Biomedical subjects

Robert E W Hancock

Publications and source records attributed to Robert E W Hancock.

At least 19 recordsLinked to original sources

PubMed2026

Pseudomonas aeruginosa mgtC gene is under the control of PhoP and CbrAB regulators, and its expression can be visualized in macrophages.

The MgtC virulence factor is important during the intramacrophage stage in both classical intracellular pathogens, such as Salmonella Typhimurium, and in extracellular bacteria that transiently encounter intracellular environments during infection, such as Pseudomonas aeruginosa. In these different pathogens, mgtC expression is induced in vitro by magnesium ion depletion, a condition reported to mimic the macrophage environment. Here, we developed an unstable GFP reporter system to monitor in real time the transcriptional activation of the P. aeruginosa mgtC promoter. After in vitro validation in magnesium-defined media, this reporter system allowed visualization of the mgtC promoter induction in a subset of bacteria when P. aeruginosa localized inside cultured macrophages. In addition, although rare under our experimental conditions, in vivo activation of the mgtC promoter was observed for the first time within macrophages of live, infected zebrafish larvae, a cutting-edge vertebrate model for real-time imaging. While MgtC regulation in Salmonella is mediated by the magnesium-responsive PhoPQ two-component system, its regulation in P. aeruginosa remained unknown. The use of mutant strains for two-component regulatory systems revealed that the PhoP regulator, but not by its cognate sensor PhoQ, was required to activate P. aeruginosa MgtC expression in vitro. Unexpectedly, CbrAB, a two-component system specific to Pseudomonas species, was also involved in P. aeruginosa MgtC regulation. Both PhoP and CbrB regulatory proteins were found to directly bind the mgtC promoter, supporting a dual transcriptional control. These findings reveal substantial differences in mgtC gene regulation in different bacterial pathogens, reflecting distinct strategies to drive appropriate expression of a shared virulence factor involved in macrophage adaptation.IMPORTANCEThe adaptation of bacterial pathogens to the host intracellular microenvironment requires tight and rapid regulation of specific genes, and investigating the in vivo transcriptional dynamics of such genes is a major challenge. Here, we focused on the expression of mgtC, a gene important for adaptation to the intramacrophage environment in classical intracellular pathogens, such as Salmonella Typhimurium, and bacteria with a transient intracellular lifestyle, such as Pseudomonas aeruginosa. An unstable GFP reporter system was designed to monitor the transcriptional dynamics of P. aeruginosa mgtC. The use of this reporter system in a state-of-the-art vertebrate model for live imaging, the zebrafish embryo, allowed in vivo tracking of P. aeruginosa mgtC promoter activation inside macrophages in a living host. Furthermore, the expression of P. aeruginosa mgtC was found to be regulated through a mechanism distinct from that of Salmonella MgtC, since it involves the PhoP regulatory protein, but not the PhoQ sensor, and the Pseudomonas-specific CbrAB two-component system, reflecting diverse, finely tuned strategies to control a virulence factor shared by several major human pathogens.

Pseudomonas aeruginosa↗

PubMed2007

Fluorescent probes alter miscibility phase boundaries in ternary vesicles.

We use 2H NMR to study the effects of probes on the miscibility transition in multilamellar vesicles of di(18:1) phosphatidylcholine (PC; DOPC), chain perdeuterated di(16:0)PC (DPPCd62), and cholesterol both with and without 0.5 mol % of the fluorescent probes DiIC12 and DiOC18. Both probes raise the miscibility transition temperature in dispersions of 1:1 DOPC/DPPCd62 + 30% cholesterol but to differing extents. In membranes containing the popular probe DiIC12, the fraction of DPPCd62 lipids in the liquid disordered phase is increased, and the ordering of that phase is reduced even at low temperatures. All findings are consistent with a probe-induced expansion of the entire miscibility phase boundary. We examine membranes with smaller DiIC12 fractions and find a significant increase in transition temperature for samples with 0.05 mol % DiIC12, demonstrating that trace components can dramatically alter membrane phase behavior.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2006

An arginine ladder in OprP mediates phosphate-specific transfer across the outer membrane.

The outer membrane protein OprP mediates the transport of essential phosphate anions into the pathogenic bacterium Pseudomonas aeruginosa. Here we report the crystallographic structure of trimeric OprP at 1.9-A resolution, revealing an unprecedented 9-residue arginine 'ladder' that spans from the extracellular surface down through a constriction zone where phosphate is coordinated. Lysine residues coat the inner periplasmic surface, creating an 'electropositive sink' that pulls the phosphates through the eyelet and into the cell.

Arginine↗

PubMed2006

Identification of genes involved in swarming motility using a Pseudomonas aeruginosa PAO1 mini-Tn5-lux mutant library.

During a screening of a mini-Tn5-luxCDABE transposon mutant library of Pseudomonas aeruginosa PAO1 for alterations in swarming motility, 36 mutants were identified with Tn5 insertions in genes for the synthesis or function of flagellin and type IV pilus, in genes for the Xcp-related type II secretion system, and in regulatory, metabolic, chemosensory, and hypothetical genes with unknown functions. These mutants were differentially affected in swimming and twitching motility but in most cases had only a minor additional motility defect. Our data provide evidence that swarming is a more complex type of motility, since it is influenced by a large number of different genes in P. aeruginosa. Conversely, many of the swarming-negative mutants also showed an impairment in biofilm formation, indicating a strong relationship between these types of growth states.

Bacterial Proteins↗

PubMed2006

IRAK-4 mutation (Q293X): rapid detection and characterization of defective post-transcriptional TLR/IL-1R responses in human myeloid and non-myeloid cells.

Innate immunodeficiency has recently been reported as resulting from the Q293X IRAK-4 mutation with consequent defective TLR/IL-1R signaling. In this study we report a method for the rapid allele-specific detection of this mutation and demonstrate both cell type specificity and ligand specificity in defective IL-1R-associated kinase (IRAK)-4-deficient cellular responses, indicating differential roles for this protein in human PBMCs and primary dermal fibroblasts and in LPS, IL-1beta, and TNF-alpha signaling. We demonstrate transcriptional and post-transcriptional defects despite NF-kappaB signaling and intact MyD88-independent signaling and propose that dysfunctional complex 1 (IRAK1/TRAF6/TAK1) signaling, as a consequence of IRAK-4 deficiency, generates specific defects in MAPK activation that could underpin this patient's innate immunodeficiency. These studies demonstrate the importance of studying primary human cells bearing a clinically relevant mutation; they underscore the complexity of innate immune signaling and illuminate novel roles for IRAK-4 and the fundamental importance of accessory proinflammatory signaling to normal human innate immune responses and immunodeficiencies.

Amino Acid Sequence↗

PubMed2006

Characterization of OpdH, a Pseudomonas aeruginosa porin involved in the uptake of tricarboxylates.

The Pseudomonas aeruginosa outer membrane is intrinsically impermeable to many classes of antibiotics, due in part to its relative lack of general uptake pathways. Instead, this organism relies on a large number of substrate-specific uptake porins. Included in this group are the 19 members of the OprD family, which are involved in the uptake of a diverse array of metabolites. One of these porins, OpdH, has been implicated in the uptake of cis-aconitate. Here we demonstrate that this porin may also enable P. aeruginosa to take up other tricarboxylates. Isocitrate and citrate strongly and specifically induced the opdH gene via a mechanism involving derepression by the putative two-component regulatory system PA0756-PA0757. Planar bilayer analysis of purified OpdH demonstrated that it was a channel-forming protein with a large single-channel conductance (230 pS in 1 M KCl; 10-fold higher than that of OprD); however, we were unable to demonstrate the presence of a tricarboxylate binding site within the channel. Thus, these data suggest that the requirement for OpdH for efficient growth on tricarboxylates was likely due to the specific expression of this large-channel porin under particular growth conditions.

Amino Acid Sequence↗

PubMed2006

Bovine and human cathelicidin cationic host defense peptides similarly suppress transcriptional responses to bacterial lipopolysaccharide.

Genomic approaches can be exploited to expose the complexities and conservation of biological systems such as the immune network across various mammalian species. In this study, temporal transcriptional expression profiles were analyzed in human and bovine monocytic cells in response to the TLR-4 agonist, LPS, in the presence or absence of their respective host defense peptides. The cathelicidin peptides, human LL-37 and bovine myeloid antimicrobial peptide-27 (BMAP-27), are homologs, yet they have diverged notably in terms of sequence similarity. In spite of their low sequence similarities, both of these cathelicidin peptides demonstrated potent, antiendotoxin activity in monocytic cells at low, physiologically relevant concentrations. Microarray studies indicated that 10 ng/ml LPS led to the up-regulation of 125 genes in human monocytes, 106 of which were suppressed in the presence of 5 mug/ml of the human peptide LL-37. To confirm and extend these data, temporal transcriptional responses to LPS were assessed in the presence or absence of the species-specific host defense peptides by quantitative real-time PCR. The transcriptional trends of 20 LPS-induced genes were analyzed in bovine and human monocytic cells. These studies demonstrated conserved trends of gene responses in that both peptides were able to profoundly suppress many LPS-induced genes. Consistent with this, the human and bovine peptides suppressed LPS-induced translocation of NF-kappaB subunits p50 and p65 into the nucleus of monocytic cells. However, there were also distinct differences in responses to LPS and the peptides; for example, treatment with 5 mug/ml BMAP-27 alone tended to influence gene expression (RELA, TNF-alpha-induced protein 2, MAPK phosphatase 1/dual specificity phosphatase 1, IkappaBkappaB, NFkappaBIL1, TNF receptor-associated factor 2) to a greater extent than did the same amount of human LL-37. We hypothesize that the immunomodulatory effects of the species-specific host defense peptides play a critical role in regulating inflammation and represent an evolutionarily conserved mechanism for maintaining homeostasis, although the sequence divergence of these peptides is substantial.

Active Transport, Cell Nucleus↗

PubMed2006

The human cationic host defense peptide LL-37 mediates contrasting effects on apoptotic pathways in different primary cells of the innate immune system.

The human cathelicidin LL-37 is a cationic host defense peptide (antimicrobial peptide) expressed primarily by neutrophils and epithelial cells. This peptide, up-regulated under conditions of inflammation, has immunomodulatory and antimicrobial functions. We demonstrate that LL-37 is a potent inhibitor of human neutrophil apoptosis, signaling through P2X(7) receptors and G-protein-coupled receptors other than the formyl peptide receptor-like-1 molecule. This process involved modulation of Mcl-1 expression, inhibition of BID and procaspase-3 cleavage, and the activation of phosphatidylinositol-3 kinase but not the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway. In contrast to the inhibition of neutrophil apoptosis, LL-37 induced apoptosis in primary airway epithelial cells, demonstrating alternate consequences of LL-37-mediated modulation of apoptotic pathways in different human primary cells. We propose that these novel immunomodulatory properties of LL-37 contribute to peptide-mediated enhancement of innate host defenses against acute infection and are of considerable significance in the development of such peptides and their synthetic analogs as potential therapeutics for use against multiple antibiotic-resistant infectious diseases.

Antimicrobial Cationic Peptides↗

PubMed2006

Host defence peptides from invertebrates--emerging antimicrobial strategies.

Cationic antimicrobial (host defence) peptides are found as potent components of the innate immune system of all invertebrates in which they have been investigated. They vary substantially in their amino acid sequences, secondary structures, inducibility, potency and antimicrobial activity spectra. This enormous diversity is providing templates for the design and development of both antibiotic peptides and peptides that selectively modulate innate immunity to increase protection against infections and sepsis.

Amino Acid Sequence↗

PubMed2006

Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: comparison with cationic antimicrobial peptides and lipopeptides.

With the steady rise in the number of antibiotic-resistant Gram-positive pathogens, it has become increasingly important to find new antibacterial agents which are highly active and have novel and diversified mechanisms of action. Two classes will be discussed here: the cationic antimicrobial peptides, which are amphiphilic in nature, targeting membranes and increasing their permeability; and lipopeptides, which consist of linear or cyclic peptides with an N-terminus that is acylated with a fatty acid side chain. One member of the cyclic lipopeptide family, the anionic molecule daptomycin, has been extensively studied and is the major focus of this review. Models will be presented on its mode of action and comparisons will be made to the known modes of action of cationic antimicrobial peptides and other lipopeptides.

Anti-Bacterial Agents↗

PubMed2006

Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37.

The sole human cathelicidin peptide, LL-37, has been demonstrated to protect animals against endotoxemia/sepsis. Low, physiological concentrations of LL-37 (< or =1 microg/ml) were able to modulate inflammatory responses by inhibiting the release of the proinflammatory cytokine TNF-alpha in LPS-stimulated human monocytic cells. Microarray studies established a temporal transcriptional profile and identified differentially expressed genes in LPS-stimulated monocytes in the presence or absence of LL-37. LL-37 significantly inhibited the expression of specific proinflammatory genes up-regulated by NF-kappaB in the presence of LPS, including NFkappaB1 (p105/p50) and TNF-alpha-induced protein 2 (TNFAIP2). In contrast, LL-37 did not significantly inhibit LPS-induced genes that antagonize inflammation, such as TNF-alpha-induced protein 3 (TNFAIP3) and the NF-kappaB inhibitor, NFkappaBIA, or certain chemokine genes that are classically considered proinflammatory. Nuclear translocation, in LPS-treated cells, of the NF-kappaB subunits p50 and p65 was reduced > or =50% in the presence of LL-37, demonstrating that the peptide altered gene expression in part by acting directly on the TLR-to-NF-kappaB pathway. LL-37 almost completely prevented the release of TNF-alpha and other cytokines by human PBMC following stimulation with LPS and other TLR2/4 and TLR9 agonists, but not with cytokines TNF-alpha or IL-1beta. Biochemical and inhibitor studies were consistent with a model whereby LL-37 modulated the inflammatory response to LPS/endotoxin and other agonists of TLR by a complex mechanism involving multiple points of intervention. We propose that the natural human host defense peptide LL-37 plays roles in the delicate balancing of inflammatory responses in homeostasis as well as in combating sepsis induced by certain TLR agonists.

Antimicrobial Cationic Peptides↗

PubMed2006

Sequence requirements and an optimization strategy for short antimicrobial peptides.

Short antimicrobial host-defense peptides represent a possible alternative as lead structures to fight antibiotic resistant bacterial infections. Bac2A is a 12-mer linear variant of the naturally occurring bovine host defense peptide, bactenecin, and demonstrates moderate, broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria as well as against the yeast Candida albicans. With the assistance of a method involving peptide synthesis on a cellulose support, the primary sequence requirements for antimicrobial activity against the human pathogen Pseudomonas aeruginosa of 277 Bac2A variants were investigated by using a luciferase-based assay. Sequence scrambling of Bac2A led to activities ranging from superior or equivalent to Bac2A to inactive, indicating that good activity was not solely dependent on the composition of amino acids or the overall charge or hydrophobicity, but rather required particular linear sequence patterns. A QSAR computational analysis was applied to analyze the data resulting in a model that supported this sequence pattern hypothesis. The activity of selected peptides was confirmed by conventional minimal inhibitory concentration (MIC) analyses with a panel of human pathogen bacteria and fungi. Circular-dichroism (CD) spectroscopy with selected peptides in liposomes and membrane depolarization assays were consistent with a relationship between structure and activity. An additional optimization process was performed involving systematic amino acid substitutions of one of the optimal scrambled peptide variants, resulting in superior active peptide variants. This process provides a cost and time effective enrichment of new candidates for drug development, increasing the chances of finding pharmacologically relevant peptides.

Amino Acid Sequence↗

PubMed2006

Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies.

Short cationic amphiphilic peptides with antimicrobial and/or immunomodulatory activities are present in virtually every life form, as an important component of (innate) immune defenses. These host-defense peptides provide a template for two separate classes of antimicrobial drugs. Direct-acting antimicrobial host-defense peptides can be rapid-acting and potent, and possess an unusually broad spectrum of activity; consequently, they have prospects as new antibiotics, although clinical trials to date have shown efficacy only as topical agents. But for these compounds to fulfill their therapeutic promise and overcome clinical setbacks, further work is needed to understand their mechanisms of action and reduce the potential for unwanted toxicity, to make them more resistant to protease degradation and improve serum half-life, as well as to devise means of manufacturing them on a large scale in a consistent and cost-effective manner. In contrast, the role of cationic host-defense peptides in modulating the innate immune response and boosting infection-resolving immunity while dampening potentially harmful pro-inflammatory (septic) responses gives these peptides the potential to become an entirely new therapeutic approach against bacterial infections.

Anti-Infective Agents, Local↗

PubMed2006

Involvement of two related porins, OprD and OpdP, in the uptake of arginine by Pseudomonas aeruginosa.

The OprD family of specific porins in Pseudomonas aeruginosa comprises 19 members, some of which have been demonstrated to facilitate the uptake of specific compounds into the cell. The members of this family share considerable amino acid sequence similarity (46-57%), which is unusual among porin molecules. In this work, we sought to establish whether this sequence conservation was the basis for other shared aspects of this family. The transcriptional profiles of eight relatively well-characterized OprD homologs were assessed in cells grown on a variety of carbon compounds. The expression of these paralogous proteins correlated with their phylogenetic distribution into two subfamilies in that the three members of the OpdK subfamily were induced by their specific (organic acid) substrates while the five members of the amino-acid/peptide-specific OprD subfamily appeared to be constitutively expressed. Functional overlap with respect to arginine transport was observed between two members of the latter subfamily, the basic amino acid-specific porin, OprD, and the glycine-glutamate-specific porin, OpdP. The impact of this apparent functional redundancy on the genetic fitness of P. aeruginosa is discussed.

Arginine↗

PubMed2006

Comparison of biophysical and biologic properties of alpha-helical enantiomeric antimicrobial peptides.

In our previous study (Chen et al. J Biol Chem 2005, 280:12316-12329), we utilized an alpha-helical antimicrobial peptide V(681) as the framework to study the effects of peptide hydrophobicity, amphipathicity, and helicity on biologic activities where we obtained several V(681) analogs with dramatic improvement in peptide therapeutic indices against gram-negative and gram-positive bacteria. In the present study, the D-enantiomers of three peptides--V(681), V13A(D) and V13K(L) were synthesized to compare biophysical and biologic properties with their enantiomeric isomers. Each D-enantiomer was shown by circular dichroism spectroscopy to be a mirror image of the corresponding L-isomer in benign conditions and in the presence of 50% trifluoroethanol. L- and D-enantiomers exhibited equivalent antimicrobial activities against a diverse group of Pseudomonas aeruginosa clinical isolates, various gram-negative and gram-positive bacteria and a fungus. In addition, L- and D-enantiomeric peptides were equally active in their ability to lyse human red blood cells. The similar activity of L- and D-enantiomeric peptides on prokaryotic or eukaryotic cell membranes suggests that there are no chiral receptors and the cell membrane is the sole target for these peptides. Peptide D-V13K(D) showed significant improvements in the therapeutic indices compared with the parent peptide V(681) by 53-fold against P. aeruginosa strains, 80-fold against gram-negative bacteria, 69-fold against gram-positive bacteria, and 33-fold against Candida albicans. The excellent stability of D-enantiomers to trypsin digestion (no proteolysis by trypsin) compared with the rapid breakdown of the L-enantiomers highlights the advantage of the D-enantiomers and their potential as clinical therapeutics.

Animals↗

PubMed2006

The antimicrobial peptide polyphemusin localizes to the cytoplasm of Escherichia coli following treatment.

The horseshoe crab peptide polyphemusin I possesses high antimicrobial activity, but its mechanism of action is as yet not well defined. Using a biotin-labeled polyphemusin I analogue and confocal fluorescence microscopy, we showed that the peptide accumulates in the cytoplasm of wild-type Escherichia coli within 30 min after addition without causing substantial membrane damage.

Amino Acid Sequence↗

PubMed2006

Peptide antimicrobial agents.

Antimicrobial host defense peptides are produced by all complex organisms as well as some microbes and have diverse and complex antimicrobial activities. Collectively these peptides demonstrate a broad range of antiviral and antibacterial activities and modes of action, and it is important to distinguish between direct microbicidal and indirect activities against such pathogens. The structural requirements of peptides for antiviral and antibacterial activities are evaluated in light of the diverse set of primary and secondary structures described for host defense peptides. Peptides with antifungal and antiparasitic activities are discussed in less detail, although the broad-spectrum activities of such peptides indicate that they are important host defense molecules. Knowledge regarding the relationship between peptide structure and function as well as their mechanism of action is being applied in the design of antimicrobial peptide variants as potential novel therapeutic agents.

Amino Acid Sequence↗

PubMed2006

Role of the novel OprD family of porins in nutrient uptake in Pseudomonas aeruginosa.

To circumvent the permeability barrier of its outer membrane, Pseudomonas aeruginosa has evolved a series of specific porins. These channels have binding sites for related classes of molecules that facilitate uptake under nutrient-limited conditions. Here, we report on the identification of a 19-member family of porins similar to the basic-amino-acid-specific porin OprD. The members of this family fell into one of two phylogenetically distinct clusters, one bearing high similarity to OprD and the other bearing most similarity to the putative phenylacetic acid uptake porin PhaK of Pseudomonas putida. Analysis of the genome context, operon arrangement, and regulation of the PhaK-like porin OpdK indicated that it might be involved in vanillate uptake. This result was confirmed by demonstrating that an opdK mutant had a deficiency in the ability to grow on vanillate as a carbon source. To extrapolate these data to other paralogues within this family, the substrate specificities of 6 of the 17 remaining OprD homologues were inferred using an approach similar to that used with opdK. The specificities determined were as follows: OpdP, glycine-glutamate; OpdC, histidine; OpdB, proline; OpdT, tyrosine; OpdH, cis-aconitate; and OpdO, pyroglutamate. Thus, members of the OprD subfamily took up amino acids and related molecules, and those characterized members most similar to PhaK were responsible for the uptake of a diverse array of organic acids. These results imply that there is a functional basis for the phylogenetic clustering of these proteins and provide a framework for studying OprD homologues in other organisms.

Amino Acids↗