Purification of a novel arthropod defensin from the American oyster, Crassostrea virginica

An antimicrobial peptide was purified from acidified gill extract of a bivalve mollusk, the American oyster (Crassostrea virginica), by preparative acid-urea--polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. The 4265.0 Da peptide had 38 amino acids, including 6 cysteines. It showed strongest activity against Gram-positive bacteria (Lactococcus lactis subsp. lactis and Staphylococcus aureus; minimum effective concentrations [MECs] 2.4 and 3.0 microg/ml, respectively) but also had significant activity against Gram-negative bacteria (Escherichia coli D31 and Vibrio parahemolyticus; MECs 7.6 and 15.0 microg/ml, respectively). Comparison of the amino acid sequence with those of other known antimicrobial peptides revealed that the novel peptide had high sequence homology to arthropod defensins, including those from other bivalves, the mussels Mytilus edulis and Mytilus galloprovincialis. This is the first antimicrobial peptide to be isolated from any oyster species and we have named it American oyster defensin (AOD).     

Cardiac function and critical swimming speed of the winter flounder (Pleuronectes americanus) at two temperatures

Using Transonic flow probes and a uniquely designed swimming flume, we directly measured cardiac parameters (Q, cardiac output; SV, stroke volume; and fH, heart rate) in winter flounder (Pleuronectes americanus) before and during critical swim speed (Ucrit) tests at 4 and 10 degrees C. Resting Q, SV and fH averaged 9.8 ml min(-1) kg(-1), 0.5 ml kg(-1) (1.0 ml g ventricle(-1)) and 21 beats min(-1) at 4 degrees C and 15.5 ml min(-1) kg(-1), 0.5 ml kg(-1) (0.95 ml g ventricle(-1)) and 34 beats min(-1) at 10 degrees C (Q10 values of 2.13, 0.91 and 2.35, for Q, SV and fH, respectively). Cardiac output, SV and fH increased by approx. 170%, 70% and 60% at both temperatures during the Ucrit test. However, cardiac parameters generally reached near maximal levels almost immediately upon swimming and remained at these levels until Ucrit (0.65 +/- 0.06 bl s(-1) at 4 degrees C and 0.73 +/ -0.07 bl s(-1) at 10 degrees C). This rapid rise in cardiac function to near maximal levels did not appear to be the result of stress alone, as Q only fell slightly when flounder were swum for 75 min at < 0.4 bl s(-1), speeds at which they appeared to swim comfortably. Our results suggest that both Q and Ucrit have been significantly overestimated in flatfishes, and that "lift-off"/slow swimming is energetically expensive. Furthermore, they show that maximum and resting stroke volume (per g of ventricle) are extremely high in the flounder as compared with other teleosts.     

Antimicrobial function of the GAPDH-related antimicrobial peptide in the skin of skipjack tuna,Katsuwonus pelamis

A 3.4 kDa of antimicrobial peptide was purified from an acidified skin extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea–polyacrylamide gel electrophoresis and C₁₈ reversed-phase HPLC. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high sequence homology with the YFGAP (Yellowfin tuna Glyceraldehyde-3-phosphate dehydrogenase-related Antimicrobial Peptide); thus, this peptide was identified as the skipjack tuna GAPDH-related antimicrobial peptide (SJGAP). SJGAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 1.2–17.0 μg/mL), Gram-negative bacteria, such as Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs, 3.1–12.0 μg/mL), and against Candida albicans (MEC, 16.0 μg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide is heat-stable but salt-sensitive. According to the secondary structural prediction and the homology modeling, this peptide consists of three secondary structural motifs, including one α-helix and two parallel β-strands, and forms an amphipathic structure. This peptide showed neither membrane permeabilization ability nor killing ability, but did display a small degree of leakage ability. These results suggest that SJGAP acts through a bacteriostatic process rather than bactericidal one. SJGAP is another GAPDH-related antimicrobial peptide isolated from skipjack tuna and likely plays an important role for GAPDH in the innate immune defense of tuna fish.     

Multiple antibacterial histone H2B proteins are expressed in tissues of American oyster

We have previously identified a histone H2B isomer (cvH2B-1) from tissue extracts of the bivalve mollusk, the American oyster (Crassostrea virginica). In this paper, we isolate an additional three antibacterial proteins from acidified gill extract by preparative acid-urea-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. Extraction of these proteins from tissue was best accomplished by briefly boiling the tissues in a weak acetic acid solution. Addition of protease inhibitors while boiling resulted in somewhat lower yields, with one protein being totally absent with this method. Via mass spectrometry, the masses of one of these purified proteins was 13607.0Da (peak 2), which is consistent with the molecular weight of histone H2B. In addition, via western-blotting using anti-calf histone H2B antibody, all three proteins were positive and were thus named cvH2B-2, cvH2B-3 and cvH2B-4. The antibacterial activity of cvH2B-2 was similar to that of cvH2B-1, with activity against a Gram-positive bacterium (Lactococcus lactis subsp. lactis; minimum effective concentration [MEC] 52-57μg/mL) but inactive against Staphylococcus aureus (MEC>250μg/mL). However, both proteins had relatively potent activity against the Gram-negative oyster pathogen Vibrio parahemolyticus (MEC 11.5-14μg/mL) as well as the human pathogen Vibrio vulnificus (MEC 21.3-25.3μg/mL). cvH2B-3 and cvH2B-4 also had similarly strong activity against Vibrio vulnificus. These data provide further evidence for the antimicrobial function of histone H2B isomers in modulating bacterial populations in oyster tissues. The combined estimated concentrations of these histone H2B isomers were far above the inhibitory concentrations for the tested vibrios, including human pathogens. Our results indicate that the highly conserved histone proteins might be important components not only of immune defenses in oysters but have the potential to influence the abundance of a ubiquitous microbial resident of oyster tissues that is the major source of seafood-borne illness in humans.     

Catesbeianin-1, a novel antimicrobial peptide isolated from the skin of <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> (American bullfrog)

Objectives

To identify and characterize a novel antimicrobial peptide, catesbeianin-1.

Results

Catesbeianin-1 is 25 amino acids long and is α-helical, cationic and amphipathic. It had antimicrobial activity against Gram-positive and Gram-negative bacteria. It was resistant against trypsin and pepsin. Catesbeianin-1 exhibited moderate hemolytic activity (approx 8%) at 100 μg/ml, and its HC₅₀ (50% hemolytic concentration) was 300 μg/ml. Its cytotoxicity was approx 10–20% at 100 μg/ml, and its CC₅₀ (50% cytotoxic concentration) was >100 μg/ml. The LD₅₀ of catesbeianin-1 in mice was 80 mg/kg. At 3.1 µg/ml, catesbeianin-1 significantly inhibited the growth of methicillin-resistant Staphylococcus aureus.

Conclusions

A new antimicrobial peptide from the skin of Lithobates catesbeianus (American bullfrog) may represent a template for the development of novel antimicrobial agents.

     

Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33

With great therapeutic potential against antibiotic-resistant bacteria, viruses, and even parasites, antimicrobial peptides (AMPs) have received increased interest as pharmaceutical agents in recent years. It is a worthy yet challenging work to carry out the implement and improvement of AMPs production using bioengineering techniques. In the present study, a novel hybrid peptide LFT33 was designed derived from LfcinB and thanatin. The cDNA fragment encoding LFT33 with preferred codons of Escherichia coli was chemically synthesized and ligated into the vector pET32a(+) to express the LFT33 fusion protein. The fusion protein was successfully expressed in soluble form in E. coli induced under optimized conditions. After purification by affinity chromatography, the fusion protein was cleaved successfully by enterokinase and released the peptide LFT33. About 0.5?mg of the recombinant LFT33 was obtained by reversed-phase high performance liquid chromatography from 1?l of culture medium. Mass spectrometry analysis of the purified recombinant LFT33 demonstrated that the molecular weight perfectly matched the calculated mass (4,195?Da). The recombinant peptide LFT33 caused an increase in antimicrobial activity (IC(50)?=?16-64?μg/ml) against given strains and did not show hemolytic activity for human erythrocytes. The results indicated that the hybrid peptide LFT33 could serve as a promising candidate for pharmaceutical agents.     

Differential mode of antimicrobial actions of arginine-rich and lysine-rich histones against Gram-positive Staphylococcus aureus

We previously reported the activities and modes of action of arginine (Arg)-rich histones H3 and H4 against Gram-negative bacteria. In the present study, we investigated the properties of the Arg-rich histones against Gram-positive bacteria in comparison with those of lysine (Lys)-rich histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against Staphylococcus aureus with minimum effective concentration values of 4.0, 4.0, and 5.6 μM, respectively. Laser confocal microscopic analyses revealed that both the Arg-rich and Lys-rich histones associated with the surface of S. aureus. However, while the morphology of S. aureus treated with histone H2B appeared intact, those treated with the histones H3 and H4 closely resembled each other, and the cells were blurred. Electrophoretic mobility shift assay results revealed these histones have binding affinity to lipoteichoic acid (LTA), one of major cell surface components of Gram-positive bacteria. Scanning electron microscopic analyses demonstrated that while histone H2B elicited no obvious changes in cell morphology, histones H3 and H4 disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. Consequently, our results suggest that bacterial cell surface LTA initially attracts both the Arg- and Lys-rich histones, but the modes of antimicrobial action of these histones are different; the former involves cell membrane disruption and the latter involves the cell integrity disruption.     

Chemical Characteristics,Antimicrobial, and Cytotoxic Activities of the Essential Oil of Egyptian Cinnamomum glanduliferum Bark

The essential oil isolated from the bark of Cinnamomum glanduliferum (Wall ) Meissn grown in Egypt was screened for its composition as well as its biological activity for the first time. The chemical composition was analyzed by GC and GC/MS. The antimicrobial activity of the oil was assessed using agar‐well diffusion method toward representatives for each of Gram‐positive bacteria, Gram‐negative bacteria, and fungi. The cytotoxic activity was checked using three human cancer cell lines. Twenty seven compounds were identified, representing 99.07% of the total detected components. The major constituents were eucalyptol (65.87%), terpinen‐4‐ol (7.57%), α‐terpineol (7.39%). The essential oil possessed strong antimicrobial activities against Escherichia coli, with an activity index of one and minimum inhibitory concentration (MIC) equaling to 0.49 μg/ml. The essential oil possessed good antimicrobial activities against methicillin‐resistant Staphylococcus aureus, Geotrichum candidum, Pseudomonas aeruginosa, Bacillus subtilis, Helicobacter pylori, Aspergillus fumigatus (MIC: 7.81, 1.95, 7.81, 0.98, 31.25, and 32.5 μg/ml, respectively). A considerable activity was reported against S. aureus and Mycobacterium tuberculosis (MIC; 32.5 and 31.25 μg/ml, respectively). The extracted oil was cytotoxic to colon (HCT‐116), liver (HepG2), and breast (MCF‐7) carcinoma cell lines with IC₅₀ of 9.1, 42.4, and 57.3 μg/ml, respectively. These results revealed that Egyptian Cinnamomum glanduliferum bark oil exerts antimicrobial and cytotoxic activities mainly due to eucalyptol and other major compounds.     

The relative roles of factor H binding protein, neisserial surface protein A, and lipooligosaccharide sialylation in regulation of the alternative pathway of complement on meningococci

Neisseria meningitidis inhibits the alternative pathway (AP) of complement using diverse mechanisms, including expression of capsule (select serogroups), Neisserial surface protein A (NspA), factor H (fH) binding protein (fHbp), and lipooligosaccharide (LOS) sialylation. The contribution of the latter three molecules in AP regulation in encapsulated meningococci was studied using isogenic mutants. When LOS was unsialylated, deleting NspA alone from group A strain A2594 (low fHbp/high NspA) significantly increased AP-mediated C3 deposition. C3 deposition further increased ～2-fold in a ΔfHbpΔNspA double mutant, indicating cooperative fHbp function. LOS sialylation of A2594 ΔfHbpΔNspA decreased the rate of C3 deposition, revealing AP inhibition by LOS sialic acid. Maximal C3 deposition on group B strain H44/76 (high fHbp/low NspA) occurred when all three molecules were absent; again, LOS sialylation attenuated the AP in the absence of both fHbp and NspA. When H44/76 LOS was unsialylated, both fHbp and NspA independently inhibited the AP. LOS sialylation enhanced binding of fH C-terminal domains 18-20 to C3 fragments deposited on bacteria. Interaction of meningococci with nonhuman complement is relevant for animal models and vaccine evaluation studies that use nonhuman complement. Consistent with their human-specific fH binding, neither fHbp nor NspA regulated the rat AP. However, LOS sialylation inhibited the rat AP and, as with human serum, enhanced binding of rat fH to surface-bound C3. These data highlight the cooperative roles of meningococcal NspA and fHbp in regulating the human AP and broaden the molecular basis for LOS sialylation in AP regulation on meningococci in more than one animal species.     

The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance

Neisseria meningitidis binds factor H (fH), a key regulator of the alternative complement pathway. A approximately 29 kD fH-binding protein expressed in the meningococcal outer membrane was identified by mass spectrometry as GNA1870, a lipoprotein currently under evaluation as a broad-spectrum meningococcal vaccine candidate. GNA1870 was confirmed as the fH ligand on intact bacteria by 1) abrogation of fH binding upon deleting GNA1870, and 2) blocking fH binding by anti-GNA1870 mAbs. fH bound to whole bacteria and purified rGNA1870 representing each of the three variant GNA1870 families. We showed that the amount of fH binding correlated with the level of bacterial GNA1870 expression. High levels of variant 1 GNA1870 expression (either by allelic replacement of gna1870 or by plasmid-driven high-level expression) in strains that otherwise were low-level GNA1870 expressers (and bound low amounts of fH by flow cytometry) restored high levels of fH binding. Diminished fH binding to the GNA1870 deletion mutants was accompanied by enhanced C3 binding and increased killing of the mutants. Conversely, high levels of GNA1870 expression and fH binding enhanced serum resistance. Our findings support the hypothesis that inhibiting the binding of a complement down-regulator protein to the neisserial surface by specific Ab may enhance intrinsic bactericidal activity of the Ab, resulting in two distinct mechanisms of Ab-mediated vaccine efficacy. These data provide further support for inclusion of this molecule in a meningococcal vaccine. To reflect the critical function of this molecule, we suggest calling it fH-binding protein.     

The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.     

Expression and Purification of the Main Component Contained in Camel Milk and Its Antimicrobial Activities Against Bacterial Plant Pathogens

Lactoferrin is the most dominant protein in milk after casein. This protein plays a crucial role in many biological processes including the regulation of iron metabolism, induction and modulation of the immune system, the primary defense against microorganisms, inhibiting lipid peroxidation and presenting antimicrobial activity against various pathogens such as parasites, fungi, bacteria, and viruses. The major antimicrobial effect of lactoferrin is related to its N-terminal tail where different peptides for instance lactoferricin and lactoferrampin which are important for their antimicrobial abilities are present. The growth rate of bacterial cells in camel milk is lower than that of the cow milk due to having more antimicrobial compounds. In this study, we have fused a codon-optimized partial camel lactoferrcin and lactoferrampin DNA sequences in order to construct a fused peptide via a lysine. This chimeric 42-mer peptide consists of complete and partial amino acid sequence of camel lactoferrampin and lactoferricin, respectively. Human embryonic kidney 293 (HEK-293) cells were used for synthesizing this recombinant peptide. Finally, the antibacterial activities of this constructed peptide were investigated under in vitro condition. The result showed that, all construction, cloning and expression processes were successfully performed in HEK-293. One His-tag tail was added to the chimera in order to optimize the isolation and purification processes and also reduce the cost of production. Additionally, His-tag retained the antimicrobial activity of the chimera. The antimicrobial tests showed that the growth rate in the majority of bacterial plant pathogens, including gram negative and positive bacteria, was inhibited by recombinant chimera as the level of MIC values were evaluated between 0.39 and 25.07 μg/ml for different bacterial isolates.