Rhesus macaque theta defensins suppress inflammatory cytokines and enhance survival in mouse models of bacteremic sepsis.

Theta-defensins (θ-defensins) are macrocyclic antimicrobial peptides expressed in leukocytes of Old World monkeys. The peptides are broad spectrum microbicides in vitro and numerous θ-defensin isoforms have been identified in granulocytes of rhesus macaques and Olive baboons. Several mammalian α- and ß-defensins, genetically related to θ-defensins, have proinflammatory and immune-activating properties that bridge innate and acquired immunity. In the current study we analyzed the immunoregulatory properties of rhesus θ-defensins 1-5 (RTDs 1-5). RTD-1, the most abundant θ-defensin in macaques, reduced the levels of TNF, IL-1α, IL-1ß, IL-6, and IL-8 secreted by blood leukocytes stimulated by several TLR agonists. RTDs 1-5 suppressed levels of soluble TNF released by bacteria- or LPS-stimulated blood leukocytes and THP-1 monocytes. Despite their highly conserved conformation and amino acid sequences, the anti-TNF activities of RTDs 1-5 varied by as much as 10-fold. Systemically administered RTD-1 was non-toxic for BALB/c mice, and escalating intravenous doses were well tolerated and non-immunogenic in adult chimpanzees. The peptide was highly stable in serum and plasma. Single dose administration of RTD-1 at 5 mg/kg significantly improved survival of BALB/c mice with E. coli peritonitis and cecal ligation-and-puncture induced polymicrobial sepsis. Peptide treatment reduced serum levels of several inflammatory cytokines/chemokines in bacteremic animals. Collectively, these results indicate that the anti-inflammatory properties of θ-defensins in vitro and in vivo are mediated by the suppression of numerous proinflammatory cytokines and blockade of TNF release may be a primary effect.

Rhesus macaque θ-defensin isoforms: expression, antimicrobial activities, and demonstration of a prominent role in neutrophil granule microbicidal activities.

Mammalian defensins are cationic, antimicrobial peptides that play a central role in innate immunity. The peptides are composed of three structural subfamilies: α-, ß-, and θ-defensins. θ-defensins are macrocyclic octadecapeptides expressed only in Old World monkeys and orangutans and are produced by the pair-wise, head-to-tail splicing of nonapeptides derived from their respective precursors. The existence of three active θ-defensin genes predicts that six different RTDs (1-6) are produced in this species. In this study, we isolated and quantified RTDs 1-6 from the neutrophils of 10 rhesus monkeys. RTD-1 was the most abundant θ-defensin, constituting ~50% of the RTD content; total RTD content varied by as much as threefold between animals. All peptides tested were microbicidal at ∼1 µM concentrations. The contribution of θ-defensins to macaque neutrophil antimicrobial activity was assessed by analyzing the microbicidal properties of neutrophil granule extracts after neutralizing θ-defensin content with a specific antibody. θ-defensin neutralization markedly reduced microbicidal activities of the corresponding extracts. Macaque neutrophil granule extracts had significantly greater microbicidal activity than those of human neutrophils, which lack θ-defensins. Supplementation of human granule extracts with RTD-1 markedly increased the microbicidal activity of these preparations, further demonstrating a prominent microbicidal role for θ-defensins.

Synthesis, structure, and activities of an oral mucosal alpha-defensin from rhesus macaque.

The oral cavity is an environment challenged by a large variety of pathogens. Consequently, the antimicrobial peptides expressed in that environment are interesting as they evolved to defend against a broad spectrum of bacteria and fungi. Here we report the discovery of new alpha-defensins from rhesus macaque oral mucosa and determine the first alpha-defensin structure from that species. The new peptides were identified by sequencing of reverse transcriptase-PCR products obtained from oral mucosal tissues, disclosing three mucosal alpha-defensins, termed rhesus macaque oral alpha-defensins (ROADs). The peptide corresponding to fully processed ROAD-1 was synthesized, subjected to folding/oxidation conditions, and purified. ROAD-1 was active against Staphylococcus aureus, Escherichia coli, and Candida albicans in a concentration-dependent manner. We determined the structure of ROAD-1 using NMR spectroscopy and find that the synthetic peptide adopts the canonical disulfide pairing and alpha-defensin fold. The antimicrobial mechanism of defensins has been correlated with their ability to disrupt and permeabilize the cell envelope, activities that depend on the surface features of the folded peptide. Although ROAD-1 maintains the defensin fold, the oral defensin displays distinct surface features when compared with other alpha-defensin structures.

Isolation, synthesis, and antimicrobial activities of naturally occurring theta-defensin isoforms from baboon leukocytes.

Theta-defensins are macrocyclic antimicrobial peptides that were previously isolated from leukocytes of a single species, the rhesus macaque. We now report the characterization of baboon theta-defensins (BTDs) expressed in bone marrow and peripheral blood leukocytes. Four cDNAs encoding theta-defensin precursors were characterized, allowing for the prediction of 10 theoretical theta-defensins (BTD-1 to BTD-10) produced by binary, head-to-tail splicing of nonapeptides excised from paired precursors. Five of the predicted theta-defensins were purified from baboon leukocytes, and synthetic versions of each were prepared. Anti-theta-defensin antibody localized the peptides in circulating neutrophils and monocytes and in immature and mature myeloid elements in bone marrow. Each of the BTDs possessed antimicrobial activity against bacterial and fungal test organisms in vitro. Peptide activities varied markedly despite a high degree of sequence conservation among the theta-defensins tested. Thus, baboons express numerous theta-defensins which appear to differentially contribute to host defense against diverse pathogens.

Microbicidal properties and cytocidal selectivity of rhesus macaque theta defensins.

Rhesus macaque theta-defensins (RTDs) are unique macrocyclic antimicrobial peptides. The three RTDs (RTD 1-3), isolated from macaque leukocytes, have broad-spectrum antimicrobial activities in vitro and share certain structural features with acyclic porcine protegrins, which are microbicidal peptides of the cathelicidin family. To understand the structural features that confer the respective cytocidal properties to theta-defensins and protegrins, we determined and compared the biological properties of RTD 1-3 and protegrin 1 (PG-1) in assays for antimicrobial activity, bacterial membrane permeabilization, and toxicity to human cells. RTD 1-3 and PG-1 had similar microbicidal potencies against Escherichia coli, Staphylococcus aureus, and Candida albicans in low-ionic-strength (10 mM) buffers at pH 7.4. The inclusion of physiologic sodium chloride partially inhibited the microbicidal activities of the RTDs, and the degree of inhibition depended on the buffer used in the assay. Similarly, the inclusion of 10% normal human serum partially antagonized the bactericidal activities of all four peptides. In contrast, the microbicidal activities of PG-1 and RTD 1-3 against E. coli were unaffected by physiologic concentrations of calcium chloride and magnesium chloride. Treatment of E. coli ML35 cells with RTD 1-3 or PG-1 rapidly rendered the bacteria permeable to omicron-nitrophenyl-beta-D-galactopyranoside, and this was accompanied by the rapid entry of the RTDs. Finally, although PG-1 was toxic to human fibroblasts and caused a marked lysis of erythrocytes, the RTDs were not cytotoxic or hemolytic. Thus, compared to PG-1, RTD 1-3 possess substantially greater cytocidal selectivity against microbes. Surprisingly, the low cytotoxicity of the RTDs did not depend on the peptides' cyclic conformation.

Antimicrobial properties of the Escherichia coli R1 plasmid host killing peptide.

The 52 amino acid host killing peptide (Hok) from the hok/sok post-segregational killer system of the Escherichia coli plasmid R1 was synthesized using Fmoc (9-fluorenylmethoxycarbonyl) chemistry, and its molecular weight was confirmed by mass spectroscopy. Hok kills cells by depolarizing the cytoplasmic membrane when it is made in the cytosol. Six microorganisms, E. coli, Bacillus subtilis, Pseudomonas aeruginosa, P. putida, Salmonella typhimurium, and Staphylococcus aureus were exposed to the purified peptide but showed no significant killing. However, electroporation of Hok (200 microgml(-1)) into E. coli cells showed a dramatic reduction (100000-fold) in the number of cells transformed with plasmid DNA which indicates that the synthetic Hok peptide killed cells. Electroporation of Hok into P. putida was also very effective with a 500-fold reduction in electrocompetent cells (100 microgml(-1)). Heat shock in the presence of Hok (380 microgml(-1)) resulted in a 5-fold reduction in E. coli cells but had no effect on B. subtilis. In addition, three Hok fragments (Hok(1-28), Hok(31-52) and Hok(16-52)) killed cells when electroporated into E. coli at 200 microgml(-1) (over 1000-fold killing for Hok(1-28), 50-fold killing for Hok(16-52) and over 1000-fold killing for Hok(31-52)). E. coli cells electroporated with Hok and visualized using transmission electron microscopy showed the same morphological changes as control cells to which Hok was induced using a plasmid inside the cell.