REPAIR OF NEAR (365 nm)- AND FAR (254 nm)-UV DAMAGE TO BACTERIOPHAGE OF ESCHERICHIA COLI

Abstract: Intact bacteriophage have been irradiated at 365 nm or at 254 nm and then analysed for DNA photoproducts or injected into their bacterial host to test susceptibility of the damage to both phage and host-cell mediated repair systems. Both thymine dimers and single-strand breaks are induced in the phage DNA by 365 nm radiation. The dimers appear to be the major lethal lesion (approximately 2 dimers per lethal event) in both repair deficient bacteriophage T4 and bacteriophage λ. after irradiation with either 254 nm or 365 nm radiation. Damage induced in T4 by either wavelength is equally susceptible to x -gene reactivation (repair sector approximately 0.5). v -gene reactivation acts on a larger fraction of the near-UV damage (repair sector of 0.82 at 365 nm as against 0.66 at 254 nm). The host-cell mediated photoreactivation system is only slightly less effective for near-UV damage but host-cell reactivation (as measured by comparing survival of phage λ. on a uvr⁺ and a uvr^- host) is effective against a far smaller sector of near-UV damage (0.35) than far-UV damage (0.85). Weigle-reactivation (far-UV induced) of near-UV damage to phage λ is not observed. The results suggest that unless the near-UV damaged phage DNA is repaired immediately after injection. the lesions rapidly lose their susceptibility to repair with a consequent loss of activity of the phage particles.     

Shotgun phage display cloning

Shotgun phage display cloning is a useful tool for studying interactions between bacterial and host proteins. Libraries are constructed by cloning randomly fragmented prokaryotic DNA into phage mid-vectors. Theoretically, these libraries will consist of phages that together display all proteins encoded by the bacterial genome. Selecting a gene III-based library, made from Staphylococcus aureus DNA, against IgG and fibronectin resulted in 20-40% positive clones after two pannings. Increasing the number of fusion proteins per phage particle by using gene VIII-based display, increased the frequency of correct clones to 75-100%.     

Studies related to the head-maturation pathway of bacteriophages T4 And T2:II. nuclear disruption, protein synthesis and particle formation with the mutant 43-.30-.46-

We describe the aberrant phage multiplication of the triple conditional lethal mutant 43-(polymerase).30-(ligase).46-(exonuclease) of bacteriophage T4D in which phage DNA replication is arrested but some late protein synthesis occurs (33). The nuclear disruption is indistinguishable from wild type. Forty-five empty small and empty large particles are assembled per cell when the multiplicity of infection (m.o.i.) is 100. This number corresponds closely to the 38 phage equivalents of cleaved major head protein determined biochemically. By reducing the m.o.i. the number of observable particles decreases, reaching 1-5 per cell at an m.o.i. of 5(+5). The total synthesis of phage related proteins is not significantly dependant on the m.o.i. The synthesis of late proteins is about 10% of that of wild type at high m.o.i. and decreases with the m.o.i. The different early and late proteins do not show the same relative proportions as in wild type and respond differently to an increased m.o.i. These and other results are discussed with respect to the role of phage DNA in prehead assembly and head maturation.     

PHOTODYNAMIC EFFECTS OF PROFLAVINE ON BACTERIOPHAGE φ times 174 AND ITS ISOLATED DNA

Abstract. Proflavine-mediated photoinactivation of φ times 174 phage and its isolated DNA was studied under identical irradiation conditions. The inactivations followed single-hit kinetics and a linear relationship was obtained in reciprocal plots of the inactivation rates vs the proflavine concentrations for both phage and isolated DNA. The phage photoinactivation rate was increased with an increase in the amount of proflavine bound to the phage DNA in a strong binding range (0.01-0.04 proflavine/ nucleotide) as the total proflavine concentration was increased or the ionic strength decreased. Further, a phage-specific factor was also found to affect the inactivation rate. The photodynamic treatment induced mutations in three phage strains from "amber" to "wild type" at a mutation rate per lethal hit of 0.3 times 10^-5 to 2.6 times 10^-5. In contrast to phage infectivity, the φ times 174 DNA infectivity was measurable only at a high multiplicity of infection, and its photoinactivation occurred only at high proflavine concentrations. The photoinactivation rate was enhanced either with a decrease in the multiplicity of infection or with the use of spheroplasts of recA mutants strains. The results are discussed in terms of the nature of and possible repair mechanisms of photodynamically induced lesions in φ times 174 phage DNA.     

Chemical genetic screening identifies critical pathways in anthrax lethal toxin-induced pathogenesis

Anthrax lethal toxin (LT)-induced cell death via mitogen-activated protein kinase kinase (MAPKK) cleavage remains questionable. Here, a chemical genetics approach was used to investigate what pathways mediate LT-induced cell death. Several small molecules were found to protect macrophages from anthrax LT cytotoxicity and MAPKK from cleavage by lethal factor (LF), without inhibiting LF enzymatic activity or cellular proteasome activity. Interestingly, the compounds activated MAPK-signaling molecules, induced proinflammatory cytokine production, and inhibited LT-induced macrophage apoptosis in a concentration-dependent manner. We propose that induction of antiapoptotic responses by MAPK-dependent or -independent pathways and activation of host innate responses may protect macrophages from anthrax LT-induced cell death. Altering host responses through a chemical genetics approach can help identify critical cellular pathways involved in the pathogenesis of anthrax and can be exploited to further explore host-pathogen interactions.     

Bacteriophage reporter technology for sensing and detecting microbial targets

Bacteriophages (phages) are bacterial viruses evolutionarily tuned to very specifically recognize, infect, and propagate within only a unique pool of host cells. Knowledge of these phage host ranges permits one to devise diagnostic tests based on phage–host recognition profiles. For decades, fundamental phage typing assays have been used to identify bacterial pathogens on the basis of the ability of phages to kill, or lyse, the unique species, strain, or serovar to which they are naturally targeted. Over time, and with a better understanding of phage–host kinetics and the realization that there exists a phage specific for nearly any bacterial pathogen of clinical, foodborne, or waterborne consequence, a variety of improved, rapid, sensitive, and easy-to-use phage-mediated detection assays have been developed. These assays exploit every stage of the phage recognition and infection cycle to yield a wide variety of pathogen monitoring, detection, and enumeration formats that are steadily advancing toward new biosensor integrations and advanced sensing technologies.     

Active antibacterial coating of cotton fabrics with antimicrobial proteins

The prevention of bacteria colonization by immobilizing proteins with antimicrobial activity onto cotton fabrics was investigated. Such coatings have potential applications in medical dressing materials used in wound care and healing. Two antimicrobial proteins lysozyme and hydramacin-1 (HM-1) were surface immobilized through two linkers (3-aminopropyl) triethoxysilane (APTES) and citric acid in the presence of the water soluble carbodiimide coupling reagent 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. Surface composition analysis by attenuated total reflection-Fourier transform infrared and X-ray photoelectron spectroscopies confirmed formation of the protein-cellulose conjugates. Antimicrobial activities of the different functionalized surfaces were found to vary between APTES and citric acid directed coatings. Citric acid immobilized lysozyme treated samples demonstrated superior activity against Gram-positive Bacillus subtilis, whereas APTES immobilized HM-1 treated samples demonstrated an advantage in inhibiting the growth of Gram-negative Escherichia coli. The antibacterial activity and stability of citric acid immobilized protein fabrics following sonication, boiling and chemical treatment were noticeably higher than that of the corresponding APTES immobilized protein fabrics. The dual coating of fibers with both antimicrobial proteins afforded efficient antimicrobial activities against both bacterial species. The results suggest that coating cotton fibers with antimicrobial proteins and peptides represents a feasible approach for developing active surfaces that prohibit growth and colonization of bacterial strains and can be potentially used in medical cotton-based fabrics.

     

Molybdenum cofactor biosynthesis in plants and humans

The transition element molybdenum (Mo) needs to be complexed by a special cofactor in order to gain catalytic activity. With the exception of bacterial Mo-nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor Moco, which in different variants is the active compound at the catalytic site of all other Mo-containing enzymes. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also requires reducing equivalents, iron, ATP and probably copper. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco–carrier/binding proteins that also participate in Moco-insertion into the cognate apoproteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. In humans, Moco deficiency is a severe inherited inborn error in metabolism resulting in severe neurodegeneration in newborns and causing early childhood death. Due to our better understanding of the chemistry of Moco synthesis, a first therapy has been brought to the clinic.     

Peroxisome proliferator-activated receptor alpha antagonism inhibits hepatitis C virus replication

Hepatitis C virus (HCV) is a global health problem and a leading cause of liver disease. Here, we demonstrate that the replication of HCV replicon RNA in Huh-7 cells is inhibited by a peroxisome proliferator-activated receptor (PPAR) antagonist, 2-chloro-5-nitro-N-(pyridyl)benzamide (BA). Downregulation of PPARgamma with RNA interference approaches had no effect on HCV replication in Huh-7 cells, whereas PPARalpha downregulation inhibited HCV replication. Fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy demonstrate a clear buildup of lipids upon treatment with BA. These observations are consistent with the misregulation of lipid metabolism, phospholipid secretion, cholesterol catabolism, and triglyceride clearance events associated with the inhibition of PPARalpha. The inhibition of HCV replication by BA may result from disrupting lipidation of host proteins associated with the HCV replication complex or, more generally, by disrupting the membranous web where HCV replicates.     

Virus Particles Monitored by Fluorescence Spectroscopy: A Potential Detection Assay for Macromolecular Assembly¶

Native fluorescence spectroscopy was used for in situ investigations of two lipid‐containing bacteriophages from the cystovirus family as well as their Pseudomonad host cells. Both the viruses φ6 and φ12 and their bacterial host proteins contain the amino acid tryptophan (trp), which is the predominant fluorophore in UV. Within proteins, trp's structural environment differs, and the differences are reflected in their spectroscopic signatures. It was observed that the peak of the trp emission from both viruses was at 330 nm, a significantly shorter wavelength than trp in either the Pseudomonad host cells or the amino acid's chemical form. This allowed us to monitor the viral attachment process and subsequent lytic release of progeny virus particles by measurement of the trp emission spectra during the infection process. This work demonstrates that fluorescence may offer a novel tool to detect viruses and monitor viral infection of cells and may be part of a biodefense application.     

A chemical reporter for protein AMPylation

Protein AMPylation is an emerging post-translational modification, which plays key roles in bacterial pathogenesis and cell biology. Enzymes with AMPylation activity, referred to as AMPylators, have been identified in several bacterial pathogens and eukaryotes. To facilitate the study of this unique modification, we developed an alkynyl chemical reporter for detection and identification of protein AMPylation substrates. Covalent functionalization of AMPylation substrates with the alkynyl reporter in lieu of adenylyl 5'-monophosphate (AMP) allows their subsequent bioorthogonal ligation with azide-fluorescent dyes or affinity enrichment tags. We show that this chemical reporter is transferred by a range of AMPylators onto their cognate protein substrates and allows rapid detection and identification of AMPylated substrates.     

Bacterial capture efficiency and antimicrobial activity of phage-functionalized model surfaces

The rise of antibiotic-resistant bacteria has directed substantial attention toward the use of bacteriophages as a means to control bacterial populations. It has been proposed that bacteriophages can be applied as a coating on surfaces in healthcare settings or on indwelling medical devices to create an antimicrobial surface. In this study, antimicrobial model surfaces functionalized with five different types of bacteriophage were prepared and characterized with X-ray photoelectron spectroscopy and atomic force microscopy. The bacterial capture efficiency of these functionalized surfaces was studied for two common bacteria, Escherichia coli and Salmonella typhimurium. Binding of the phages to a solid surface affected their biofunctionality as expressed by the capture efficiency and rate of host membrane disruption. Moreover, the size and shape of the bacteriophage and positioning of its specific binding proteins significantly affected its bacterial capture capability in the immobilized state. Symmetric bacteriophages were found to be a better choice for antibacterial surfaces compared to more asymmetric tailed bacteriophages. Immobilized phages were found to disrupt the membranes of attached bacteria and are thus proposed as a candidate for antimicrobial surfaces.     

Designing cytokine variants by phage-display

Cytokines are important mediators of many cellular functions including coordination of the immune system and regulation of regenerative processes. Therefore, cytokines can be exploited for therapeutic strategies. Cytokines can be altered in a way that their biologic activity is enhanced or antagonized. This can be accomplished by changing the interaction of cytokines with their cognate cytokine receptor complexes. Therefore, many research groups tried to design cytokines, which bind with higher affinity to their receptors. Alternatively, cytokine variants have been created which do bind to their receptors but do not elicit a signal. Such strategies have been followed using high throughput techniques like error-prone polymerase chain reaction and phage display. Designer cytokines can be used to specifically inhibit cytokine functions. Moreover, peptides have been generated with the help of phage display techniques, which exhibit cytokine activity. Surprisingly, such mimetic peptides do not show any sequence similarity to the parental cytokines. Such peptide mimetics can be used as lead structures for the generation of non-peptidic chemical compounds with cytokine activity.     

Antibody-scanning and epitope-tagging methods; molecular mapping of proteins using antibodies

Because synthetic short peptides bearing critical binding residues, can chemically mimic the folded antigenic determinants on proteins, short synthetic peptides can generate antibodies that react with cognate sequences in intact folded proteins. According to this mimotope theory, we produced site-specific antibodies by immunization with short peptides which overlapped each other and covered the entire protein, and used them for domain mapping of influenza virus RNA polymerase (antibody-scanning method). We also used a tagged-epitope and its monoclonal antibodies for topology mapping of clathrin light chains in clathrin triskelions by electron microscopy. Both methods using specific epitopes in combination with their antibodies enable us to determine the domains of interesting proteins systematically without the need to generate monoclonal antibodies or mutant proteins.     

Maximizing the therapeutic window of an antimicrobial drug by imparting mitochondrial sequestration in human cells

The number of antimicrobial agents available for use in humans is limited by the difficulty of discovering chemical agents with selective toxicity to bacterial targets. Numerous small molecule inhibitors have potential as antimicrobial agents, yet their use has been prevented by high levels of toxic cross-reactivity in human cells. For example, methotrexate (Mtx) is an effective antimetabolite that exerts its effects by inhibiting DHFR. It is a potent antibacterial when accumulated intracellularly, but toxicity in human cells limits clinical utility in infectious disease treatment. Here, we describe peptide conjugates of Mtx that are sequestered into the mitochondria of human cells (mt-Mtx). This alteration in localization of Mtx, which directs it away from its enzyme target, decreases its toxicity in human cells by a factor of 10(3). Mt-Mtx, however, maintains activity against a variety of pathogenic gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA). The results from this proof-of-principle study describe a novel methodology for augmenting the antibacterial efficacy of drugs amenable to peptide conjugation while simultaneously decreasing their toxicity to the host organism.     

Silica–silver core–shell particles for antibacterial textile application

The silica–silver core–shell particles were synthesized by simple one pot chemical method and were employed on the cotton fabric as an antibacterial agent. Extremely small (1–2 nm) silver nanoparticles were attached on silica core particles of average 270 nm size. The optimum density of the nano silver particles was found which was sufficient to show good antibacterial activity as well as the suppression in their surface plasmon resonance responsible for the colour of the core–shell particle for antibacterial textile application. The change in the density and size of the particles in the shell were monitored and confirmed by direct evidence of their transmission electron micrographs and by studying surface plasmon resonance characteristics. The colony counting method of antibacterial activity testing showed excellent results and even the least silver containing core–shell particles showed 100% activity against bacterial concentration of 10⁴ colony counting units (cfu). The bonding between core–shell particles and cotton fabric was examined by X-ray photoelectron spectroscopy. The antibacterial activity test confirmed the firm attachment of core–shell particles to the cotton fabric as a result 10 times washed sample was as good antibacterial as that of unwashed sample. The bacterial growth was inhibited on and beneath the coated fabric, at the same time no zone of inhibition which occurs due to the migration of silver ions into the medium was observed indicating immobilization of silver nanoparticles on silica and core–shell particles on fabric by strong bonding.     

Action of chitosan against Xanthomonas pathogenic bacteria isolated from Euphorbia pulcherrima

The antibacterial activity and mechanism of two kinds of chitosan were investigated against twelve Xanthomonas strains recovered from Euphorbia pulcherrima. Results indicated that both chitosans markedly inhibited bacterial growth based on OD loss. Furthermore, the release of DNA and RNA from three selected strains was increased by both chitosans. However, the release of intracellular proteins was inhibited by both chitosans at different concentration and incubation times, except chitosan A at 0.1 mg/mL for 0.5 h incubation and 0.2 mg/mL for 2.0 h incubation increased the release of proteins, indicating the complexity of the interaction and cell membranes, which was affected by incubation time, bacterial species, chitosan type and concentration. Transmission electron microscopic observations revealed that chitosan caused changes in protoplast concentration and surface morphology. In some cells, the membranes and walls were badly distorted and disrupted, while other cells were enveloped by a thick and compact ribbon-like layer. The contrary influence on cell morphology may explain the differential effect in the release of material. In addition, scanning electron microscope and biofilm formation test revealed that both chitosans removed biofilm biomass. Overall, this study showed that membrane and biofilm play an important role in the antibacterial mechanism of chitosan.     

HOST-CELL REACTIVATION OF NON-LETHAL ULTRAVIOLET-EFFECTS

Abstract— Delay of intracellular growth of u.v.-irradiated bacteriophage T1 and Λ was compared in host-cell reactivating [HCR(+)] and non-host-cell reactivating [HCR(—)] bacterial strains. At a given phage survival level, intracellular growth delay occurs to the same extent in HCR (+) and HCR (-) strains; at a given absolute u.v.-dose, this delay is considerably more expressed in HCR (-) than in HCR (+) strains. Therefore, it does not reflect the time required for the HCR repair of otherwise lethal U.V. lesions. The results rather suggest that U.V. causes, besides lethal lesions, stable photoproducts in the DNA, which are a priori non-lethal, and which are recognized and efficiently eliminated by the HCR repair system. The HCR enzymes likewise act on (non-lethal) u.v.-photoproducts causing prophage induction in lysogenic cells. Consequently, one obtains the maximum induction effect in a lysogenic HCR (-) strain at a much lower u.v.-dose than in the corresponding lysogenic HCR (+) strain. In contrast, u.v.-damage causing loss of the host cell's capacity to support growth of unirradiated phage is not affected by HCR.