Decrease of α-defensin impairs intestinal metabolite homeostasis via dysbiosis in mouse chronic social defeat stress model.

Psychological stress has been reported to relate to dysbiosis, imbalance of the intestinal microbiota composition, and contribute to the onset and exacerbation of depression, though, underlying mechanisms of psychological stress-related dysbiosis have been unknown. It has been previously established that α-defensins, which are effector peptides of innate enteric immunity produced by Paneth cells in the small intestine, play an important role in regulation of the intestinal microbiota. However, the relationship between disruption of intestinal ecosystem and α-defensin under psychological stress is yet to be determined. Here we show using chronic social defeat stress (CSDS), a mouse depression model that (1) the exposure to CSDS significantly reduces α-defensin secretion by Paneth cells and (2) induces dysbiosis and significant composition changes in the intestinal metabolites. Furthermore, (3) they are recovered by administration of α-defensin. These results indicate that α-defensin plays an important role in maintaining homeostasis of the intestinal ecosystem under psychological stress, providing novel insights into the onset mechanism of stress-induced depression, and may further contribute to discovery of treatment targets for depression.

Development of a multiplex and sensitive lateral flow immunoassay for the diagnosis of periprosthetic joint infection.

The diagnosis of periprosthetic joint infection (PJI) remains a challenge. However, recent studies showed that synovial fluid biomarkers have demonstrated greater diagnostic accuracy than the currently used PJI diagnostic tests. In many diagnostic tests, combining several biomarkers into panels is critical for improving diagnostic efficiency, enhancing the diagnostic precision for specific diseases, and reducing cost. In this study, we prove that combining alpha-defensin and C-reactive protein (CRP) as biomarkers possesses the potential to provide accurate PJI diagnosis. To further verify the result, we developed a multi-target lateral flow immunoassay strip (msLFIA) with staking pad design to obtain on-site rapid response for clinical diagnosis of PJI. A total of 10 synovial fluid samples were tested using the msLFIA, and the results showed that the combined measurements of synovial fluid alpha-defensin and CRP levels were consistent with those obtained from a commercial enzyme-linked immunosorbent assay kit. In addition, we developed a multi-target lateral flow immunoassay strip (msLFIA) with staking pad design to obtain on-site rapid response for clinical diagnosis of PJI, which the multi-target design is used to increase specificity and the stacking pad design is to enhance detection sensitivity. As a result, the turnaround time of the highly sensitive test can be limited from several hours to 20 min. We expect that the developed msLFIA possesses the potential for routine monitoring of PJI as a convenient, low-cost, rapid and easy to use detection device for PJI.

Human neutrophil peptide 3 could be functionally expressed in Rhodobacter sphaeroides.

Human neutrophil peptides (HNPs) possess high antimicrobial activities against a broad spectrum of microorganisms. Rhodobacter sphaeroides is the best-characterized photosynthetic bacterium and exhibits potential as a novel expression system. Up to date, no literature has been reported regarding expression of HNP3 in Rb. sphaeroides. In the present study, the HNP3 gene fragment was amplified by SOE PCR and ligated into photosynthetic bacteria light-harvesting complex 2 (LH2) expression vector leading to HNP3 fusion protein expression vector. The HNP3 fusion protein was successfully expressed as rapidly evaluated by the LH2 characteristic peaks at ~800 nm and ~850 nm before purification and SDS/PAGE. Subsequently, the HNP3 fusion protein was purified by one-step affinity chromatography, and could be rapidly detected by the color and the spectral absorption at ~800 nm and ~850 nm before SDS/PAGE. Antimicrobial activity assay suggested that the HNP3 fusion protein exhibited high antimicrobial activity towards E. coli. The present study may supply an insight into employing the novel Rb. sphaeroides expression system, exhibiting dramatic advantages over currently used commercial expression system, to heterologously express human neutrophil peptides.

Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization.

Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed, attempts to obtain large amounts of α-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse α-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coli expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies. Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of α-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step.

An innovative biologic recycling process of leukoreduction filters to produce active human antimicrobial peptides.

BACKGROUND: Human neutrophil peptides (HNPs) 1 to 3 are the major antimicrobial peptides of the azurophilic granules of neutrophils. They represent an important arm of the innate immune system. Their production by chemical synthesis and recombinant technologies is expensive and limited by technical constraints due to their composition and the presence of three disulfide bonds. STUDY DESIGN AND METHODS: We have developed an original approach based on the purification of the natural human defensins HNPs 1 to 3 from neutrophils trapped on leukoreduction filters used in blood processing. The purification of HNPs 1 to 3 from these filters is performed in two steps: extraction of HNPs 1 to 3 retained in the filters followed by their immunoprecipitation. Studies were performed to determine the stability of defensins in the filters stored at room temperature. The activity of HNPs 1 to 3 obtained by our rapid protocol was validated by determining minimal inhibitory concentrations (MICs) against six reference bacterial strains and 12 clinical isolates. RESULTS: The human defensins HNPs 1 to 3 extracted from leukoreduction filters displayed high antimicrobial activity against tested strains, with MIC values between 0.12 and 1 µg/mL. Kinetics assays showed the appearance of activity 15 minutes after peptide addition. Moreover, we found that the HNPs 1 to 3 purified from leukoreduction filters that had been stored for 45 days at room temperature remained active. CONCLUSION: Leukoreduction filters provide a rich and safe source of active human defensins HNPs 1 to 3. Moreover, the stability of the peptides in filters stored at room temperature allows envisaging a large-scale development of the process.

An optimized Fmoc synthesis of human defensin 5.

Human α-defensin 5 (DEF5), expressed by the Paneth cells of human small intestine, plays an important role in host defense against microbial infections. DEF5, a 32-residue peptide adopting a three-stranded ß-sheet fold stabilized by three internal disulfide bonds, is not efficiently produced by recombinant expression techniques and is, therefore, an interesting goal for chemical synthesis. While DEF5 production by Boc-based solid-phase synthesis has been described, to date no synthetic account by the more convenient Fmoc method has been published. Herein, we report an optimized solid-phase synthesis of DEF5 using the Fmoc strategy. Starting from a rather problematic initial synthesis using standard Wang resin and coupling protocols, the sequence elongation process has been monitored by mini-cleavage and MS analysis at strategic points, to identify problematic spots and act accordingly. For expediency, some of the optimization rounds have been run on defensin 5 amide. Main modifications have included the ChemMatrix(®) resin, known to decrease chain aggregation, and the use of pseudoproline dipeptide units at selected positions. Combination of some of these improvements results in a significantly purer product, to the extent that it can undergo in situ anaerobic oxidative folding to the native form without the need of an intermediate purification step. A typical synthesis run yielded about 15 mg of >95 % pure material. This approach should facilitate production of DEF5 and of selected analogs for structure-activity studies and other applications.

Activation of neutrophils by the two-component leukotoxin LukE/D from Staphylococcus aureus: proteomic analysis of the secretions.

Staphylococcus aureus is responsible for severe bacterial infections in hospitals and healthcare facilities. It produces single and bicomponent toxins (leukotoxins and hemolysins) that hinder innate immune function. Leukotoxin subunits bind to leukocyte cell membrane thus inducing transmembrane pores and subsequently, cell lysis. Leukotoxin LukE/D is a member of the bicomponent toxin family, but to date, no study concerning its involvement in host-pathogen interactions has been reported. In the present study, we performed the proteomic analysis of the secretions recovered after activation of human neutrophils by leukotoxin LukE/D. The neutrophil secretions were purified by RP-HPLC and different fractions were analyzed by Edman sequencing, LC-MS/MS, immunoblotted for chromogranin-derived peptides and further analyzed for antimicrobial properties. Proteomic analysis revealed that neutrophil secretions constitute a large number of proteins related with immune boosting mechanisms, proteolytic degradation, inflammatory process and antioxidant reactions.

[Enteric alpha defensin 5 is secreted into the blood stream by colon tumors].

It was demonstrated that enteric alpha-defensin 5 is undetectable in five blood serum samples of healthy donors, whereas its processed form is present in two out of five serum samples of colon cancer patients. Obtained results open a possibility of serological diagnosis of colon tumors in high risk cancer patients.

Proteinase 3 carries small unusual carbohydrates and associates with αlpha-defensins.

The neutrophil granulocyte is an important first line of defense against intruding pathogens and it contains a range of granules armed with antibacterial peptides and proteins. Proteinase 3 (PR3) is one among several serine proteases of the azurophilic granules in neutrophil granulocytes. Here, we characterize the glycosylation of PR3 and its association with antimicrobial human neutrophil peptides (HNPs, α-defensins) and the effect of these on the mechanism of inhibition of the major plasma inhibitor of PR3, α1-antitrypsin. The glycosylation of purified, mature PR3 showed some heterogeneity with carbohydrates at Asn 102 and 147 carrying unusual small moieties indicating heavy processing. Mass spectrometric analysis and immuno blotting revealed strong association of highly purified PR3 with α-defensins and oligomers hereof. Irreversible inhibition of PR3 by α1-antitrypsin did not affect its association with defensins. Other proteins from neutrophil granules were also found to be associated with defensins, whereas purified plasma proteins did not carry defensins. These results point to a role of defensins in controlling and targeting the activity of neutrophil granule proteins.

Specificity in killing pathogens is mediated by distinct repertoires of human neutrophil peptides.

Neutrophil-derived antimicrobial peptides and proteins (AMPs) play an important role in the defense against microbes. Absence of defense is illustrated by neutropenic patients with frequent bacterial and fungal infections. However, the specificity of the antimicrobial effects has not been adequately described. We set out to determine the specific antimicrobial pattern of polypeptides in neutrophils (polymorphonuclear leukocytes, PMNs) against 4 potential human pathogens: Moraxella catarrhalis, Staphylococcus aureus, Haemophilus influenzae and Candida albicans. Protein extracts of human PMNs were separated using high-performance liquid chromatography and fractions were assayed for antimicrobial activity. Fractions displaying antimicrobial activity were separated on SDS-PAGE and characterized using MALDI-MS. Depletion experiments were utilized to determine the contribution of each AMP to the antimicrobial effect. Among the identified AMPs, α-defensins 1-3, azurocidin, LL-37, lysozyme, calprotectin and lactotransferrin were studied in detail. We found a divergent pattern of killing, that is, certain peptides and proteins exhibited selective activity against specific pathogens, while others displayed a broader antimicrobial activity. α-Defensins, LL-37 and calprotectin were active against all species, while lactotransferrin exclusively inhibited growth of S. aureus. Conversely, azurocidin was active against all species except S. aureus. Our observations may shed light on bacterial resistance to AMPs and on the elimination of specific bacterial communities on mucosal surfaces.

High efficiency preparation of bioactive human alpha-defensin 6 in Escherichia coli Origami(DE3)pLysS by soluble fusion expression.

Human alpha-defensin 6 (HD(6)), a small cysteine-rich cationic peptide specially expressed in epithelial cells of digestive tract, may play a crucial role in mucosal immunity. This is the first report on efficient production of bioactive HD(6) through a gene-engineering approach in Escherichia coli. The recombinant plasmid pET32a-omHD(6) was primarily constructed by inserting a PCR fragment encoding mature HD(6) peptide (mHD(6)) preceded by an enterokinase recognition sequence into the expression vector pET32a(+), in frame with the upstream thioredoxin (TrxA) gene. Under optimized expression conditions, a high percentage (>60%) of soluble TrxA-omHD(6) fusion protein was obtained with a yield of about 1.69 g/l, and the theoretical productivity of recombinant mHD(6) (rmHD(6)) reached 0.38 g/l. A feasible three-step purification strategy involving nickel-sepharose chromatography, enterokinase-cleavage and cation exchange chromatography was developed to purify rmHD(6), followed by characteristic identifications by Western blot, mass spectrometry and sequencing. About 102 mg/l of rmHD(6) with its intact N-terminal amino acid sequence was finally achieved. The in vitro experiments showed that rmHD(6) possesses high potency to inhibit herpes simplex virus-2 infection. This work settles substantial foundation for further functional study of HD(6).

Expression and purification of recombinant alpha-defensins and alpha-defensin precursors in Escherichia coli.

Recombinant expression of alpha-defensins can be obtained at efficient levels in Escherichia coli. Amplified alpha-defensin or pro-alpha-defensin coding cDNA sequences are cloned directionally between EcoRI and SalI sites of the pET-28a expression vector and expressed in E. coli BL21 RIS cells. Cells growing exponentially in nutrient-rich liquid medium are induced to express the recombinant protein by addition of 50 mM isopropyl beta-D-1-thiogalactopyranoside for 3-6 h. After bacterial cells collected by centrifugation are lysed in 6 M guanidine-HCl under non-reducing conditions, the expressed defensin fused to its 6xHis-34 amino acid N-terminal fusion partner is purified by affinity chromatography on nickel-NTA columns. A Met codon introduced at the N terminus of expressed Met-free peptides provides a unique CNBr cleavage site, enabling release of the alpha-defensin free of ancillary residues by sequential C18 RP-HPLC. Molecular masses of C18 RP-HPLC purified peptides are confirmed by MALDI-TOF mass spectrometry, and peptide homogeneity is assessed using analytical RP-HPLC and acid-urea polyacrylamide gel electrophoresis. alpha-Defensins prepared in this manner are biochemically equivalent to the natural molecules.

Alpha-defensins in enteric innate immunity: functional Paneth cell alpha-defensins in mouse colonic lumen.

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric alpha-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric alpha-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative alpha-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as alpha-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1-4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell alpha-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.

High level expression and purification of bioactive human alpha-defensin 5 mature peptide in Pichia pastoris.

Human alpha-defensin 5 (HD(5)), a small cysteine-rich peptide expressed predominantly in small intestine and female reproductive tissues, plays an important role in innate and adaptive immunity. It is a worthy yet challenging work to produce bioactive recombinant HD(5) through the use of bioengineering techniques. Here, we present the expression and purification of recombinant HD(5) mature peptide (rmHD(5)) in Pichia pastoris. To avoid generating unfavorable extra N-terminal amino acids, Red/ET homologous recombination was applied to construct the expression vector pPIC9K-mHD(5) by insertion of a polymerase chain reaction-amplified DNA fragment coding for mHD(5) into the plasmid pPIC9K, at a position right after the cleavage sequence of Kex2. The pPIC9K-mHD(5) vector was transformed into P. pastoris GS115 cells, and positive colonies harboring genomic integration of the multicopy mHD(5) nucleotide sequence were screened out and used for fermentation. After high-cell density fermentation of P. pastoris GS115-HD(5), a two-step purification strategy of macroporous resin adsorption chromatography followed by cation exchange chromatography was performed to obtain purified rmHD(5). The results showed that about 165.0 mg/l of rmHD(5) with its intact N-terminal amino acid sequence as revealed by mass spectrometry analysis and amino acid sequencing was produced under optimal bioreactor-culture conditions and that approximately 50% of the initial rmHD(5) was recovered after purification. The in vitro experiments revealed that rmHD(5) exhibited a prominent antibacterial activity and potency to block human papillomavirus infection. This is the first report on the production and purification of bioactive rmHD(5) in P. pastoris. This study also provides considerations for production of other antimicrobial peptides using the P. pastoris expression system.

The capsule sensitizes Streptococcus pneumoniae to alpha-defensins human neutrophil proteins 1 to 3.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Its polysaccharide capsule causes resistance to phagocytosis and interferes with the innate immune system's ability to clear infections at an early stage. Nevertheless, we found that encapsulated pneumococci are sensitive to killing by a human neutrophil granule extract. We fractionated the extract by high-performance liquid chromatography and identified alpha-defensins by mass spectrometry as the proteins responsible for killing pneumococci. Analysis of sensitivity to the commercial alpha-defensins human neutrophil proteins 1 to 3 (HNP1-3) confirmed these findings. We analyzed the sensitivities of different pneumococcal strains to HNP1-3 and found that encapsulated strains are efficiently killed at physiological concentrations (7.5 microg/ml). Surprisingly, nonencapsulated, nonvirulent pneumococci were significantly less sensitive to alpha-defensins. The proposed mechanisms of alpha-defensin resistance in nonencapsulated pneumococci is surface charge modification, e.g., by introduction of positive charge by D-alanylation of surface-exposed lipoteichoic acids. These mechanisms are surmounted by the presence of the capsule, which we hypothesize is masking these charge modifications. Hence, alpha-defensins in the phagolysosome of neutrophils possibly contribute to intracellular killing after antibody-mediated opsonophagocytosis of encapsulated pneumococci.

[Comparative analysis of the effect of endogenous antibiotic defensin NP-1 and aminoglycoside antibiotic gentamicin on synaptic transmission in receptors of the frog vestibular apparatus].

The effect of human and rabbit neutrophilic defensins NP-1 and amonoglycoside antibiotic gentamicin on the synaptic transmission in the afferent synapse of isolated vestibular apparatus of the frog has been comparatively studied. Both defensins proved active in the concentration range of 0.0001 to 1 nM and efficiently decreased the impulse frequency in the afferent nerve fibers in a concentration-dependent manner. No significant differences in the efficiency of rabbit and human defensin NP-1 have been revealed in these experiments. Gentamicin also had an inhibitory effect on the afferent discharge in the concentration range of 10-500 microM (0.5-25 mg/kg). The inhibitory effect of gentamicin on the impulse activity of the vestibular nerve was observed at therapeutic doses. The excitatory effect of the putative neurotransmitter L-glutamate was considerably inhibited by defensin NP-1. These findings suggest that the mechanism of defensin action involves a modification of the synaptic transmission the hair receptor and is mediated by L-glutamate.

alpha-Defensins from blood leukocytes of the monkey Papio hamadryas.

Three antimicrobial peptides named PHD1-3 (Papio hamadryas defensin) have been isolated from hamadryas baboon blood leukocytes using preparative electrophoresis and reverse-phase HPLC. The primary structures of these peptides have been determined by automated Edman degradation and mass-spectrometry. The results suggest that the peptides belong to the alpha-defensin family. Structural homology analysis reveals that among alpha-defensins from other animal species, PHD3 is the most closely related to RMAD5 (rhesus macaque alpha-defensin) (90% homology) from rhesus macaque leukocytes and also highly similar to human alpha-defensin HD5 (60% homology), which is produced by intestinal Paneth cells. The homology of PHD3 with human neutrophil alpha-defensin HNP1 (human natural peptide) was 30%. The primary structures of PHD1 and PHD2 are most similar to RED1 (rhesus enteral defensin), one of six enteral alpha-defensins of rhesus monkeys. PHD1-3 have been shown to be active against the Gram-positive bacteria Listeria monocytogenes and Staphylococcus aureus, the Gram-negative bacterium Escherichia coli, and the fungus Candida albicans, similarly to the human HNP1 defensin.

Expression and purification of recombinant human alpha-defensins in Escherichia coli.

Different strategies have been developed to produce small antimicrobial peptides (AMPs) using recombinant techniques. Up to now, all efforts to obtain larger quantities of active recombinant human alpha-defensins have been only moderately successful. Here we report an effective method of biosynthesis of human alpha-defensins (hNP-1 to hNP-3 and hD-5 and hD-6) in the Escherichia coli. All the peptides, expressed as insoluble fusions with the peptide encoded by a portion of E. coli tryptophan operon (trp DeltaLE 1413 polypeptide), were isolated from the inclusion bodies by immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by chemical cleavage. Fully reduced peptides that were purified according to a straightforward protocol were subsequently folded, oxidized, and subjected to functional and structural analyses. With the exception of hD-6, all recombinant alpha-defensins exhibit expected anti-E. coli activity, as measured by the colony counting method. The method described in this report is a low-cost, efficient way of generating alpha-defensins in quantities ranging from milligrams to grams.

[Fusion expression, purification and bioactivity detection of human defensinalpha (HDalpha)].

AIM: To obtain recombinant human defensin alpha(HDalpha) and detect its biological activity, so as to facilitate further research. METHODS: The HDalpha gene fragment with hydroxylamine cleavage site was synthesized, and then cloned into pBV220-IL-4 vector to construct pBV220-IL-4-HDalpha. The constructed vector which was confirmed to be correct by sequencing was transformed into E.coli DH5alpha and the IL-4-HDalpha fusion protein was expressed under temperature induction. After fusion protein was cleaved to remove IL-4 by hydroxylamine, purification and renaturation was performed. HDalpha's characteristics were identified by SDS-PAGE and bioactivity detection. RESULTS: After temperature induction, the expressed fusion protein which accounted for about 20% of total bacterial protein existed mainly in the form of inclusion body. After cleaving by hydroxylamine, the purity of obtained HDalpha was about 99.8%. Bacteriostatic test and clone forming test showed that recombinant HDalpha could obviously inhibit the growth of bacteria. CONCLUSION: The recombinant expression plasmid for HDalpha gene has been constructed successfully and obtained engineering bacteria can stably express target protein. Furthermore, techniques of purification and renaturation was set out, which lays the foundation for further functional study and application of HDalpha.

Peptides of human gingival crevicular fluid determined by HPLC-ESI-MS.

The acidic-soluble protein content of human gingival crevicular fluid was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), and the eluent deriving from the chromatography separation was directly introduced into an ion-trap mass spectrometer through electrospray ionization (ESI-IT MS). By this technique the molecular weight of peptides/proteins was determined with a precision of approximately 1/10,000 amu. On the basis of the chromatographic behavior and the knowledge of the molecular mass value, some peptides and proteins soluble in acidic solution were unambiguously recognized. Besides high quantities of human serum albumin, alpha-defensins 1-4 and minor amounts of cystatin A, statherin, basic PB salivary peptide and other unidentified components were detected. The presence of alpha-defensins in gingival crevicular fluid is in agreement with their relevant contribution to protein composition deriving from granulocyte secretions. Other peptides and proteins abundant in human saliva, such as proline-rich proteins (PRPs) and histatins, were not detected in gingival crevicular fluid. Further investigations will be necessary to establish the origin of statherin and PB salivary peptide in gingival crevicular fluid.