Cooperative strategies for pairwise secure communication channels in sensor networks

Establishing secure communication channels in sensor networks is made especially difficult because of low power resources, hostile environments, and wireless communication. The power requirements of traditional cryptographic methods create the need for alternative strategies for secure communication in sensor networks. This thesis explores key distribution techniques in sensor networks. Specifically, we study in depth one method that enables sensors to establish pairwise secure communication channels. This strategy relies on a cooperative set of peer sensors to construct a unique key between two sensors. We built a unique network simulator to test secure communication parameters in a typical deployment scenario. This research tests the strategy by which the cooperative set of sensors is chosen. The results demonstrate that a strategy favoring neighbor nodes consumes significantly less energy than other alternatives at the expense of vulnerability to geographically localized attacks

Hydroxyl groups in the ββ sandwich of metallo-β-lactamases favor enzyme activity: a computational protein design study

Metallo-β-lactamases challenge antimicrobial therapies by their ability to hydrolyze and inactivate a broad spectrum of β-lactam antibiotics. The potential of these enzymes to acquire enhanced catalytic efficiency through mutation is of great concern. Here, we explore the potential of computational protein design to predict mutants of the imipenemase IMP-1 that modulate the catalytic efficiency of the enzyme against a range of substrates. Focusing on the four amino acid positions 69, 121, 218, and 262, we carried out a number of design calculations. Two mutant enzymes were predicted: the single mutant S262A and the double mutant F218Y-S262A. Compared to IMP-1, the single mutant (S262A) results in the loss of a hydroxyl group and the double mutant (F218Y-S262A) results in a hydroxyl transfer from position 262 to position 218. The presence of both hydroxyl groups at positions 218 and 262 was tested by examining the mutant F218Y. Kinetic constants of IMP-1, the two computationally designed mutants (S262A and F218Y-S262A), and the hydroxyl addition mutant (F218Y) were determined with seven substrates. Catalytic efficiencies are highest for the enzyme with both hydroxyl groups (F218Y) and lowest for the enzyme lacking both hydroxyl groups (S262A). The catalytic efficiencies of the two enzymes with one hydroxyl group each are intermediate, with the F218Y-S262A double mutant exhibiting enhanced hydrolysis of nitrocefin, cephalothin, and cefotaxime relative to IMP-1

Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity

Computational protein design methods were used to predict five variants of monofunctional Escherichia coli chorismate mutase expected to maintain catalytic activity. The variants were tested experimentally and three active site mutants exhibited catalytic activity similar to or greater than the wild-type enzyme. One mutant, Ala32Ser, showed increased catalytic efficiency

E. coli Nissle 1917 Affects Salmonella Adhesion to Porcine Intestinal Epithelial Cells

BACKGROUND: The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN. CONCLUSIONS: We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion

Modeling Domino Effects in Enzymes: Molecular Basis of the Substrate Specificity of the Bacterial Metallo-β-lactamases IMP-1 and IMP-6

Metallo-β-lactamases can hydrolyze a broad spectrum of β-lactam antibiotics and thus confer resistance to bacteria. For the Pseudomonas aeruginosa enzyme IMP-1, several variants have been reported. IMP-6 and IMP-1 differ by a single residue (glycine and serine at position 196, respectively), but have significantly different substrate spectra; while the catalytic efficiency toward the two cephalosporins cephalothin and cefotaxime is similar for both variants, IMP-1 is up to 10-fold more efficient than IMP-6 toward cephaloridine and ceftazidime. Interestingly, this biochemical effect is caused by a residue remote from the active site. The substrate-specific impact of residue 196 was studied by molecular dynamics simulations using a cationic dummy atom approach for the zinc ions. Substrates were docked in an intermediate structure near the transition state to the binding site of IMP-1 and IMP-6. At a simulation temperature of 100 K, most complexes were stable during 1 ns of simulation time. However, at higher temperatures, some complexes became unstable and the substrate changed to a nonactive conformation. To model stability, six molecular dynamics simulations at 100 K were carried out for all enzyme−substrate complexes. Stable structures were further heated to 200 and 300 K. By counting stable structures, we derived a stability ranking score which correlated with experimentally determined catalytic efficiency. The use of a stability score as an indicator of catalytic efficiency of metalloenzymes is novel, and the study of substrates in a near-transition state intermediate structure is superior to the modeling of Michaelis complexes. The remote effect of residue 196 can be described by a domino effect:  upon replacement of serine with glycine, a hole is created and a stabilizing interaction between Ser196 and Lys33 disappears, rendering the neighboring residues more flexible; this increased flexibility is then transferred to the active site

Impact of remote mutations on metallo-β-lactamase substrate specificity: implications for the evolution of antibiotic resistance

Metallo-β-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of β-lactam antibiotics. The G262S point mutation distinguishing the metallo-β-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally

Insight into the mechanism of the IMP‐1 metallo‐β‐lactamase by molecular dynamics simulations

Two models, a purely nonbonded model and a cationic dummy atom approach, were examined for the modeling of the binuclear zinc‐containing IMP‐1 metallo‐β‐lactamase in complex with a mercaptocarboxylate inhibitor. The cationic dummy atom approach had substantial advantages as it maintained the initial, experimentally determined geometry of the metal‐containing active site during molecular dynamics simulations in water. The method was extended to the modeling of the free enzyme and the enzyme in complex with a cephalosporin substrate docked in an intermediate structure. For all three systems, the modeled complexes and the tetrahedral coordination of the zinc ions were stable. The average zinc–zinc distance increased by ~1 Å in the substrate complex compared with the inhibitor complex and the free enzyme in which a hydroxide ion acts as a bridging ligand. Thus, the zinc ions are predicted to undergo a back and forth movement upon the cycle of hydrolysis. In contrast to previous assumptions, no interaction of the Asn167 side chain with the bound cephalosporin substrate was observed. Our observations are in agreement with quantum‐mechanical calculations and experimental data and indicate that the cationic dummy atom approach is useful to model zinc‐containing metallo‐β‐lactamases as free proteins, in complex with inhibitors and in complex with substrates

Modeling Domino Effects in Enzymes: Molecular Basis of the Substrate Specificity of the Bacterial Metallo-β-lactamases IMP-1 and IMP-6

Metallo-β-lactamases can hydrolyze a broad spectrum of β-lactam antibiotics and thus confer resistance to bacteria. For the Pseudomonas aeruginosa enzyme IMP-1, several variants have been reported. IMP-6 and IMP-1 differ by a single residue (glycine and serine at position 196, respectively), but have significantly different substrate spectra; while the catalytic efficiency toward the two cephalosporins cephalothin and cefotaxime is similar for both variants, IMP-1 is up to 10-fold more efficient than IMP-6 toward cephaloridine and ceftazidime. Interestingly, this biochemical effect is caused by a residue remote from the active site. The substrate-specific impact of residue 196 was studied by molecular dynamics simulations using a cationic dummy atom approach for the zinc ions. Substrates were docked in an intermediate structure near the transition state to the binding site of IMP-1 and IMP-6. At a simulation temperature of 100 K, most complexes were stable during 1 ns of simulation time. However, at higher temperatures, some complexes became unstable and the substrate changed to a nonactive conformation. To model stability, six molecular dynamics simulations at 100 K were carried out for all enzyme−substrate complexes. Stable structures were further heated to 200 and 300 K. By counting stable structures, we derived a stability ranking score which correlated with experimentally determined catalytic efficiency. The use of a stability score as an indicator of catalytic efficiency of metalloenzymes is novel, and the study of substrates in a near-transition state intermediate structure is superior to the modeling of Michaelis complexes. The remote effect of residue 196 can be described by a domino effect:  upon replacement of serine with glycine, a hole is created and a stabilizing interaction between Ser196 and Lys33 disappears, rendering the neighboring residues more flexible; this increased flexibility is then transferred to the active site