In this study, using the mouse model of intraperitoneal (i.p.) mucin-enhanced A. baumannii infection, we characterized the kinetics of bacterial replication and dissemination and the host innate immune responses, as well as their potential contribution to mucin-enhanced bacterial virulence. We found that mucin, either admixed with or separately injected with the challenge bacterial inoculum, was able to enhance the tissue and blood burdens of A. baumannii strains of different virulence. Intraperitoneal injection of A. baumannii-mucin or mucin alone induced a significant but comparable reduction of peritoneal macrophages and lymphocytes, accompanied by a significant neutrophil recruitment and early interleukin-10 (IL-10) responses, suggesting that the resulting inflammatory cellular and cytokine responses were largely induced by the mucin. Depletion of peritoneal macrophages or neutralization of endogenous IL-10 activities showed no effect on the mucin-enhanced infectivity. However, pretreatment of mucin with iron chelator DIBI, but not deferoxamine, partially abolished its virulence enhancement ability, and replacement of mucin with iron significantly enhanced the bacterial burdens in the peritoneal cavity and lung. Taken together, our results favor the hypothesis that iron at least partially contributes to the mucin-enhanced infectivity of A. baumannii in this model.

Acute intraperitoneal infection with a hypervirulent Acinetobacter baumannii isolate in mice.

Acinetobacter baumannii infection has become a major cause of healthcare-associated infection and a critical pathogen in the WHO antimicrobial resistance research and development priority list. Catheter-related septicemia is one of the major clinical manifestations of A. baumannii infection associated with high morbidity and mortality. In this study, we used a clinical A. baumannii strain (LAC-4) that is hypervirulent to immunocompetent C57BL/6 and BALB/c mice and established a mouse model of intraperitoneal (i.p.) A. baumannii infection. Our study showed that i.p. LAC-4 infection of C57BL/6 and BALB/c mice induces a lethal or sublethal infection with high bacterial burdens in peritoneal cavity, blood and tissues and the infected mice either succumbed to the infection within 24 hours or completely recovered from the infection. The infection induces acute peritoneal recruitment of neutrophils and other innate immune cells, and the local and systemic production of proinflammatory cytokines and chemokines (IL-1ß, IL-5, IL-6, TNF-α, RANTES, MIP-1ß, MCP-1, KC and IL-10). Mechanistic studies suggest an important role of macrophages in the host innate defense in this model in that in vitro stimulation of peritoneal macrophages with killed LAC-4 induced a similar pattern of cytokine/chemokine responses to those in the infected mice, and depletion of peritoneal macrophages rendered the mice significantly more susceptible to the infection. Thus, this mouse infection model will provide an alternative and useful tool for future pathogenesis studies of A. baumannii-associated septicemia and identification and characterization of important virulence factors, as well as serve as a surrogate model for rapid evaluation of novel therapeutics and vaccines for this emerging infectious agent.

Identification and characterization of novel isothiazolones with potent bactericidal activity against multi-drug resistant Acinetobacter baumannii clinical isolates.

Acinetobacter baumannii has emerged as a globally important nosocomial pathogen characterized by an increased multi-drug resistance (MDR), leaving limited options for treating its infection. To identify novel antibacterial compounds with activity against clinical isolates of A. baumannii, we performed high-throughput screening against a chemical library of 42,944 compounds using nonpathogenic Escherichia coli MG1655 and identified 55 hit compounds. The antibacterial activities of 30 pure compounds were determined against MDR clinical isolates of A. baumannii obtained from Los Angeles County hospitals. Two isothiazolones identified, 5-chloro-2-(4-chloro-3-methylphenyl)-4-methyl-3(2H)-isothiazolone (Compound 6) and 5-chloro-2-(4-chlorophenyl)-4-methyl-3(2H)-isothiazolone (Compound 7), possess novel structure and exhibited consistent, potent and cidal activity against all 46 MDR A. baumannii clinical isolates and reference strains. Additionally, structure-activity relationship analysis involving several additional isothiazolones supports the link between a chloro-group on the heterocyclic ring or a fused benzene ring and the cidal activity. Attempts to obtain isothiazolone resistant mutants failed, consistent with the rapid cidal action and indicative of a complex mechanism of action. While cytotoxicity was observed with Compound 7, it had a therapeutic index value of 28 suggesting future therapeutic potential. Our results indicate that high-throughput screening of compound libraries followed by in vitro biological evaluations is a viable approach for the discovery of novel antibacterial agents to contribute in the fight against MDR bacterial pathogens.

Mouse Models of Acinetobacter baumannii Infection.

This unit describes basic protocols for infecting mice through intranasal and intraperitoneal routes with Acinetobacter baumannii to induce associated pneumonia and sepsis, the two most common manifestations of clinical infections with this pathogen. By selecting the appropriate protocols and bacterial strains of different virulence, these mouse models provide an opportunity to study the infection pathogenesis and host-immune responses, and to evaluate the efficacies of prophylactic and therapeutic anti-A. baumannii candidates. © 2017 by John Wiley & Sons, Inc.

Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis.

Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.

Identification of cellular targets of a series of boron heterocycles using TIPA II-A sensitive target identification platform.

One of the hurdles in the discovery of antibiotics is the difficulty of linking antibacterial compounds to their cellular targets. Our laboratory has employed a genome-wide approach of over-expressing essential genes in order to identify cellular targets of antibacterial inhibitors. Our objective in this project was to develop and validate a more sensitive disk diffusion based platform of target identification (Target Identification Platform for Antibacterials version 2; TIPA II) using a collection of cell clones in an Escherichia coli mutant (AS19) host with increased outer membrane permeability. Five known antibiotics/inhibitors and 28 boron heterocycles were tested by TIPA II assay, in conjunction with the original assay TIPA. The TIPA II was more sensitive than TIPA because eight boron heterocycles previously found to be inactive to AG1 cells in TIPA assays exhibited activity to AS19 cells. For 15 boron heterocycles, resistant colonies were observed within the zones of inhibition only on the inducing plates in TIPA II assays. DNA sequencing confirmed that resistant clones harbor plasmids with fabI gene as insert, indicating that these boron heterocycles all target enoyl ACP reductase. Additionally, cell-based assays and dose response curved obtained indicated that for two boron heterocycle inhibitors, the fabI cell clone in AG1 (wild-type) host cells exhibited at least 11 fold more resistance under induced conditions than under non-induced conditions. Moreover, TIPA II also identified cellular targets of known antibacterial inhibitors triclosan, phosphomycin, trimethoprim, diazaborine and thiolactomycin, further validating the utility of the new system.

Molecular characterization and antimicrobial susceptibility of Acinetobacter baumannii isolates obtained from two hospital outbreaks in Los Angeles County, California, USA.

BACKGROUND: Antibiotic resistant strains of Acinetobacter baumannii have been responsible for an increasing number of nosocomial infections including bacteremia and ventilator-associated pneumonia. In this study, we analyzed 38 isolates of A. baumannii obtained from two hospital outbreaks in Los Angeles County for the molecular epidemiology, antimicrobial susceptibility and resistance determinants. METHODS: Pulsed field gel electrophoresis, tri-locus multiplex PCR and multi-locus sequence typing (Pasteur scheme) were used to examine clonal relationships of the outbreak isolates. Broth microdilution method was used to determine antimicrobial susceptibility of these isolates. PCR and subsequent DNA sequencing were employed to characterize antibiotic resistance genetic determinants. RESULTS: Trilocus multiplex PCR showed these isolates belong to Global Clones I and II, which were confirmed to ST1 and ST2, respectively, by multi-locus sequence typing. Pulsed field gel electrophoresis analysis identified two clonal clusters, one with 20 isolates (Global Clone I) and the other with nine (Global Clone II), which dominated the two outbreaks. Antimicrobial susceptibility testing using 14 antibiotics indicated that all isolates were resistant to antibiotics belonging to four or more categories of antimicrobial agents. In particular, over three fourth of 38 isolates were found to be resistant to both imipenem and meropenem. Additionally, all isolates were found to be resistant to piperacillin, four cephalosporin antibiotics, ciprofloxacin and levofloxacin. Resistance phenotypes of these strains to fluoroquinolones were correlated with point mutations in gyrA and parC genes that render reduced affinity to target proteins. ISAba1 was detected immediately upstream of the bla OXA-23 gene present in those isolates that were found to be resistant to both carbapenems. Class 1 integron-associated resistance gene cassettes appear to contribute to resistance to aminoglycoside antibiotics. CONCLUSION: The two outbreaks were found to be dominated by two clonal clusters of A. baumannii belonging to MLST ST1 and ST2. All isolates were resistant to antibiotics of at least four categories of antimicrobial agents, and their antimicrobial susceptibility profiles correlate well with genetic determinants. The results of this study will facilitate our understanding of the molecular epidemiology, antimicrobial susceptibility and mechanisms of resistance of A. baumannii obtained from Los Angeles hospitals.

Host resistance to intranasal Acinetobacter baumannii reinfection in mice.

Acinetobacter baumannii is a major causative agent of healthcare-associated infection and develops multidrug resistance rapidly. However, little is known in the host defense mechanisms against this infection. In this study, we examined if mice recovered from a previous intranasal A. baumannii infection (recovered mice) are fully protected against a subsequent reinfection. We found that, despite the presence of specific serum IgG and mucosal IgA responses prior to the reinfection, the recovered mice were only marginally better protected against intranasal challenge with low doses of homologous or heterologous A. baumannii strains than the naïve mice. Post-challenge immune and inflammatory (cells and cytokines) responses were generally comparable between recovered and naïve mice although the recovered mice produced significantly higher amounts of IFN-γ and IL-17 and had higher percentages and numbers of resident lung CD44(hi)CD62L(-)CD4(+) and CD19(+) B lymphocytes. Taken together, our results suggest that mice recovered from a previous A. baumannii infection remain susceptible to reinfection, indicating the complexity of immune protection mechanism for this Gram-negative, multidrug-resistant emerging pathogen.

Crystallographic insights into the structure-activity relationships of diazaborine enoyl-ACP reductase inhibitors.

Enoyl-ACP reductase, the last enzyme of the fatty-acid biosynthetic pathway, is the molecular target for several successful antibiotics such as the tuberculosis therapeutic isoniazid. It is currently under investigation as a narrow-spectrum antibiotic target for the treatment of several types of bacterial infections. The diazaborine family is a group of boron heterocycle-based synthetic antibacterial inhibitors known to target enoyl-ACP reductase. Development of this class of molecules has thus far focused solely on the sulfonyl-containing versions. Here, the requirement for the sulfonyl group in the diazaborine scaffold was investigated by examining several recently characterized enoyl-ACP reductase inhibitors that lack the sulfonyl group and exhibit additional variability in substitutions, size and flexibility. Biochemical studies are reported showing the inhibition of Escherichia coli enoyl-ACP reductase by four diazaborines, and the crystal structures of two of the inhibitors bound to E. coli enoyl-ACP reductase solved to 2.07 and 2.11 Šresolution are reported. The results show that the sulfonyl group can be replaced with an amide or thioamide without disruption of the mode of inhibition of the molecule.

Complete genome sequence of hypervirulent and outbreak-associated Acinetobacter baumannii strain LAC-4: epidemiology, resistance genetic determinants and potential virulence factors.

Acinetobacter baumannii is an important human pathogen due to its multi-drug resistance. In this study, the genome of an ST10 outbreak A. baumannii isolate LAC-4 was completely sequenced to better understand its epidemiology, antibiotic resistance genetic determinants and potential virulence factors. Compared with 20 other complete genomes of A. baumannii, LAC-4 genome harbors at least 12 copies of five distinct insertion sequences. It contains 12 and 14 copies of two novel IS elements, ISAba25 and ISAba26, respectively. Additionally, three novel composite transposons were identified: Tn6250, Tn6251 and Tn6252, two of which contain resistance genes. The antibiotic resistance genetic determinants on the LAC-4 genome correlate well with observed antimicrobial susceptibility patterns. Moreover, twelve genomic islands (GI) were identified in LAC-4 genome. Among them, the 33.4-kb GI12 contains a large number of genes which constitute the K (capsule) locus. LAC-4 harbors several unique putative virulence factor loci. Furthermore, LAC-4 and all 19 other outbreak isolates were found to harbor a heme oxygenase gene (hemO)-containing gene cluster. The sequencing of the first complete genome of an ST10 A. baumannii clinical strain should accelerate our understanding of the epidemiology, mechanisms of resistance and virulence of A. baumannii.

SecReT6: a web-based resource for type VI secretion systems found in bacteria.

SecReT6 (http://db-mml.sjtu.edu.cn/SecReT6/) is an integrated database providing comprehensive information on type VI secretion systems (T6SSs) in bacteria. T6SSs are a class of sophisticated cell contact-dependent apparatuses involved in mediating antagonistic or synergistic communications between bacteria and/or bacteria and eukaryotes. These apparatuses have recently been found to be widely distributed among Gram-negative bacterial species. SecReT6 offers a unique, readily explorable archive of known and putative T6SSs, and cognate effectors found in bacteria. It currently contains data on 11 167 core T6SS components mapping to 906 T6SSs found in 498 bacterial strains representing 240 species, as well as a collection of over 600 directly relevant references. Also collated and archived were 1340 diverse candidate secreted effectors which were experimentally shown and/or predicted to be delivered by T6SSs into target eukaryotic and/or prokaryotic cells as well as 196 immunity proteins. A broad range of T6SS gene cluster detection and comparative analysis tools are readily accessible via SecReT6, which may aid identification of effectors and immunity proteins around the T6SS core components. This database will be regularly updated to ensure its ongoing maximal utility and relevance to the scientific research community.

Intranasal immunization protects against Acinetobacter baumannii-associated pneumonia in mice.

Multidrug-resistant Acinetobacter baumannii has become an important causative agent of healthcare associated infections. Hospital- and community-acquired pneumonia is the most common clinical manifestation of A. baumannii infection worldwide and is often associated with high mortality. Most experimental vaccine studies to date have evaluated vaccines against systemic A. baumannii infections following systemic immunization. We recently demonstrated that a mouse model of respiratory A. baumannii infection using the strain LAC-4 results in disease progression that is similar to that observed in humans. Here we used this model in conjunction with an inactivated whole cell vaccine to evaluate the feasibility of developing protective mucosal vaccines against respiratory A. baumannii infection and to investigate the potential mechanism of protection of such vaccines. Our results showed that intranasal immunization with formalin-killed whole cells of the LAC-4 strain elicited mucosal and systemic antigen-specific immune responses, and protected mice against lethal intranasal or intraperitoneal challenges. Compared to naïve mice, immunized mice had significantly fewer bacteria in their lungs, and the pathogen was barely detectable in blood and spleens at 24h post challenge, indicating the ability of immunized mice to control extrapulmonary dissemination of the pathogen. Mechanistic studies using gene-deficient mice, neutropenic mice, or passive immunization showed that B cells and neutrophils, but not FcRγ, played crucial roles in the protection against respiratory A. baumannii challenge of intranasally immunized mice whereas passive transfer of hyperimmune sera only prolonged the survival time of challenged mice by 48 h. These results provide immunological insights for the rational design of novel mucosal vaccines to protect against respiratory A. baumannii infection and demonstrate the feasibility to develop such vaccines.

Synthesis, characterization, and antibacterial activity of structurally complex 2-acylated 2,3,1-benzodiazaborines and related compounds.

A set of 2-acylated 2,3,1-benzodiazaborines and some related boron heterocycles were synthesized, characterized, and tested for antibacterial activity against Escherichia coli and Mycobacterium smegmatis. By high-field solution NMR, the heretofore unknown class of 2-acyl-1-hydroxy-2,3,1-diazaborines has been found to be able to exist in several interconvertable structural forms along a continuum comprised of an open hydrazone a, a monomeric B-hydroxy diazaborine b, and an anhydro dimer c. X-Ray crystallography of one of the anhydro dimers, 17c, revealed it to have an unprecedented structure featuring a double intramolecular O→B chelation. The crystal structure of another compound, 37, showed it to be based on a new pentacyclic B heterocycle framework. Nine compounds were found to possess activities against E. coli, and two others were active against M. smegmatis. The finding that these two contain isoniazid covalently embedded in their structures suggests that they might possibly be acting as prodrugs of this well-known antituberculosis agent in vivo.

The structure of the polysaccharide isolated from Acinetobacter baumannii strain LAC-4.

The structure of the surface polysaccharide from a hypervirulent for mice Acinetobacter baumannii strain LAC-4 was studied. The polysaccharide was built of trisaccharide repeating units containing α-l-fucosamine, α-d-glucosamine, and α-8-epi-legionaminic acid. The structure interpretation was based mostly on NMR data. Polysaccharide was obtained using a procedure of LPS O-chain preparation, although whether it is an LPS O-chain or capsular polysaccharide remained unclear.

A disk-diffusion-based target identification platform for antibacterials (TIPA): an inducible assay for profiling MOAs of antibacterial compounds.

One of the challenges in antibiotic lead discovery is the difficulty and time-consuming task of determining the mechanism of action (MOA) of antibacterial compounds. In this report, we describe the development and validation of a facile and inexpensive assay system utilizing disk diffusion of inhibitors on solid agar medium embedded with mixed pools of a comprehensive collection of Escherichia coli clones each containing a plasmid-borne inducible essential gene from E. coli. From individual clones, pilot small-scale (48 or 50 clones) assays, to full-scale target identification platform for antibacterials (TIPA) system, involving a variety of assay formats (liquid vs solid media, individual vs mix clones), we demonstrate that elevated resistance phenotypes of relevant cell clones were highly specific. In particular, the TIPA system was able to reveal cellular targets of several known antibacterial inhibitors: cerulenin, diazaborine, indolmycin, phosphomycin, and triclosan. Complementary to several existing MOA profiling schemes, the TIPA system offers a simple and low-cost method for elucidating the target proteins of antibacterial inhibitors, thus will facilitate discovery and development of novel antibacterial compounds to combat multidrug-resistant bacterial pathogens.

Serum resistance, gallium nitrate tolerance and extrapulmonary dissemination are linked to heme consumption in a bacteremic strain of Acinetobacter baumannii.

Bacteremia caused by Acinetobacter baumannii is a highly lethal complication of hospital-acquired pneumonia. In the present study, we investigated the serum resistance, gallium nitrate tolerance and heme consumption of A. baumannii strain LAC-4 which was recently reported to display high virulence in a mouse pneumonia model with extrapulmonary dissemination leading to fatal bacteremia. This strain showed enhanced growth in mouse and fetal bovine serum that was independent of complement and was not observed with regular growth media. The LAC-4 strain was found to possess a high tolerance to gallium nitrate (GaN), whereas serum synergized with GaN in inhibiting A. baumannii strain ATCC 17978. We found that LAC-4 contains a heme oxygenase gene and expresses a highly efficient heme consumption system. This system can be fully blocked in vitro and in vivo by gallium protoporphyrin IX (GaPPIX). Inhibition of heme consumption by GaPPIX completely abrogated the growth advantage of LAC-4 in serum as well as its tolerance to GaN. More importantly, GaPPIX treatment of mice intranasally infected with LAC-4 prevented extrapulmonary dissemination and death. Thus, we propose that heme provides an additional source of iron for LAC-4 to bypass iron restriction caused by serum transferrin, lactoferrin or free gallium salts. Heme consumption systems in A. baumannii may constitute major virulence factors for lethal bacteremic isolates.

Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway.

BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe-4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe-4S](2+) cluster. Mutagenesis studies demonstrated that Cys31, Cys35, Cys38 in the C×××C×MC motif and Gly73, Gly74, Glu75, Pro76 in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met37 had little effect on its function. Our results indicate that BlsE represents a typical [4Fe-4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.

A mouse model of Acinetobacter baumannii-associated pneumonia using a clinically isolated hypervirulent strain.

Acinetobacter baumannii is an important emerging pathogen in health care-acquired infections and is responsible for severe nosocomial and community-acquired pneumonia. Currently available mouse models of A. baumannii pneumonia show poor colonization with little to no extrapulmonary dissemination. Here, we describe a mouse model of A. baumannii pneumonia using a clinical isolate (LAC-4 strain) that reliably reproduces the most relevant features of human pulmonary A. baumannii infection and pathology. Using this model, we have shown that LAC-4 infection induced rapid bacterial replication in the lungs, significant extrapulmonary dissemination, and severe bacteremia by 24 h postintranasal inoculation. Infected mice showed severe bronchopneumonia and dilatation and inflammatory cell infiltration in the perivascular space. More significantly, 100% of C57BL/6 and BALB/c mice succumbed to 10(8) CFU of LAC-4 inoculation within 48 h. When this model was used to assess the efficacy of antimicrobials, all mice treated with imipenem and tigecycline survived a lethal intranasal challenge, with minimal clinical signs and body weight loss. Moreover, intranasal immunization of mice with formalin-fixed LAC-4 protected 40% of mice from a lethal (100× 100% lethal dose) intraperitoneal challenge. Thus, this model offers a reproducible acute course of A. baumannii pneumonia without requiring additional manipulation of host immune status, which will facilitate the development of therapeutic agents and vaccines against A. baumannii pneumonia in humans.

Kinetic studies of inhibition of the amyloid beta (1-42) aggregation using a ferrocene-tagged ß-sheet breaker peptide.

The aggregation of amyloidogenic proteins/peptides has been closely linked to the neuropathology of several important neurological disorders. In Alzheimer's disease, amyloid beta (Aß) peptides and their aggregation are believed to be at least partially responsible for the etiology of Alzheimer's disease. The aggregate-inflicted cellular toxicity can be inhibited by short peptides whose sequences are homologous to segments of the Aß(1-42) peptide responsible for ß-sheet stacking (referred to as the ß-sheet breaker peptides). Here, a water-soluble ferrocene (Fc)-tagged ß-sheet breaker peptide, Fc-KLVFFK(6), was used as an electrochemical probe for kinetic studies of the inhibition of the Aß(1-42) fibrillation process and for determination of the optimal concentration of ß-sheet breaker peptide for efficient inhibition. Our results demonstrate that Fc-KLVFFK(6) interacts with the Aß aggregates instantaneously in solution, and a sub-stoichiometric amount of Fc-KLVFFK(6) is sufficient to inhibit the formation of the Aß oligomers and fibrils and to reduce the toxicity of Aß(1-42). The interaction between Fc-KLVFFK(6) and Aß(1-42) follows a pseudo-first-order reaction, with a rate constant of 1.89 ± 0.05 × 10(-4) s(-1). Tagging ß-sheet breaker peptides with a redox label facilitates design, screening, and rational use of peptidic inhibitors for impeding/altering Aß aggregation.

A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes.

Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published reports describing the application of asRNA gene silencing for comprehensive analyses of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250 000 library transformants for conditional growth inhibitory recombinant clones from two shotgun genomic libraries of E. coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes, while 18 originated from nonessential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12-fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E. coli using regulated asRNA expression.