Gadd45α activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo

Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues. Shigella spp. are human pathogens that invade colonic mucosa, where they provoke lesions caused by their ability to manipulate the host cell responses. Shigella spp. induce various types of cell death in different cell populations. However, they are equally able to protect host cells from death. Here, we have investigated on the molecular mechanisms and cell effectors governing the balance between survival and death in epithelial cells infected with Shigella. To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis. Our results definitely show that Shigella induces a rapid intrinsic apoptosis of infected cells, via mitochondrial depolarization and the ensuing caspase-9 activation. Moreover, for the first time we identify the eukaryotic stress-response factor growth arrest and DNA damage 45α as a key player in the induction of the apoptotic process elicited by Shigella in epithelial cells, revealing an unexplored role of this molecule in the course of infections sustained by invasive pathogens

Interaction of melittin with endotoxic lipid

Several amphipathic and cationic substances are known to bind lipid A, the toxic component of bacterial lipopolysaccharides. In this report, we have characterized, by fluorescence methods, the interaction of melittin, an amphipathic and basic 26-residue polypeptide isolated from bee venom, with lipid A. The stoichiometry of the complex appears to be two molecules of melittin to one of lipid A with a dissociation constant of 2.5 × 10 −6 M. The binding of melittin not only modifies the endotoxic properties of lipid A in a number of biological assays, but also results in abrogation of the hemolytic activity of melittin. A model of the complex is proposed based on the known structures of lipid A and melittin, and the observed stoichiometry of binding

Interactions of linear dicationic molecules with lipid A: structural requisites for optimal binding affinity

The structural determinants of the binding affinity of linear dicationic molecules toward lipid A have been examined with respect to the distance between the terminal cationic functions, the basicity, and the type of cationic moieties using a series of spermidine derivatives and pentamidine analogs by fluorescence spectroscopic methods, The presence of two terminal cationic groups corresponds to enhanced affinity, A distinct sigmoidal relationship between the intercationic distance and affinity was observed with a sharp increase at 11 Angstrom, levelling off at about 13 Angstrom. The basicity (pK) and nature of the cationic functions are poor correlates of binding potency, since molecules bearing primary amino, imidazolino, or guanido termini are equipotent, The interaction of pentamidine, a bisamidine drug, with lipid A, characterized in considerable detail employing the putative intermolecular excimerization of the drug, suggests a stoichiometry of 1:1 in the resultant complex, The binding is driven almost exclusively by electrostatic forces, and is dependent on the ionization states of both lipid A and the drug, Under conditions when lipid A is highly disaggregated, pentamidine binds specifically to bis-phosphoryl- but not to monophosphoryl-lipid A indicating that both phosphate groups of lipid A are necessary for electrostatic interactions by the terminal amidininium groups of the drug, Based on these data, a structural model is proposed for the pentamidine-lipid A complex, which may be of value in designing endotoxin antagonists from first principles

Characterization of the interaction of lipid A and lipopolysaccharide with human serum albumin: implications for an endotoxin carrier function for albumin

The interactions of lipid A and lipopolysaccharide (LPS) with human serum albumin (HSA) were examined using fluorescence methods. Lipid A binds HSA with a stoichiometry of 2:1 with dissociation constants of 1.0 µM and 6.0 µM for the high- and low-affinity interactions, respectively. Lipid A displaces HSA-bound dansylsarcosine competitively, but not HSA-bound warfarin, suggesting that domain III-A, and not domain 11-A, is a lipid A binding site. Domain I does not contribute a site for lipid A. Based on these data, and the structural similarity between subdomains III-A and III-B, it is proposed that these two regions of HSA represent the high- and low-affinity sites of interaction of lipid A. Whole LPS also binds HSA, displacing dansylsarcosine, and its lipid A moiety appears to be the interaction site. However, there are differences between LPS and free lipid A. Polymyxin B forms ternary complexes with LPS bound to HSA, suggesting that the regions on LPS recognized by HSA and polymyxin B are different. The observed affinity of lipid A for HSA and mass action effects due to its abundance in the circulation would imply a major LPS carrier function for HSA

Analysis of the binding of polymyxin B to endotoxic lipid A and core glycolipid using a fluorescent displacement probe

Dansylcadaverine, a cationic fluorescent probe binds to bacterial lipopolysaccharide and lipid A, and is displaced competitively by other compounds which possess affinity toward endotoxins. The binding parameters of dansylcadaverine for lipid A were determined by Scatchard analysis to be two apparently equivalent sites with apparent dissociation constants (Kd) ranging between 16 μM to 26 μM, while that obtained for core glycolipid from Salmonella minnesota Re595 yielded a Kd of 22 μM to 28 μM with three binding sites. The Kd of polymyxin B for lipid A was computed from dansylcadaverine displacement by the method of Horovitz and Levitzki (Horovitz, A., and Levitzki, A. (1987) Proc. Natl. Acad. Sci. USA 84, 6654–6658). The applicability of this method for analyzing fluorescence data was validated by comparing the Kds of melittin for lipid A obtained by direct Scatchard analysis, and by the Horovitz-Levitzki method. The displacement of dansylcadaverine from lipid A by polymyxin B was distinctly biphasic with Kds for polymyxin B-lipid A interactions corresponding to 0.4 μM and 1.5 μM, probably resulting as a consequence of lipid A being a mixture of mono- and di-phosphoryl species. This was not observed with core glycolipid, for which the Kd for polymyxin was estimated to range from 1.1 μM to 5.8 μM. The use of dansylcadaverine as a displacement probe offers a novel and convenient method of quantitating the interactions of a wide variety of substances with lipid A

Polymyxin B Nonapeptide: Conformations in Water and in the Lipopolysaccharide-Bound State Determined by Two-Dimensional NMR and Molecular Dynamics

Polymyxin B nonapeptide (PMN) is a derivative of polymyxin B, an alpha, gamma-diaminobutyric acid-rich decapeptide from Bacillus polymyxa that displays antimicrobial and lipopolysaccharide (LPS)-antagonistic activities. The conformations of PMN in aqueous solution as well as in the LPS-bound state have been studied by 1H two-dimensional nmr methods in conjunction with molecular dynamics techniques. The aqueous structure of the free peptide is characterized by a type II beta-turn centered around D-Phe5 and Leu6, and an inverse -turn at Thr9. The LPS-bound conformation of PMN was studied by transferred nuclear Overhauser effect experiments. The essential features of the cyclic portion of free aqueous PMN are mostly preserved when the peptide is bound to LPS; however, the linear dipeptide fragment as well as the side chains of the heptatpeptide ring show a conformational change and a reduction in mobility. The LPS-bound PMN structure was used to construct a model of the lipid A-polymyxin B (decapeptide) complex, which allows the rationalization of several experimental observations concerning the binding of polymyxin to, and consequent neutralization of, the toxicity of LPS, and may be of value in the rational therapeutic targeting of endotoxin

Interaction of cationic amphiphilic drugs with lipid A: Implications for development of endotoxin antagonists

This report presents evidence for the interactions of several classes of cationic amphiphilic drugs including the phenothiazines, aminoquinolines, biguanides, and aromatic diamidines, with lipid A, the endotoxic principle of lipopolysaccharides. The interactions of the drugs were quantitatively assessed by fluorescence methods. The affinities of the drugs for lipid A parallel their endotoxin-antagonistic effects in the Limulus gelation assay. Dicationic compounds bind lipid A with greater affinity; the affinity of such molecules increases exponentially as a function of the distance between the basic moieties. The bis-amidine drug - pentamidine - examined in greater detail, binds lipid A with high affinity (apparent K-d: 0.12 mu M), and LPS, probably due to simultaneous interactions of the terminal amidine groups with the anionic phosphates on lipid A. The sequestration of endotoxin by pentamidine reduces its propensity to bind to cells, and the complex exhibits attenuated toxicity in biological assays. These results have implications in the development of therapeutic strategies against endotoxin-related disease states