Bordetella pertussis and Bordetella bronchiseptica contain nearly identical BvgAS signal-transduction systems that mediate a biphasic transition between virulent (Bvg+) and avirulent (Bvg-) phases. In the Bvg+ phase, the two species express a similar set of adhesins and toxins, and in both organisms the transition to the Bvg- phase occurs in response to the same environmental signals (low temperature or the presence of nicotinic acid or sulphate anion). These two species differ, however, with regard to Bvg(-)-phase phenotypes, host specificity, the severity and course of the diseases they cause, and also potentially in their routes of transmission. To investigate the contribution of the virulence-control system to these phenotypic differences, we constructed a chimeric B. bronchiseptica strain containing bvgAS from B. pertussis and compared it with wild-type B. bronchiseptica in vitro and in vivo. The chimeric strain was indistinguishable from the wild type in its ability to express Bvg(+)- and Bvg(-)- phase-specific factors. However, although the chimeric strain responded to the same signals as the wild type, it differed dramatically in sensitivity to these signals; significantly more nicotinic acid or MgSO4 was required to modulate the chimeric strain compared with the wild-type strain. Despite this difference in signal sensitivity, the chimeric strain was indistinguishable from the wild type in its ability to cause respiratory-tract infections in rats, indicating that the bvgAS loci of B. pertussis and B. bronchiseptica are functionally interchangeable in vivo. By exchanging discrete fragments of bvgAS, we found that the periplasmic region of BvgS determines signal sensitivity.
Systemic bacterial infections are associated with high mortality. The access of bacteria or constituents thereof to systemic circulation induces the massive release of immunomodulatory mediators, ultimately causing tissue hypoperfusion and multiple-organ failure despite adequate antibiotic treatment. Lipid A, the "endotoxic principle" of bacterial lipopolysaccharide (LPS), is one of the major bacterial immunostimuli. Here we demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the Limulus anti-LPS factor for systemic application. We show efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock in vivo, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. The molecular mechanism of LPS neutralization was elucidated by biophysical techniques. The lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure, and the binding affinity of the peptide to LPS exceeds those of known LPS-binding proteins, such as LPS-binding protein (LBP). Our results thus delineate a novel therapeutic strategy for the clinical management of patients with septic shock.The life-threatening clinical consequences of sepsis and septic shock arise from recognition of microbial immunostimulatory molecules by the hosts' professional immune cells and the release of hemodynamically active mediators. The most potent immunostimulatory constituents are part of the microbial cell envelope, such as lipopolysaccharide (LPS) or lipoproteins. They are released continuously due to cell growth and division and massively liberated as a consequence of the attack of the immune system. In the case of Gram-negative bacteria, the most potent factor is LPS, which, therefore, is also called an endotoxin. LPS concentrations in blood serum as low as 1 ng/ml are able to cause sepsis. Septic shock resulting from bacterial infection remains a frequent cause of death, particularly in intensive care units, with more than 200,000 people dying each year in the United States alone. Death by septic shock can happen despite appropriate broad-range antibiotic treatment, which may kill bacteria but is not only incapable of neutralizing immunostimulatory LPS but also may promote its release into circulation (11).The response of mammalian cells to LPS is initiated by its interaction with serum proteins such as lipopolysaccharidebinding protein (LBP) and specific receptors and/or binding proteins of immune cells such as soluble CD14 (sCD14) and membrane-bound CD14 (mCD14), which finally leads to cell activation through the Toll-like receptor 4 (TLR4)-MD-2 pathway (31). The hydrophobic moiety of LPS, lipid A, anchoring LPS to the bacterial outer membrane, constitutes the "endotoxic principle" of LPS (24). Enterobacterial lipid A consists of a diglucosamine backbone phosphorylated at positions 1 and 4Ј, to which six acyl chains are linked at positions 2,3 a...
Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.
Bacterial infections are known to cause severe health-threatening conditions, including sepsis. All attempts to get this disease under control failed in the past, and especially in times of increasing antibiotic resistance, this leads to one of the most urgent medical challenges of our times. We designed a peptide to bind with high affinity to endotoxins, one of the most potent pathogenicity factors involved in triggering sepsis. The peptide Pep19-2.5 reveals high endotoxin neutralization efficiency in vitro, and here, we demonstrate its antiseptic/anti-inflammatory effects in vivo in the mouse models of endotoxemia, bacteremia, and cecal ligation and puncture, as well as in an ex vivo model of human tissue. Furthermore, we show that Pep19-2.5 can bind and neutralize not only endotoxins but also other bacterial pathogenicity factors, such as those from the Gram-positive bacterium Staphylococcus aureus. This broad neutralization efficiency and the additive action of the peptide with common antibiotics makes it an exceptionally appropriate drug candidate against bacterial sepsis and also offers multiple other medication opportunities.
The actions of polymyxin B, rabbit polymorphonuclear lysosome extracts, 14 polycationic peptides (including defensin NP-2, cecropin P1, lactoferricin B, and active peptides from cationic protein 18 and bactenecin), EDTA, and Tris on Brucella spp. were studied, with other gram-negative bacteria as controls. Brucella spp. were comparatively resistant to all of the agents listed above and bound less polymyxin B, and their outer membranes (OMs) were neither morphologically altered nor permeabilized to lysozyme by polymyxin B concentrations, although both effects were observed for controls. EDTA and peptides increased or accelerated the partition of the hydrophobic probe N-phenyl-naphthylamine into Escherichia coli and Haemophilus influenzae OMs but had no effect on Brucella OMs. Since Brucella and H. influenzae OMs are permeable to hydrophobic compounds (G. Martínez de Tejada and I. Moriyón, J. Bacteriol. 175:5273-5275, 1993), the results show that such unusual permeability is not necessarily related to resistance to polycations. Although rough (R) B. abortus and B. ovis were more resistant than the controls were, there were qualitative and quantitative differences with smooth (S) brucellae; this may explain known host range and virulence differences. Brucella S-lipopolysaccharides (LPSs) had reduced affinities for polycations, and insertion of Brucella and Salmonella montevideo S-LPSs into the OM of a Brucella R-LPS mutant increased and decreased, respectively, its resistance to cationic peptides. The results show that the core lipid A of Brucella LPS plays a major role in polycation resistance and that O-chain density also contributes significantly. It is proposed that the features described above contribute to Brucella resistance to the oxygen-independent systems of phagocytes.
The peptide NK-2 is an effective antimicrobial agent with low hemolytic and cytotoxic activities and is thus a promising candidate for clinical applications. It comprises the ␣-helical, cationic core region of porcine NK-lysin a homolog of human granulysin and of amoebapores of pathogenic amoeba. Here we visualized the impact of NK-2 on Escherichia coli by electron microscopy and used NK-2 as a template for sequence variations to improve the peptide stability and activity and to gain insight into the structure/ function relationships. We synthesized 18 new peptides and tested their activities on seven Gram-negative and one Gram-positive bacterial strains, human erythrocytes, and HeLa cells. Although all peptides appeared unordered in buffer, those active against bacteria adopted an ␣-helical conformation in membrane-mimetic environments like trifluoroethanol and negatively charged phosphatidylglycerol (PG) liposomes that mimick the cytoplasmic membrane of bacteria. This conformation was not observed in the presence of liposomes consisting of zwitterionic phosphatidylcholine (PC) typical for the human cell plasma membrane. The interaction was paralleled by intercalation of these peptides into PG liposomes as determined by FRET spectroscopy. A comparative analysis between biological activity and the calculated peptide parameters revealed that the decisive factor for a broad spectrum activity is not the peptide overall hydrophobicity or amphipathicity, but the possession of a minimal positive net charge plus a highly amphipathic anchor point of only seven amino acid residues (two helical turns).
Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8-to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ؍ 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals.
The patterns of susceptibility to hydrophobic and hydrophilic drugs and the uptake of the fluorescent probe N-phenyl-naphthylamine in Brucefla spp., Haemophilus influenzae, Escherichia coli, and deep rough Salmonella minnesota mutants were compared. The results show that the outer membranes of smooth and naturally rough Brucefla spp. do not represent barriers to hydrophobic permeants and that this absence of a barrier relates at least in part to the properties of Brucefla lipopolysaccharide.Gram-negative bacteria have a cell envelope with a cytoplasmic membrane, a periplasmic compartment, and an outer membrane (OM). The OM contains phospholipids, proteins, and a characteristic lipopolysaccharide (LPS) located in the outer leaflet. The distribution and molecular properties of LPS, phospholipids, and porin proteins play a key role in both the protection against some harmful agents and the access of substances to the periplasmic space and transport systems (14). Hydrophilic solutes of appropriate molecular characteristics penetrate the OM through porins, and harmful agents that bind to lipid A of the LPS weaken the OM barrier and penetrate via the self-promoted pathway (6,14). Moreover, uptake of hydrophobic substances can happen by partition into the lipid phase of the OM, but in most gram-negative bacteria this hydrophobic pathway is not accessible (6,7,14). The few exceptions described include the genera with O-chain-lacking LPSs (i.e., Neisseria and Haemophilus) (6-8) and some deep rough mutants of enteric bacteria (14).Brucella spp. are facultative intracellular pathogens of both humans and animals. The genus includes species with typical smooth LPS (Brucella abortus, B. melitensis, B. suis, and B. neotomae) along with others (B. ovis and B. canis) that lack the 0 chain and that are usually designated naturally rough brucellae because of their similarity with the true rough mutants (3). Porins in Brucella spp. have been characterized and shown to have sieving properties similar to those of Escherichia coli OmpF (4), but evidence for other uptake pathways is only indirect (4,10 These results can be taken as indirect evidence for a functional hydrophobic pathway (7).Direct proof was obtained by using viable cells and N-phenyl-naphthylamine (NPN), a fluorescent probe whose quantum yield increases upon transfer from a hydrophilic to a hydrophobic environment (17) and which has been used in OM permeation studies (16). Exponentially growing cells were harvested (centrifuged for 10 min at 5,000 x g at 20'C), resuspended in 1 mM KCN-10 mM phosphate-buffered saline (pH 7.2) at an optical density (600 nm) of 0.48 (370 pug [dry weight]/ml), and transferred to 1-cm-diameter fluorimetric cuvettes. NPN (500 ,uM in acetone) was added after 15 to 20 s to a final concentration of 10 ,uM. Fluorescence was monitored at 20'C with an LS-50 fluorimeter (Perkin-Elmer Ltd., Beaconsfield, England) set as follows: excitation, 350 nm; emission, 420 nm; and slit widths, 2.5 nm (16). Whereas only a small increase in fluorescence (up to 25...
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