Killing Of Bacterial Cells Research Articles

Reaction of protein chloramines with DNA and nucleosides: evidence for the formation of radicals, protein-DNA cross-links and DNA fragmentation.

Stimulated phagocyte cells produce the oxidant HOCl, via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is important in bacterial cell killing, but excessive or misplaced generation can damage the host tissue and may lead to the development of certain diseases such as cancer. The role of HOCl in the oxidation of isolated proteins, DNA and their components has been investigated extensively, but little work has been performed on the protein-DNA (nucleosome) complexes present in eukaryotic cell nuclei. Neither the selectivity of damage in such complexes nor the possibility of transfer of damage from the protein to DNA or vice versa, has been studied. In the present study, kinetic modelling has been employed to predict that reaction occurs predominantly with the protein and not with the DNA in the nucleosome, using molar HOCl excesses of up to 200-fold. With 50-200-fold excesses, 50-80% of the HOCl is predicted to react with histone lysine and histidine residues to yield chloramines. The yield and stability of such chloramines predicted by these modelling studies agrees well with experimental data. Decomposition of these species gives protein-derived, nitrogen-centred radicals, probably on the lysine side chains, as characterized by the EPR and spin-trapping experiments. It is shown that isolated lysine, histidine, peptide and protein chloramines can react with plasmid DNA to cause strand breaks. The protection against such damage afforded by the radical scavengers Trolox (a water-soluble alpha-tocopherol derivative) and 5,5-dimethyl-1-pyrroline-N-oxide suggests a radical-mediated process. The EPR experiments and product analyses have also provided evidence for the rapid addition of protein radicals, formed on chloramine decomposition, to pyrimidine nucleosides to give nucleobase radicals. Further evidence for the formation of such covalent cross-links has been obtained from experiments performed using (3)H-lysine and (14)C-histidine chloramines. These results are consistent with the predictions of the kinetic model and suggest that histones are major targets for HOCl in the nucleosome. Furthermore, the resulting protein chloramines and the radicals derived from them may act as contributing agents in HOCl-mediated DNA oxidation.

Antibacterial activity of Harungana madagascariensis leaf extracts.

Aqueous extracts of the leaf of Harungana madagascariensis were analysed phytochemically and evaluated for antimicrobial activity against strains of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa. Glycosides, tannins, saponins, flavonoids and alkaloids were detected in the plant material. B. subtilis, E. coli and S. typhi, but not Ps. aeruginosa, showed susceptibility at MICs of 2.0 and 15.6 mg/mL; and MBCs of 2.0-3.9 mg/mL and 15.6-31.3 mg/mL, respectively, for the cold and hot extracts. Staph. aureus showed susceptibility only to the hot extract. Concentrations of 2.5-10.0 mg/mL of the cold extract killed over 7 log(10) of the test bacterial population within 30-60 min of exposure. The hot extract needed higher concentrations and longer treatment to achieve similar levels of bacterial cell killing. The results provide a rationalization for the traditional use of H. madagascariensis leaf extracts for the treatment of gastrointestinal disorders.

CRAMP Analogues Having Potent Antibiotic Activity against Bacterial, Fungal, and Tumor Cells without Hemolytic Activity

CRAMP-18 (GEKLKKIGQKIKNFFQKL) is the antibacterial sequence derived from CRMAP, a member of cathelicidin-derived antimicrobial peptides. To develop the novel antibiotic peptides useful as therapeutic drugs requires strong antibiotic activity against bacterial and fungal cells without hemolytic effect. To this goal, the analogues were designed to increase only net positively charge by Lys-substitution of positions 2, 9, 13, or 16 at the hydrophilic helix face of CRAMP-18 without any change at the hydrophobic helix face. In particular, Lys-substitution (K2-CRAMP-18) of position 2 in CRAMP-18 induced the enhanced antibiotic activity without any increase in hemolysis. Thus, this peptide may provide a useful template for the design novel antibiotic peptides for the treatment of infectious diseases. Additional CD spectra studies suggested that the α-helical structure of the peptides plays an important role in killing bacterial and fungal cells, but the increase of α-helical content is less connected with the enhanced antibiotic activity.

Hypochlorite-induced oxidation of thiols: Formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.

Microbial methods for assessment of cleaning and disinfection of food-processing surfaces cleaned in a low-pressure system

An assessment system using various microbial methods was developed for the detection of residual, surface-attached microbes and soil on surfaces after sanitation. The microbial methods tested were: conventional cultivation, microscopy using image analysis after staining with acridine orange (AO), 5-cyano-2,3-ditolyl tetrazolium chloride/4′, 6-diamidino-2-phenylindole (CTC-DAPI) and LIVE/DEAD stains, impedance measurements, viable counts with 2,3,5-triphenyltetrazolium chloride (TTC) agar and ATP measurements. A test rig was used for the trials with four different low-pressure cleaning procedures (pressure <20 bar flow rate 27 l min–1). A strong alkaline foam cleaner, Trippel, and a mild alkaline foam cleaner, Topax 12, in combination with two disinfectants, respectively, were used. The disinfectants were peroxide-peracetic-acid-based Oxonia Aktiv and potassium-persulphate-based Virkon. Conventional cultivation combined with impedance measurements and image analysis of surfaces stained with AO, as well as CTC-DAPI, gave results that were comparable and complementary. The combination of these methods enabled a total evaluation of both the removal of biofilm and the killing of bacterial cells. The low-pressure cleaning system did not remove all of the bacterial cells from the surfaces and did not kill the bacteria even after use of the strong alkaline foam cleaner. The above-mentioned protocol carried out on the test rig can also be used to evaluate the sensitivity of microbial methods for use in certain industrial premises.

Enhancement of macrophage-mediated bactericidal activity by macrophage-mannose receptor-ligand interaction.

Neutrophils represent one of the host's primary defenses against invading organisms. These cells often arrive at the site of infection prior to macrophages (M phi). Neutrophils release myeloperoxidase (MPO) into the micro-environment during phagocytosis. Previous studies by the present investigators have shown that M phi bactericidal activity is enhanced by exposure to MPO. A recent report suggests that as much as 40% of this protein is enzymatically inactive once it is released into the micro-environment. In the present study, exposure of M phi to an enzymatically inactive form of MPO (iMPO) or another mannosylated protein, mannosylated bovine serum albumin (mBSA), can induce the same enhanced Mø-mediated bacterial cell killing observed with the active form of MPO. Furthermore, this phenomenon is limited as galactosylated BSA (gBSA) did not induce enhancement of bacterial killing. The data suggest that interaction of either enzymatically active or inactive mannosylated proteins with the M phi mannose receptor (MMR), is sufficient to enhance M phi bactericidal activity and further underscores the binding of the MMR and resultant responses as a major host defense mechanism.

Poultry Scientists and Agromedicine:

Since the discovery by Glick approximately 40 years ago of the role of bursa of Fabricius in antibody production, research by poultry scientists has continued to add to our knowledge and understanding of basic immunological concepts. The role of nonlymphoid cells--namely cells belonging to the mononuclear phagocytic system lineage in antigen uptake and processing and mediating cellular interactions--is currently an active area of research in animal and human immunology. The mechanisms and pathways are being defined which enable chicken macrophages to perform their effector functions such as phagocytosis, bacterial and tumor cell killing. In addition, nutritional, physiological and genetic parameters are being examined to potentiate the first line of immunological defense by enhancing macrophage functions. These research efforts would benefit the poultry industry in raising chickens with improved disease resistance potential. At the same time, the knowledge of macrophage function pathways would broaden our understanding of the overall role which the immune system plays both in human and animal health.

Killing of Bacillus subtilis by Cell Suicide through Autolysis Induction

A novel method of killing bacterial cells by cell autolysis induction, ‘the suicide induction method, ’ was proposed. Cold shock treatment and the addition of a surfactant or a high concentration of monovalent cations used for autolysis induction were found to cause the substantial cell death of Bacillus subtilis. Enumeration with the conventional colony counting of the wild-type B. subtilis and its autolysis-defective mutant suggests that the autolysis induction is responsible for the induced cell death, namely bacterial suicide. Although no direct relationship between the cell lysis detected by the optical density decrease and the reduction in colony counts was observed, the evaluation of cell death by the respiratory activity measurement supported the above suggestion. In a continuous tube-flow system consisting of a cooling-warming cycle, bacterial suicide of B. subtilis in substantial numbers was observed. We suggest that ‘the suicide induction method’ may be applicable for practical control of some bacteria.

Mucoid Pseudomonas aeruginosa growing in a biofilm in vitro are killed by opsonic antibodies to the mucoid exopolysaccharide capsule but not by antibodies produced during chronic lung infection in cystic fibrosis patients.

Serum opsonophagocytic-killing titers often indicate the level of immune resistance to bacterial pathogens, yet in almost all cystic fibrosis (CF) patients that have chronic lung infections with mucoid Pseudomonas aeruginosa, high titers of opsonic-killing Abs can be measured and the infectious pathology still progresses through pulmonary failure and death. This anomalous finding may be due to the use of suspended cells of P. aeruginosa to evaluate phagocytic killing, whereas in the lungs of CF patients the organisms grow in a microcolony or biofilm, encased in mucoid exopolysaccharide (MEP, also called alginate). To determine whether the microcolony mode of growth contributes to bacterial resistance to host defenses, we evaluated opsonophagocytic killing of mucoid P. aeruginosa growing in a biofilm. Abs from infected CF patients were poorly able to mediate opsonic killing of biofilm, but not suspended, mucoid P. aeruginosa cells. Bacterial resistance to killing could be overcome by disruption of the biofilm layer with an enzyme that degrades MEP. Chronically infected CF patients also fail to produce opsonic-killing Abs specific to MEP, and when these Abs were evaluated in sera of older, noninfected CF patients and humans vaccinated with MEP, comparable killing of P. aeruginosa in biofilms and suspensions was obtained. In this case, C3 was deposited onto the MEP layer and could be visualized by fluorescence microscopy deposited throughout the biofilm. We conclude that opsonic Abs made by CF patients in response to chronic infection are ineffective at mediating phagocytic killing and elimination of bacterial cells growing as microcolonies in their lungs.

Action of cystine in the cytotoxic response of Escherichia coli cells exposed to hydrogen peroxide.

Cystine markedly enhanced the cytotoxic response of Escherichia coli cells to concentrations of hydrogen peroxide resulting in mode one killing, but displayed little effect in mode two killed cells. The effect of cystine was concentration-dependent over a range of 5-50 microM and did not further increase at higher levels. Cystine had similar effects in other bacterial systems. In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells. In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant. The enhancing effect of cystine on oxidative injury of E. coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide. Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant. The effect of the disulfides was not concentration-dependent over a range of 200-800 microM and was statistically significant only for cystamine. Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide. This effect may involve a thiol-disulfide exchange reaction at the cell membrane level.

C9-mediated killing of bacterial cells by transferred C5b-8 complexes: transferred C5b-9 complexes are nonbactericidal

The formation of the C5b-9 complex on the outer membrane of complement-sensitive cells of Escherichia coli results in inhibition of inner membrane function and the death of the cell. Cells bearing a precursor of the C5b-9 site, the C5b-8 complex, suffer no loss in viability. Antibiotic-sensitive, complement-sensitive donor cells bearing precursor C5b-8 complexes were incubated with equal numbers of antibiotic-resistant, complement-sensitive acceptor cells that had not been exposed to a complement source. This cell mixture was incubated with 5 mM EDTA for 5 min and then with calcium chloride (20 mM) for various times. The excess calcium ion concentration was effectively reduced with additional EDTA, and the cell mixture was washed and resuspended in buffer. The viability of the acceptor cells was assayed by plating on antibiotic-containing media. C9 was added to the mixture, and the mixture was incubated for 10 min at 37 degrees C and then plated as described above. It was found that the acceptor cells were killed by the addition of purified C9 only after incubation with donor cells bearing C5b-8 sites during the transfer procedure. This indicates that precursor C5b-8 sites that support C9-mediated killing could be transferred between cells. No loss in viability was detected for acceptor cells subjected to the procedure described above in the presence of donor cells bearing complete C5b-9 complexes, formed prior to mixing with acceptor cells for the transfer procedure.

Differential effects of histidine on hydrogen peroxide-induced bacterial killing and DNA nicking in vitro.

The hydrogen peroxide dose-response curves for Escherichia coli killing and DNA nicking in vitro display remarkably similar bimodal patterns. The concentrations of the oxidant resulting in maximum mode one killing, however, exceeds by two orders of magnitude those resulting in the mode one DNA nicking response. Addition of histidine differentially affects the experimental curves describing the dose-dependency for bacterial killing and DNA damage in vitro. Indeed, the lethal effect elicited by the oxidant in the presence of the amino acid is strictly concentration-dependent and thus the inactivation curve loses its bimodal character. In marked contrast, histidine abolishes DNA damage generated by low concentrations of hydrogen peroxide (< 100 microM) in the in vitro system (the mode one DNA nicking response) but greatly increases DNA damage produced by concentrations of the oxidant higher than 1 mM (the mode two DNA nicking response). Experimental results also suggest that treatment of covalently closed circular double-stranded super-coiled DNA with hydrogen peroxide, in the presence of both histidine and iron, may result in the formation of DNA double strand breakage, a type of lesion which is not efficiently produced by the oxidant in the absence of the amino acid. Taken together, the above results indicate that histidine differentially affects the in vitro DNA cleavage and E. coli lethality induced by hydrogen peroxide and suggest that different molecular events mediate mode one DNA nicking in vitro and mode one killing of bacterial cells.

Bacterial cell killing by antibody targeted photolysis: enhanced effect by OH radical generation

Two structurally distinct immunoconjugates were used for photolysis of bacterial cells. One contained a dextran carbazate (DC) polymer as a linker between the photosensitizer molecules and the Fc oligosaccharide moiety of the monoclonal antibody, while the other linked the photosensitizer molecules and the Fc oligosaccharide by a short ethylenediamine (ED) spacer. The two immunoconjugates exhibited remarkably different photophysical properties and cell killing potential with respect to their abilities to generate singlet oxygen. The DC conjugate exhibited poor singlet oxygen ( 1Δ g) yields, yet was shown to produce more efficient cell killing on the basis of 1Δ g dose than did the ED conjugate. In light of the enhanced cell killing capacity of the DC conjugate, a search for other toxic photoproducts was initiated. It was found that the DC conjugate was capable of generating hydroxyl radicals (OH.) upon light illumination. Quantum yields for OH. generation were evaluated. The DC-photosensitizer polymer appeared to initiate a cascade addition reaction presumably by adding peroxides and hydroperoxides to the glucose residues of the dextran carbazate linker. These results suggest that this radical formation could propagate down the DC polymer and account for the superior cell killing exhibited by the DC conjugate.

Electric depolarization of photosensitized cells: lipid vs. protein alterations

We have monitored several photosensitized reactions in proteins, liposomes and cells under similar conditions. We found that the depolarization of K +-diffusion potential of liposomes or the leakage of an entrapped molecule, calcein, progress at a much slower rate than the photosensitized damage to proteins and the photosensitized killing of bacterial and leukemic cells. X-ray microanalysis revealed that upon light exposure of HP-treated leukemic cells and bacteria, they totally lost their cellular potassium. We deduce that the direct photosensitized oxidation of lipid components cannot cause the depolarization of cells, which in turn could be responsible for their death. A photosensitized damage to protein sites in the cell, probably in the membrane, is a more likely reason for the depolarization, the loss of potassium ions and cell death that is caused in light-activated photodynamic processes.

An Interrelatedness of the Potentiation of Radiation-Induced Bacterial Cell Killing by Cisplatin and Binuclear Rhodium Carboxylates

Cisplatin and binuclear rhodium (Rh2) carboxylates appear to potentiate radiation-induced killing of Salmonella typhimurium cells largely as a consequence of one-electron reduction that leads to an active radiolytic product. This conclusion is supported by results from experiments wherein the hydrated electron and the hydroxyl radical are competed for in the presence of cisplatin and Rh2 carboxylates, and by the similarly shaped radiation survival curves for cisplatin and Rh2 carboxylates wherein potentiation is expressed beyond variable thresholds of radiation dose. Increasing concentrations of phosphate and chloride also inhibit radiation potentiation by both cisplatin and Rh2 carboxylates, further supporting the contention for similar mechanisms. Radiation potentiation by cisplatin is relatively much more sensitive to the inhibition by chloride.

Antibody‐Targeted Photolysisa

Antibody-targeted photolysis is a technique for damaging or killing cells using light and an antibody-bound photosensitizer. In the present study, immunoconjugates were constructed to selectively kill Pseudomonas aeruginosa bacteria using tin (IV) chlorin e6 which was linked to dextran and then bound to the carbohydrate moiety of a monoclonal antibody specific for Pseudomonas aeruginosa Fisher type I polysaccharide antigen. Killing of Pseudomonas during mid-log phase growth was shown to be dependent upon light dose with complete bacterial cell killing observed at an irradiation dose of 80 J/cm2. Individual components of the immunoconjugates (e.g., antibody or chlorin alone) showed no bacterial cytotoxicity and immunoconjugates constructed with nonbinding antibodies were also ineffective as cytotoxic agents. These studies demonstrate that killing of gram negative bacteria using photoradiation is feasible and suggest that this methodology may be applicable in treatment of infections in man.

Potentiation of Radiation-Induced Bacterial Cell Killing by Binuclear Rhodium(II) Complexes and Their Amines

Rhodium(II) binuclear complexes were surveyed for potentiation of radiation-induced cell killing of hypoxic and fully oxic Salmonella typhimurium cells. The Rh2 tetracarbonate ion substantially potentiated hypoxic cell radiation sensitivity. Phosphate interfered with this potentiation. In the latter two respects, radiation potentiation by Rh2 tetracarbonate is similar to that found for Rh2 tetracarboxylates. Amines such as ammonia, methylamine, ethylamine, n-propylamine, and n-butylamine were examined with both Rh2 tetracarbonate and tetraacetate complexes. With Rh2 tetraacetate in phosphate-buffered saline, these amines variably increased radiation potentiation to a maximum of nearly that seen by Rh2 tetraacetate alone in the absence of phosphate. With Rh2 tetracarbonate, particular amines were found to either enhance or restrict radiation potentiation. Results as a whole support the hypothesis that a radiolytic Rh species initiated in a one-electron reduction process external to the cell is responsible for the potentiation by Rh2 complexes in bacteria. Phosphate interference of potentiation by Rh2 tetracetate appears to be limited competitively by amines, suggesting that axial associations of phosphate with the Rh2 center may be involved in the inhibition of radiation potentiation. Of interest in this regard is the finding that 5'-adenosinemonophosphate eliminates the potentiation seen with Rh2 tetraacetate.

Potentiation of Radiation-Induced Bacterial Cell Killing by Binuclear Rhodium(II) Carboxylates

A series of binuclear rhodium(II) tetracarboxylate complexes was examined for potentiation of radiation-induced killing of Salmonella typhimurium cells. Carboxylate bridging ligands were varied as formate, acetate, trifluoroacetate, and propionate. All complexes caused hypoxic non-dose-modifying radiation potentiation in that variable thresholds were obtained with the radiation dose response. In phosphate-buffered saline (PBS), decreasing threshold doses, i.e., increasing potentiating efficiencies, were seen in the order of acetate = trifluoroacetate less than propionate less than formate. Beyond the threshold dose, the degree of potentiation for all complexes in PBS approximated 12 times the degree of radiation sensitivity seen for the N2 baseline of the radiation dose-response curve. No radiation potentiation by Rh2 carboxylates was seen for fully oxic suspensions. Irradiation of cells in the absence of phosphate increased the efficiency as well as the degree of radiation potentiation. It is hypothesized that bacterial radiation potentiation is initiated by one-electron reduction of the Rh2 carboxylates, most likely involving the hydrated electron, followed then by formation of an active product. These events likely occur outside the bacterial cell.

Deficiency of leukocyte adhesion molecules

Study of patients congenitally deficient in expression of the CD11/CD18 dimer has clarified the fundamental role of these transmembrane glycoproteins of the integrin family in multiple functions of polymorphonuclear leukocytes (PMNL). Deficient expression is associated with severe deficiency of PMNL aggregation, adhesion to protein-coated surfaces and endothelial monolayers, locomotion, chemotaxis, phagocytosis and bacterial cell killing. Severely affected patients develop leukocytosis and die in infancy of overwhelming infection. A patient we studied at autopsy died from septicemia and pneumonia. There was striking lack of any acute inflammatory response near sites of necrosis and infection. In the lungs, in contrast, a distinct but deficient acute focal inflammatory response was observed.We used colloidal gold immunolabeling to study the subcellular localization of the CD11/CD18 adhesion molecules in human PMNL and their expression and movement on cell surfaces after stimulation with the chemotactic peptide formyl-met-leu-phe (fMLP). Human PMNL in pyrogen-free PBS were fixed for lh in 1% glutaraldehyde, dehydrated to 70% ethanol and embedded in LR White resin at 55°C. A polyclonal rabbit antiserum recognizing CD18 and absorbed with CD18-deficient PMNL was used to label sites of storage in thin sections, using 5-nm goat-anti-rabbit-IgG gold. We compared sites labeling for CD18, lactoferrin and gelatinase in adjacent thin sections. For surface labeling, cells were fixed in 1% glutaraldehyde for lh, exposed to OKM1 monoclonal antibody, washed and labeled using 36-nm rabbit-anti-mouse-IgG-gold. Controls for both studies included substitution of other or nonspecific primary and secondary antibody. Cells were attached to polylysine-coated coverslips, critical-point dried in CO2, carbon coated and examined by scanning EM using backscattered electron imaging. Other cells were exposed to f(ab’)2 fragments of OKMl and to f (ab’)2-rabbit-anti-mouse prior to fixation, followed by labeling with 36-nm goat-anti-rabbit-gold, or exposed to f(ab’)2-OKMl conjugated to 36-nm gold particles prior to fixation.

Inhibitors of DNA gyrase

Since the action of the two major classes of gyrase inhibitor, the coumarins and the quinolones, differ mechanistically, their primary physiological effects are described separately. These discussions include aspects of inhibition of eukaryotic topoisomerases to facilitate a comparison of prokaryotic and eukaryotic organisms. We then address the repair of drug-induced lesions and factors involved in the killing of bacterial cells by the drugs