Bow Ho

The ancient origin of the complement system

The complement system has been thought to originate exclusively in the deuterostomes. Here, we show that the central complement components already existed in the primitive protostome lineage. A functional homolog of vertebrate complement 3, CrC3, has been isolated from a ‘living fossil’, the horseshoe crab (Carcinoscorpius rotundicauda). CrC3 resembles human C3 and shows closest homology to C3 sequences of lower deuterostomes. CrC3 and plasma lectins bind a wide range of microbes, forming the frontline innate immune defense system. Additionally, we identified CrC2/Bf, a homolog of vertebrate C2 and Bf that participates in C3 activation, and a C3 receptor‐like sequence. Furthermore, complement‐mediated phagocytosis of bacteria by the hemocytes of horseshoe crab was also observed. Thus, a primitive yet complex opsonic complement defense system is revealed in the horseshoe crab, a protostome species. Our findings demonstrate an ancient origin of the critical complement components and the opsonic defense mechanism in the Precambrian ancestor of bilateral animals.

De novo design of potent antimicrobial peptides.

ABSTRACT Lipopolysaccharide (LPS), shed by gram-negative bacteria during infection and antimicrobial therapy, may lead to lethal endotoxic shock syndrome. A rational design strategy based on the presumed mechanism of antibacterial effect was adopted to design cationic antimicrobial peptides capable of binding to LPS through tandemly repeated sequences of alternating cationic and nonpolar residues. The peptides were designed to achieve enhanced antimicrobial potency due to initial bacterial membrane binding with a reduced risk of endotoxic shock. The peptides designed displayed binding affinities to LPS and lipid A (LA) in the low micromolar range and by molecular modeling were predicted to form amphipathic β-hairpin-like structures when they bind to LPS or LA. They also exhibited strong effects against gram-negative bacteria, with MICs in the nanomolar range, and low cytotoxic and hemolytic activities at concentrations significantly exceeding their MICs. Quantitative structure-activity relationship (QSAR) analysis of peptide sequences and their antimicrobial, cytotoxic, and hemolytic activities revealed that site-directed substitutions of residues in the hydrophobic face of the amphipathic peptides with less lipophilic residues selectively decrease the hemolytic effect without significantly affecting the antimicrobial or cytotoxic activity. On the other hand, the antimicrobial effect can be enhanced by substitutions in the polar face with more polar residues, which increase the amphipathicity of the peptide. On the basis of the QSARs, new analogs that have strong antimicrobial effects but that lack hemolytic activity can be proposed. The findings highlight the importance of peptide amphipathicity and allow a rational method that can be used to dissociate the antimicrobial and hemolytic effects of cationic peptides, which have potent antimicrobial properties, to be proposed.

C‐reactive protein collaborates with plasma lectins to boost immune response against bacteria

Although human C‐reactive protein (CRP) becomes upregulated during septicemia, its role remains unclear, since purified CRP showed no binding to many common pathogens. Contrary to previous findings, we show that purified human CRP (hCRP) binds to Salmonella enterica, and that binding is enhanced in the presence of plasma factors. In the horseshoe crab, Carcinoscorpius rotundicauda, CRP is a major hemolymph protein. Incubation of hemolymph with a range of bacteria resulted in CRP binding to all the bacteria tested. Lipopolysaccharide‐affinity chromatography of the hemolymph co‐purified CRP, galactose‐binding protein (GBP) and carcinolectin‐5 (CL5). Yeast two‐hybrid and pull‐down assays suggested that these pattern recognition receptors (PRRs) form pathogen recognition complexes. We show the conservation of PRR crosstalk in humans, whereby hCRP interacts with ficolin (CL5 homologue). This interaction stabilizes CRP binding to bacteria and activates the lectin‐mediated complement pathway. We propose that CRP does not act alone but collaborates with other plasma PRRs to form stable pathogen recognition complexes when targeting a wide range of bacteria for destruction.

Polycaprolactone-based fused deposition modeled mesh for delivery of antibacterial agents to infected wounds

Infections represent a significant source of site morbidity following tissue trauma. Scarring and tissue adhesion remain the challenging issues yet to be solved. Prolonged inflammation and morphology of the re-epithelisated layer are important considerations. We hypothesized that the solution lies not only in the biochemistry of biomaterial but also the micro-architecture of the scaffold used as the matrix for wound healing. Targeted delivery of antibiotics may provide an efficacious means of infection control through adequate release. Here, we study the use of 3-dimensional polycaprolactone-tricalcium phosphate (PCL-TCP) mesh for the delivery of gentamicin sulphate (GS) fabricated using a solvent-free method. PCL-TCP meshes incorporated with varying loads of GS were evaluated in vitro for elution profile, antimicrobial efficacy and cytotoxicity. Results showed that PCL-TCP meshes incorporated with 15 wt% GS (PT15) efficiently eliminate bacteria within 2 h and demonstrate low cytotoxicity. Subsequently, PT15 meshes were evaluated using an infected full thickness wound mice model, and observed to eliminate bacteria in the wounds effectively. Additionally, mice from the PT15 treatment group (TG) showed no observable signs of overall infection through neutrophil count by day 7 and displayed efficient wound healing (94.2% wound area reduction) by day 14. Histology also showed significantly faster healing in TG through neo-collagen deposition and wound re-epithelisation. The meshes from TG were also observed to be expelled from wounds while gauze fibers from CG were integrated into wounds during healing.

Definition of endotoxin binding sites in horseshoe crab Factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides

Three truncated fragments, harboring different sushi domains, namely, sushi123, sushil, and sushi3 domains, of Factor C were produced as biologically active secreted recombinant proteins. Sushil and 3 each has a high‐affinity LPS binding site with Kd of 10−9 to 10−10 M. Positive cooperativity in sushi123 resulted in a 1000‐fold increase in Kd2. The core LPS binding region of sushi1 and 3 reside in two 34‐mer peptides, Sl and S3. A rigidly held disulfide‐bonded structure is not essential but is important for LPS binding, as confirmed by a 100‐ to 10000‐fold decrease in affinity. Both S1 and S3 can inhibit LAL reaction and LPS‐induced hTNF‐α secretion with different potency. LAL assay revealed that at least two molecules of S1 bind cooperatively to one LPS molecule, with Hills coefficient of 2.42. The LPS binding by S3 is independent and noncooperative. The modified SΔ1 and SΔ3 peptides exhibited increased LPS neutralization potential although its LPS binding affinities indicated only a 10‐fold improvement. Hence, the structural difference of the four sushi peptides conferred different efficiencies in LPS neutralization without altering their binding affinity for LPS. Circular dichroism spectrometry revealed that the four peptides underwent conformational change in the presence of lipid A, transitioning from a random coil to either an α‐helical or β‐sheet structure. Two factors are critical for the sensitivity of Factor C to LPS: 1) the presence of multiple binding sites for LPS on a single Factor C molecule; and 2) high positive cooperativity in LPS binding. The results showed that in the design of an improved LPS binding and neutralizing peptide, charge balance of the peptide is a critical parameter in addition to its structure.—Tan, N. S., Ng, M. L. P., Yau, Y. H., Chong, P. K. W., Ho, B., Ding, J. L. Definition of endotoxin binding sites in horseshoe crab Factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides. FASEB J. 14, 1801–1813 (2000)

A new era in pyrogen testing

Abstract Pyrogens are substances (usually of biological origin) that cause fever after injection. The best-studied pyrogen is lipopolysaccharide (LPS, also known as endotoxin), found in the membrane of Gram-negative bacteria. During Gram-negative sepsis, endotoxin stimulates host macrophages to release inflammatory cytokines and excessive inflammation causes multiple organ failure and death. Endotoxins are thus ubiquitous pathogenic molecules that are a bane to the pharmaceutical industry and medical community. Limulus amoebocyte lysate (LAL) has been widely used for ∼25 years for the detection of endotoxin in quality control of injectable drugs and medical devices. However, variations in sensitivity and specificity of LAL to endotoxin, and the limited supply of limulus (horseshoe crabs) has called for an alternative pyrogen test. Recombinant Factor C (rFC), the endotoxin-inducible coagulation enzyme in LAL, forms the basis of a novel micro-enzymatic assay for high-throughput screens of endotoxin and opens a new era in endotoxin testing. Endotoxin activates the rFC zymogen, which catalytically hydrolyses synthetic substrates to form measurable products, thus quantifying the endotoxin.

Local inflammation induces complement crosstalk which amplifies the antimicrobial response.

By eliciting inflammatory responses, the human immunosurveillance system notably combats invading pathogens, during which acute phase proteins (CRP and cytokines) are elevated markedly. However, the Pseudomonas aeruginosa is a persistent opportunistic pathogen prevalent at the site of local inflammation, and its acquisition of multiple antibiotic-resistance factors poses grave challenges to patient healthcare management. Using blood samples from infected patients, we demonstrate that P. aeruginosa is effectively killed in the plasma under defined local infection-inflammation condition, where slight acidosis and reduced calcium levels (pH 6.5, 2 mM calcium) typically prevail. We showed that this powerful antimicrobial activity is provoked by crosstalk between two plasma proteins; CRP∶L-ficolin interaction led to communication between the complement classical and lectin pathways from which two amplification events emerged. Assays for C4 deposition, phagocytosis, and protein competition consistently proved the functional significance of the amplification pathways in boosting complement-mediated antimicrobial activity. The infection-inflammation condition induced a 100-fold increase in CRP∶L-ficolin interaction in a pH- and calcium-sensitive manner. We conclude that the infection-induced local inflammatory conditions trigger a strong interaction between CRP∶L-ficolin, eliciting complement-amplification pathways which are autonomous and which co-exist with and reinforce the classical and lectin pathways. Our findings provide new insights into the host immune response to P. aeruginosa infection under pathological conditions and the potential development of new therapeutic strategies against bacterial infection.

Helicobacter pylori γ-Glutamyl Transpeptidase Is a Pathogenic Factor in the Development of Peptic Ulcer Disease

BACKGROUND & AIMS gamma-Glutamyl transpeptidase (GGT) has been reported to be a virulence factor of Helicobacter pylori associated with bacterial colonization and cell apoptosis. But its mechanism of pathogenesis is not firmly established. This study aims to examine its role in H pylori-mediated infection. METHODS Various H pylori isogenic mutants were constructed by a polymerase chain reaction (PCR) approach. H pylori native GGT protein (HP-nGGT) was purified with ion-exchange and gel-filtration chromatography. Generation of H2O2 was measured with fluorimetric analysis, whereas nuclear factor-kappaB (NF-kappaB) activation was determined by luciferase assay and Western blot. Cytokine production was examined by enzyme-linked immunoabsorbent assay and real-time PCR. DNA damage was assessed with comet assay and flow cytometry. The GGT activity of 98 H pylori isolates was analyzed by an enzymatic assay. RESULTS Purified HP-nGGT generated H2O2 in primary gastric epithelial cells and AGS gastric cancer cells, resulting in the activation of NF-kappaB and up-regulation of interleukin-8 (IL-8) production. In addition, HP-nGGT caused an increase in the level of 8-OH-dG, indicative of oxidative DNA damage. In contrast, Deltaggt showed significantly reduced levels of H2O2 generation, IL-8 production, and DNA damage in cells compared with the wild type (P<.05). The clinical importance of GGT was indicated by significantly higher (P<.001) activity in H pylori isolates obtained from patients with peptic ulcer disease (n=54) than isolates from patients with nonulcer dyspepsia (n=44). CONCLUSION Our findings provide evidence that GGT is a pathogenic factor associated with H pylori-induced peptic ulcer disease.

SARM inhibits both TRIF- and MyD88-mediated AP-1 activation.

SARM (sterile α‐ and armadillo‐motif‐containing protein), the fifth identified TIR (Toll–interleukin 1 receptor (IL‐1R)) domain‐containing adaptors in humans, downregulates NF‐κB and IRF3 (interferon‐regulatory factor 3)‐mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR‐domain‐containing adaptor protein inducing IFN‐β)‐dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF‐ and MyD88‐mediated, as well as basal MAPK activity, were repressed, indicating that SARM‐mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP‐1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N‐terminal and sterile alpha motif domains in the autoregulation of SARM activity.

Interaction of an artificial antimicrobial peptide with lipid membranes.

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.