Antibacterial activity of a synthetic peptide that mimics the LPS binding domain of Indian mud crab, Scylla serrata anti-lipopolysaccharide factor (SsALF) also involved in the modulation of vaginal immune functions through NF-kB signaling.

Recently the cDNA coding for anti-lipopolysaccharide factor (ALF) has been identified from the Indian mud crab, Scylla serrata and has been named S. serrata anti-lipopolysaccharide factor (SsALF). SsALF protein sequence demonstrated the presence of two highly conserved cystine residues between which the putative lipopolysaccharide (LPS) binding domain is known to be located. In this study, we have designed and synthesized a 24 amino acid linear (lSsALF24) and a cyclic (cSsALF24) peptides based on this putative LPS binding domain and demonstrated the ability of these peptides to bind to LPS. The peptides were active against vaginal pathogens demonstrated by MIC, CFU and phagocytosis assays. cSsALF24 did not show toxicity to human vaginal epithelial cells (HeLa-S3), macrophages and rabbit erythrocytes even at high concentration (64.64 µM). Flow cytometry results demonstrated that cSsALF24 peptide suppressed LPS induced phagocytosis of FITC labeled E. coli. HeLa cells were stimulated with LPS (10 µg/ml) alone for 6 h or after two washings with PBS, treated for 1 h with cSsALF24 (64.64 µM). After washing, the cells were cultured for 24 h in fresh media. The spent media as well as cells were collected for the determination of cytokine/chemokine levels such as interleukin-6 (IL-6) interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and interleukin-1α (IL-1α) using ELISA and RT-PCR respectively. Similar results were obtained with LPS stimulated cells treated with c/nSsALF24 or unstimulated cells treated with c/nSsALF24. The expression of cytokine/chemokines and mRNA's coding these proteins were unaffected in c/nSsALF24 treated cells. In contrast, in LPS stimulated cells, the expression levels of these molecules were up-regulated via the induction of nuclear factor kappa-B (NF-kB) levels. However, the expression of these pro-inflammatory markers was decreased in LPS stimulated cells following the treatment with cSsALF24, attributing anti-inflammatory potential of the peptide. Collectively, these findings suggest that cSsALF24 might regulate the vaginal epithelial cell immune responses indirectly through modulation of LPS-TLR4 binding in NF-kB pathway.

Antimicrobial peptides as microbicidal contraceptives: prophecies for prophylactics--a mini review.

The global increase in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) and sexually transmitted infections (STIs) has led to the introduction of barrier methods, such as condoms. However, drawbacks associated with condoms, such as men being reluctant to use them and women being unable to negotiate their use, have led to the search for better and acceptable alternatives, namely the microbicides. These are gel formulations that, when used prior to sexual intercourse, protect against the transmission of HIV and other STIs. However, after observing the side-effects of nonoxynol-9, a component of the microbicidal formulations available on the market, the focus has shifted to natural available compounds demonstrating the preferred protective effects. Antimicrobial peptides (AMPs) are one such group of compounds present in a wide range of organisms from bacteria to humans. The existing 750 or so, low-molecular-weight, cationic charged peptides are classified into five major groups based on their three-dimensional structure obtained by nuclear magnetic resonance studies. The hypothesized mode of action seems to be the interaction of the positively charged peptides with the negatively charged phospholipids present on the surface of the cell membrane. Various studies have demonstrated the effect of several AMPs, namely, defensins, protegrins, cathelicidins, cecropins, polyphemusins, magainins and melittins, against various STI-causing pathogens and HIV/herpes simplex virus, both in vitro and in vivo. The contraceptive efficacies of magainin and nisin in vitro and in vivo are worth mentioning. We believe these peptides are suitable candidates in the development of newer mechanism-based microbicides in future.

Antimicrobial peptides: premises and promises.

Antimicrobial peptides (AMPs) are an important component of the natural defences of most living organisms against invading pathogens. These are relatively small (< 10kDa), cationic and amphipathic peptides of variable length, sequence and structure. During the past two decades several AMPs have been isolated from a wide variety of animals, both vertebrates and invertebrates, and plants as well as from bacteria and fungi. Most of these peptides are obtained from different sources like macrophages, neutrophils, epithelial cells, haemocytes, fat body, reproductive tract, etc. These peptides exhibit broad-spectrum activity against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, protozoa, yeast, fungi and viruses. A few peptides have also been found to be cytotoxic to sperm and tumour cells. AMPs are classified based on the three dimensional structural studies carried out with the help of NMR. The peptides are broadly classified into five major groups namely (a) peptides that form alpha-helical structures, (b) peptides rich in cysteine residues, (c) peptides that form beta-sheet, (d) peptides rich in regular amino acids namely histatin, arginine and proline and (e) peptides composed of rare and modified amino acids. Most of these peptides are believed to act by disrupting the plasma membrane leading to the lysis of the cell. AMPs have been found to be excellent candidates for developing novel antimicrobial agents and a few of these peptides show antimicrobial activity against pathogens causing sexually transmitted infection (STI), including HIV/HSV. Peptides, namely magainin and nisin have been shown to demonstrate contraceptive properties in vitro and in vivo. A few peptides have already entered clinical trials for the treatment of impetigo, diabetic foot ulcers and gastric helicobacter infections. In this review, we discuss the source, structures and mode of action with special reference to therapeutic considerations of various AMPs.

Evaluation of antimicrobial peptide nisin as a safe vaginal contraceptive agent in rabbits: in vitro and in vivo studies.

In the midst of the global epidemics of both unwanted pregnancies and sexually transmitted infections (STIs), options that provide protection are ideal. In the present study, nisin, a known antimicrobial peptide, was evaluated for safety and contraceptive potential in vitro and in vivo in the rabbit. A concentration of 400 microg nisin per ml was found to be spermicidal in vitro, and the effect was dose and time dependent. In vivo studies indicated that intravaginal application of 1 mg nisin blocked conception in rabbits. Repeated application of nisin (50 mg/animal per day) in rabbits for 14 consecutive days did not cause local inflammation or damage to the vaginal epithelium. In addition, the rate of diffusion of nisin into the blood via the vaginal mucosal epithelium, and its clearance from the circulation was found to be rapid. No treatment-related changes were observed in the reproductive performance of rabbits after cessation of treatment. Furthermore, no changes were observed in the gestation period, subsequent growth and survival of neonates in these animals. When male rats were given nisin orally for 13 consecutive weeks, no effect was observed on reproductive performance. The number of pups born, survival and growth of pups were unaltered. The affinity studies of nisin revealed that spermatozoa are more susceptible to nisin than red blood cells and vaginal epithelial cells. We suggest that nisin with spermicidal and antimicrobial properties could serve as a safe vaginal contraceptive for future therapeutic interventions in STIs.