Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria

ABSTRACT Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used β-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and β-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-α levels, resulting in the highest survival rates.

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Differential Tumor Necrosis Factor Alpha Expression and Release from Peritoneal Mouse Macrophages In Vitro in Response to Proliferating Gram-Positive versus Gram-Negative Bacteria

Infection and Immunity ◽

10.1128/iai.68.8.4422-4429.2000 ◽

2000 ◽

Vol 68 (8) ◽

pp. 4422-4429 ◽

Cited By ~ 18

Author(s):

Wei Cui ◽

David C. Morrison ◽

Richard Silverstein

Keyword(s):

Tumor Necrosis ◽

Necrosis Factor Alpha ◽

Gram Negative ◽

E Coli ◽

Tnf Α ◽

Mouse Macrophages ◽

Factor Alpha ◽

Necrosis Factor

ABSTRACT Viable Escherichia coli and Staphylococcus aureus bacteria elicited markedly different in vitro tumor necrosis factor alpha (TNF-α) responses when placed in coculture with peritoneal murine macrophages. These include quantitative differences in TNF-α mRNA expression and corresponding protein product secretion as well as kinetic differences in the profiles of the TNF-α responses. Further, lipopolysaccharide (from E. coli) is a major contributing factor to these differences, as revealed by comparative experiments with endotoxin-responsive (C3Heb/FeJ) and endotoxin-hyporesponsive (C3H/HeJ) macrophages. Nevertheless, the eventual overall magnitude of the TNF-α secretion of macrophages in response to S. aureus was at least equivalent to that observed with E. coli, while appearing at time periods hours later than the E. coli-elicited TNF-α response. Both the magnitude and kinetic profile of the TNF-α responses were found to be relatively independent of the rate of bacterial proliferation, at least to the extent that similar results were observed with both viable and paraformaldehyde-killed microbes. Nevertheless, S. aureus treated in culture with the carbapenem antibiotic imipenem manifests markedly altered profiles of TNF-α response, with the appearance of an early TNF-α peak not seen with viable organisms, a finding strikingly similar to that recently reported by our laboratory from in vivo studies (R. Silverstein, J. G. Wood, Q. Xue, M. Norimatsu, D. L. Horn, and D. C. Morrison, Infect. Immun. 68:2301–2308, 2000). In contrast, imipenem treatment of E. coli-cocultured macrophages does not significantly alter the observed TNF-α response either in vitro or in vivo. In conclusion, our data support the concept that the host inflammatory response of cultured mouse macrophages in response to viable gram-positive versus gram-negative microbes exhibits distinctive characteristics and that these distinctions are, under some conditions, altered on subsequent bacterial killing, depending on the mode of killing. Of potential importance, these distinctive in vitro TNF-α profiles faithfully reflect circulating levels of TNF-α in infected mice. These results suggest that coculture of peritoneal macrophages with viable versus antibiotic-killed bacteria and subsequent assessment of cytokine response (TNF-α) may be of value in clarifying, and ultimately controlling, related host inflammatory responses in septic patients.

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High-Affinity Interaction between Gram-Negative Flagellin and a Cell Surface Polypeptide Results in Human Monocyte Activation

Infection and Immunity ◽

10.1128/iai.68.10.5525-5529.2000 ◽

2000 ◽

Vol 68 (10) ◽

pp. 5525-5529 ◽

Cited By ~ 113

Author(s):

Patrick F. McDermott ◽

Federica Ciacci-Woolwine ◽

James A. Snipes ◽

Steven B. Mizel

Keyword(s):

Cell Surface ◽

Mutational Analysis ◽

Hypervariable Region ◽

Gram Negative Bacteria ◽

Human Monocytes ◽

Necrosis Factor Alpha ◽

Potent Inducer ◽

Gram Negative ◽

High Affinity ◽

Tnf Α

ABSTRACT Flagella from diverse gram-negative bacteria induce tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) synthesis by human monocytes (F. Ciacci-Woolwine, P. F. McDermott, and S. B. Mizel, Infect. Immun. 67:5176–5185, 1999). In this study, we establish that purified flagellin (FliC or FljB), the major filament protein from Salmonella enterica serovar Enteritidis,S. enterica serovar Typhimurium, and Pseudomonas aeruginosa, is an extremely potent inducer of TNF-α production by human monocytes and THP-1 myelomonocytic cells. Fifty percent of maximal TNF-α production (EC50) was obtained with 1.5 × 10−11 M flagellin (0.75 ng/ml). Mutagenesis studies revealed that the central hypervariable region of flagellin is essential for the TNF-α-inducing activity of the protein. Although less active than the wild-type protein, a Salmonellaflagellin mutant composed of only the central hypervariable region retained substantial TNF-α-inducing activity at nanomolar concentrations. In contrast, the conserved amino- and carboxy-terminal regions are inactive. Mutational analysis of the hypervariable region revealed that it contains two equally active TNF-α-inducing domains. The ability of THP-1 cells to respond to purified flagellins is dramatically reduced by mild trypsin treatment of the cells. Taken together, our results demonstrate that the cytokine-inducing activity of flagellins from gram-negative bacteria results from the interaction of these proteins with high-affinity cell surface polypeptide receptors on monocytes.

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Synthesis and in vitro Biological Activity of New 4,6-Disubstituted 3(2H)-Pyridazinone-acetohydrazide Derivatives

Zeitschrift für Naturforschung C ◽

10.1515/znc-2012-5-604 ◽

2012 ◽

Vol 67 (5-6) ◽

pp. 257-265

Author(s):

Murat Sukuroglu ◽

Tijen Onkol ◽

Fatma Kaynak Onurdağ ◽

Gulsen Akalın ◽

M. Fethi Şahin

Keyword(s):

Biological Activity ◽

Antimicrobial Activities ◽

Cytotoxic Activities ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

Preliminary Results ◽

H Nmr ◽

Pyridazinone Derivatives

New 3(2H)-pyridazinone derivatives containing a N’-benzyliden-acetohydrazide moiety at position 2 were synthesized. The structures of these newly synthesized compounds were confi rmed by IR, 1H NMR, and MS data. These compounds were tested for their antibacterial, antifungal, antimycobacterial, and cytotoxic activities. The compounds 2-[4-(4-chlorophenyl)- 6-(morpholin-4-yl)-3-oxo-(2H)-pyridazin-2-yl]-N’-(4-tert-butylbenzyliden)acetohydrazide and 2-[4-(4-chlorophenyl)-6-(morpholin-4-yl)-3-oxo-(2H)-pyridazin-2-yl]-N’-(4-chlorobenzyliden) acetohydrazide exhibited activity against both Gram-positive and Gram-negative bacteria. Most of the compounds were active against E. coli ATCC 35218. The preliminary results of this study revealed that some target compounds exhibited promising antimicrobial activities

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Induction of Cytokine Synthesis by Flagella from Gram-Negative Bacteria May Be Dependent on the Activation or Differentiation State of Human Monocytes

Infection and Immunity ◽

10.1128/iai.67.10.5176-5185.1999 ◽

1999 ◽

Vol 67 (10) ◽

pp. 5176-5185 ◽

Cited By ~ 42

Author(s):

Federica Ciacci-Woolwine ◽

Patrick F. McDermott ◽

Steven B. Mizel

Keyword(s):

Mononuclear Cells ◽

Human Peripheral Blood ◽

Substantial Effect ◽

Gram Negative Bacteria ◽

Peripheral Blood Mononuclear ◽

Necrosis Factor Alpha ◽

Gram Negative ◽

E Coli ◽

Cytokine Synthesis ◽

Normal Human Peripheral Blood

ABSTRACT We have previously demonstrated that salmonellae, but notEscherichia coli or Yersinia enterocolitica, stimulates tumor necrosis factor alpha (TNFα) production in the human promonocytic cell line U38. Subsequent analysis revealed that the TNFα-inducing activity of salmonellae was associated with flagellin, a major component of flagella from gram-negative bacteria. In the present study, we have explored the basis for the apparent specificity of action of Salmonella flagella on TNFα expression in U38 cells and have extended this analysis to normal human peripheral blood mononuclear cells (PBMC). Flagella from the enteropathogenicE. coli strain E2348/69, Y. enterocoliticaJB580, and Pseudomonas aeruginosa PAO1, which did not induce significant levels of TNFα production in U38 cells, were as potent as Salmonella flagella in terms of TNFα and interleukin 1β activation in PBMC. However, TNFα production in U38 cells was greatly enhanced when these cells were stimulated with flagella from E. coli, Y. enterocolitica, andP. aeruginosa in the presence of a costimulant, phorbol 13-myristate acetate. These findings are consistent with the hypothesis that the activation or differentiation state of a monocyte may have a substantial effect on the cell’s responsiveness to flagellum stimulation of cytokine synthesis. Furthermore, these results indicate that cytokine induction in monocytes may be a general property of flagella from gram-negative bacteria.

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Activation of Interleukin-1 Receptor-Associated Kinase by Gram-Negative Flagellin

Infection and Immunity ◽

10.1128/iai.69.7.4424-4429.2001 ◽

2001 ◽

Vol 69 (7) ◽

pp. 4424-4429 ◽

Cited By ~ 61

Author(s):

Marlena A. Moors ◽

Liwu Li ◽

Steven B. Mizel

Keyword(s):

Cell Line ◽

Salmonella Enteritidis ◽

Mutant Cell ◽

Interleukin 1 ◽

Central Component ◽

No Production ◽

Gram Negative Bacteria ◽

Necrosis Factor Alpha ◽

Gram Negative ◽

Tnf Α

ABSTRACT Flagellin from various species of gram-negative bacteria activates monocytes to produce proinflammatory cytokines. We have analyzed the pathway by which Salmonella enteritidis flagellin (FliC) activates murine and human monocyte/macrophage-like cell lines. Since lipopolysaccharide (LPS), the principal immune stimulatory component of gram-negative bacteria, is known to signal through Toll-like receptor 4 (TLR4), we tested the possibility that FliC also signals via TLR4. When murine HeNC2 cells were stimulated with LPS in the presence of a neutralizing anti-TLR4 monoclonal antibody, tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production were markedly reduced. In contrast, FliC-mediated TNF-α and NO production were minimally affected by the anti-TLR4 antibody. Furthermore, FliC, unlike LPS, stimulated TNF-α production in the TLR4 mutant cell line, GG2EE, indicating that TLR4 is not essential for FliC-mediated signaling. To test the possibility that FliC signals via another TLR, we measured FliC-mediated activation of interleukin-1 (IL-1) receptor-associated kinase (IRAK), a central component in IL-1R/TLR signaling. FliC induced IRAK activation in HeNC2 and GG2EE cells as well as in the human promonocytic cell line THP-1. IRAK activation was rapid in HeNC2 cells, with maximal activity observed after 5 min of treatment with FliC. In addition, FliC-mediated IRAK activation exhibited the same concentration dependence as was demonstrated for the induction of TNF-α. These results represent the first demonstration of IRAK activation by a purified bacterial protein and strongly suggest that a TLR distinct from TLR4 is involved in the macrophage inflammatory response to FliC.

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Bacterial Induction of Beta Interferon in Mice Is a Function of the Lipopolysaccharide Component

Infection and Immunity ◽

10.1128/iai.68.3.1600-1607.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1600-1607 ◽

Cited By ~ 65

Author(s):

Andreas Sing ◽

Thomas Merlin ◽

Hans-Peter Knopf ◽

Peter J. Nielsen ◽

Harald Loppnow ◽

...

Keyword(s):

Mouse Strains ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Necrosis Factor Alpha ◽

Gram Negative ◽

Protein Levels ◽

Gram Positive Bacteria ◽

Beta Interferon

ABSTRACT We investigated the reason for the inability of lipopolysaccharide (LPS)-resistant (Lps-defective [Lpsd ]) C57BL/10ScCr mice to produce beta interferon (IFN-β) when stimulated with bacteria. For this purpose, the IFN-β and other macrophage cytokine responses induced by LPS and several killed gram-negative and gram-positive bacteria in LPS-sensitive (Lps-normal [Lpsn ]; C57BL/10ScSn and BALB/c) and Lpsd (C57BL/10ScCr and BALB/c/l) mice in vitro and in vivo were investigated on the mRNA and protein levels. In addition, double-stranded RNA (dsRNA) was used as a nonbacterial stimulus. LPS and all gram-negative bacteria employed induced IFN-β in the Lpsn mice but not in theLpsd mice. All gram-positive bacteria tested failed to induce significant amounts of IFN-β in all four of the mouse strains used. As expected, all other cytokines tested (tumor necrosis factor alpha, interleukin 1α [IL-1α], IL-6, and IL-10) were differentially induced by gram-negative and gram-positive bacteria. Stimulation with dsRNA induced IFN-β and all other cytokines mentioned above in all mouse strains, regardless of their LPS sensitivities. The results suggest strongly that LPS is the only bacterial component capable of inducing IFN-β in significant amounts that are readily detectable under the conditions used in this study. Consequently, in mice, IFN-β is inducible only by gram-negative bacteria, but not in C57BL/10ScCr or other LPS-resistant mice.

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Population Reductions of Gram-Negative Pathogens Following Treatments with Nisin and Chelators under Various Conditions

Journal of Food Protection ◽

10.4315/0362-028x-58.9.977 ◽

1995 ◽

Vol 58 (9) ◽

pp. 977-983 ◽

Cited By ~ 94

Author(s):

CATHERINE N. CUTTER ◽

GREGORY R. SIRAGUSA

Keyword(s):

Divalent Cations ◽

Escherichia Coli O157 ◽

Gram Negative Bacteria ◽

Bacterial Populations ◽

Gram Negative ◽

E Coli ◽

Transmission Electron ◽

Colony Forming Units ◽

Citrate Phosphate

When used in combination with chelating agents (EDTA, EGTA, citrate, phosphate), the bacteriocin nisin is effective for reducing populations of gram-negative bacteria in vitro. This study examined parameters (buffers, temperature presence of divalent cations) that affect nisin inhibition of Escherichia coli O157:H7 and Salmonella typhimurium. Approximately 7 log10 colony-forming units (CFU) per ml of E. coli and S. typhimurium were treated in PBS or MOPS buffers containing 50 μg/ml of purified nisin, alone or in combination with 500 mM lactate, 100 mM citrate, 50 mM EDTA, and 1% (wt/vol) sodium hexametaphosphate (pH 7.0) at 37°C for 60 min or 5°C for 30 min. Surviving bacterial populations were compared to untreated controls (buffers without nisin). Data indicated that treatments with nisin in buffers resulted in reductions of 4.30 and 2.30 log10 CFU/ml of E. coli and S. typhimurium, respectively, as compared to untreated controls. Population reductions ranging from 2.29 to 5.49 log10 CFU/ml were observed when cells were treated with nisin and chelator combinations at either 37°C for 60 min or 5°C for 30 min. The addition of magnesium and calcium to buffers with nisin decreased inhibition. Data obtained from spectrophotometric experiments indicated that treatments were causing the release of cellular constituents. However, transmission electron microscopy (TEM) analyses were inconclusive, since cellular membranes did not appear to be disrupted.

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Comparative Effects of Ciprofloxacin and Ceftazidime on Cytokine Production in Patients with Severe Sepsis Caused by Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.8.2793-2798.2004 ◽

2004 ◽

Vol 48 (8) ◽

pp. 2793-2798 ◽

Cited By ~ 21

Author(s):

C. A. Gogos ◽

A. Skoutelis ◽

A. Lekkou ◽

E. Drosou ◽

I. Starakis ◽

...

Keyword(s):

Severe Sepsis ◽

Group Versus ◽

Gram Negative Bacteria ◽

Proinflammatory Response ◽

Necrosis Factor Alpha ◽

Gram Negative ◽

Tnf Α ◽

Factor Alpha ◽

Anti Inflammatory ◽

Necrosis Factor

ABSTRACT In the present study the effect of ciprofloxacin versus ceftazidime on concentrations of pro- and anti-inflammatory cytokines in the sera of patients with severe sepsis was evaluated. The study included 58 previously healthy patients suffering from severe sepsis caused by gram-negative bacteria, treated with either ciprofloxacin or ceftazidime after thorough clinical and microbiological evaluation and followed up for clinical outcome. Levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1b (IL-1b), IL-6, and IL-8 and of the anti-inflammatory cytokine IL-10, as well as of IL-1 receptor antagonist and soluble TNF receptors I and II, in serum were measured at baseline and 24 and 48 h after the first antimicrobial dose. Mean SAPS-II scores, development of septic shock, and mortality rates were similar in the two groups (43.2 ± 9.2, 21.4%, and 14.3% in the ceftazidime group versus 49.8 ± 11.3, 20%, and 13.3% in the ciprofloxacin group). Serum TNF-α and IL-6 levels at 24 and 48 h were significantly lower in the ciprofloxacin group, while the IL-10/TNF-α ratio was significantly higher, than those for the ceftazidime group. Among patients with high baseline TNF-α levels, there were significant increases in the IL-10/TNF-α ratio at both 24 and 48 h over that at admission for the ciprofloxacin group, while no differences were noted in the ceftazidime group. These results indicate that ciprofloxacin may have an immunomodulatory effect on septic patients by attenuating the proinflammatory response, while there is no evidence that differences in the cytokines measured have any impact on the final outcome.

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Efficacy of the Combination of Tachyplesin III and Clarithromycin in Rat Models of Escherichia coli Sepsis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00086-08 ◽

2008 ◽

Vol 52 (12) ◽

pp. 4351-4355 ◽

Cited By ~ 3

Author(s):

Oscar Cirioni ◽

Roberto Ghiselli ◽

Carmela Silvestri ◽

Wojciech Kamysz ◽

Fiorenza Orlando ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Activity ◽

Bacterial Infections ◽

Plasma Concentrations ◽

Necrosis Factor Alpha ◽

Rat Models ◽

E Coli ◽

Tnf Α ◽

Male Wistar Rats ◽

Isotonic Sodium Chloride

ABSTRACT We investigated the efficacy of tachyplesin III and clarithromycin in two experimental rat models of severe gram-negative bacterial infections. Adult male Wistar rats were given either (i) an intraperitoneal injection of 1 mg/kg Escherichia coli 0111:B4 lipopolysaccharide or (ii) 2 × 1010 CFU of E. coli ATCC 25922. For each model, the animals received isotonic sodium chloride solution, 1 mg/kg tachyplesin III, 50 mg/kg clarithromycin, or 1 mg/kg tachyplesin III combined with 50 mg/kg clarithromycin intraperitoneally. Lethality, bacterial growth in the blood and peritoneum, and the concentrations of endotoxin and tumor necrosis factor alpha (TNF-α) in plasma were evaluated. All the compounds reduced the lethality of the infections compared to that for the controls. Tachyplesin III exerted a strong antimicrobial activity and achieved a significant reduction of endotoxin and TNF-α concentrations in plasma compared to those of the control and clarithromycin-treated groups. Clarithromycin exhibited no antimicrobial activity but had a good impact on endotoxin and TNF-α plasma concentrations. A combination of tachyplesin III and clarithromycin resulted in significant reductions in bacterial counts and proved to be the most-effective treatment in reducing all variables measured.

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Multiple Mechanisms of the Synthesized Antimicrobial Peptide TS against Gram-Negative Bacteria for High Efficacy Antibacterial Action In Vivo

Molecules ◽

10.3390/molecules26010060 ◽

2020 ◽

Vol 26 (1) ◽

pp. 60

Author(s):

Rui Zhang ◽

Xiaobo Fan ◽

Xinglu Jiang ◽

Mingyuan Zou ◽

Han Xiao ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Bacterial Infections ◽

Bacterial Resistance ◽

Divalent Cations ◽

Resistant Bacteria ◽

Antibacterial Drug ◽

Gram Negative Bacteria ◽

Gram Negative

The emergence of drug-resistant bacteria emphasizes the urgent need for novel antibiotics. The antimicrobial peptide TS shows extensive antibacterial activity in vitro and in vivo, especially in gram-negative bacteria; however, its antibacterial mechanism is unclear. Here, we find that TS without hemolytic activity disrupts the integrity of the outer bacterial cell membrane by displacing divalent cations and competitively binding lipopolysaccharides. In addition, the antimicrobial peptide TS can inhibit and kill E. coli by disintegrating the bacteria from within by interacting with bacterial DNA. Thus, antimicrobial peptide TS’s multiple antibacterial mechanisms may not easily induce bacterial resistance, suggesting use as an antibacterial drug to be for combating bacterial infections in the future.

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