Tribolium castaneum defensin 1 kills Moraxella catarrhalisin an in vitro infection model but does not harm commensal bacteria.

Moraxella catarrhalis is a bacterial pathogen that causes respiratory tract infections in humans. The increasing prevalence of antibiotic-resistant M. catarrhalis strains has created a demand for alternative treatment options. We therefore tested 23 insect antimicrobial peptides (AMPs) for their activity against M. catarrhalis in a human in vitro infection model with primary macrophages, and against commensal bacteria. Effects on bacterial growth were determined by colony counting and growth curve analysis. The inflammatory macrophage response was characterized by qPCR and multiplex ELISA. Eleven of the AMPs were active against M. catarrhalis. Defensin 1 from the red flour beetle Tribolium castaneum significantly inhibited bacterial growth and reduced the number of colony forming units. This AMP also showed antibacterial activity in the in vitro infection model, reducing cytokine expression and release by macrophages. Defensin 1 had no effect on the commensal bacteria Escherichia coli and Enterococcus faecalis. However, sarcotoxin 1 C from the green bottle fly Lucilia sericata was active against M. catarrhalis and E. coli, but not against E. faecalis. The ability of T. castaneum defensin 1 to inhibit M. catarrhalis but not selected commensal bacteria, and the absence of cytotoxic or inflammatory effects against human blood-derived macrophages, suggests this AMP may be suitable for development as a new therapeutic lead against antibiotic-resistant M. catarrhalis.

Succinylated casein-coated peptide-mesoporous silica nanoparticles as an antibiotic against intestinal bacterial infection.

Increasing drug resistance necessitates the discovery of novel bactericides. Human defensin (HD) peptides can eliminate resistant bacteria and are promising candidates for next-generation antibiotics. T7E21R-HD5 is a potent bactericide designed by site mutations at enteric HD5. To facilitate the development of T7E21R-HD5 into an intestinal antibiotic, we employed a mesoporous silica nanoparticle (MSN) as the peptide carrier. Despite its ineffectiveness at killing bacteria, the MSN intensified the outer membrane penetration and inner membrane permeabilization abilities of T7E21R-HD5 and thus enhanced its antibacterial action against multidrug resistant (MDR) E. coli, which broadened the role of MSNs in drug delivery. For the reduction in T7E21R-HD5 losses in the stomach, we further modified MSN@T7E21R-HD5 with succinylated casein (SCN), a milk protein that can be specifically degraded by intestinal protease. SCN coating decreased T7E21R-HD5 release from the MSNs, especially in a highly acidic environment. The controlled release of MSN@T7E21R-HD5 from SCN encapsulation was confirmed in the presence of trypsin. MSN@T7E21R-HD5@SCN was nontoxic to host cells, and it was capable of inactivating MDR E. coli in vivo and alleviating intestinal inflammation by suppressing the production of inflammatory factors TNF-α, IL-1ß, and MMP-9. This study provides a peptide-based nanobiotic with efficacy to combat intestinal infection, especially against drug-resistant bacteria. The biocompatible and readily prepared MSN/SCN delivery system may benefit further intestinal antibiotic design and promote the drug transformation of additional enterogenic functional molecules.

Tandem combination of Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) generates a more potent antifungal protein.

Plant defensins are small (45 to 54 amino acids) positively charged antimicrobial peptides produced by the plant species, which can inhibit the growth of a broad range of fungi at micro-molar concentrations. These basic peptides share a common characteristic three-dimensional folding pattern with one α-helix and three ß-sheets that are stabilized by eight disulfide-linked cysteine residues. Instead of using two single-gene constructs, it is beneficial when two effective genes are made into a single fusion gene with one promoter and terminator. In this approach, we have linked two plant defensins namely Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) genes by a linker peptide sequence (occurring in the seeds of Impatiens balsamina) and made into a single-fusion gene construct. We used pET-32a+ vector system to express Tfgd2-RsAFP2 fusion gene with hexahistidine tag in Escherichia coli BL21 (DE3) pLysS cells. Induction of these cells with 1 mM IPTG achieved expression of the fusion protein. The solubilized His6-tagged recombinant fusion protein was purified by immobilized-metal (Ni2+) affinity column chromatography. The final yield of the fusion protein was 500 ng/µL. This method produced biologically active recombinant His6-tagged fusion protein, which exhibited potent antifungal action towards the plant pathogenic fungi (Botrytis cinerea, Fusarium moniliforme, Fusarium oxysporum, Phaeoisariopsis personata and Rhizoctonia solani along with an oomycete pathogen Phytophthora parasitica var nicotianae) at lower concentrations under in vitro conditions. This strategy of combining activity of two defensin genes into a single-fusion gene will definitely be a promising utility for biotechnological applications.

Brevinin-2R(1) semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway.

Brevinin-2R is a novel non-hemolytic defensin that was isolated from the skin of the frog Rana ridibunda. It exhibits preferential cytotoxicity towards malignant cells, including Jurkat (T-cell leukemia), BJAB (B-cell lymphoma), HT29/219, SW742 (colon carcinomas), L929 (fibrosarcoma), MCF-7 (breast adenocarcinoma), A549 (lung carcinoma), as compared to primary cells including peripheral blood mononuclear cells (PBMC), T cells and human lung fibroblasts. Jurkat and MCF-7 cells overexpressing Bcl2, and L929 and MCF-7 over-expressing a dominant-negative mutant of a pro-apoptotic BNIP3 (DeltaTM-BNIP3) were largely resistant towards Brevinin-2R treatment. The decrease in mitochondrial membrane potential (DeltaPsim), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin-2R-triggered death. Brevinin-2R interacts with both early and late endosomes. Lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin-B and cathepsin-L prevented Brevinin-2R-induced cell death. Autophagosomes have been detected upon Brevinin-2R treatment. Our results show that Brevinin-2R activates the lysosomalmitochondrial death pathway, and involves autophagy-like cell death.

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.