Antiherpes simplex virus type 2 activity of the antimicrobial peptide subtilosin

In the present study we evaluated the antiviral activity of subtilosin, a cyclical peptide isolated from Bacillus amyloliquefaciens, against herpes simplex virus type 2 (HSV-2) in cell cultures and we investigated subtilosin mode of action. We determined, using a virus yield inhibition assay, that non cytotoxic concentrations of subtilosin inhibit HSV-2 replication in Vero cell cultures. Subtilosin strongly inhibited extracellular and total virus production even when it was added at 8 h post-infection indicating that not only virus release but also viral particle formation is impeded by the antiviral peptide. Although viral glycoprotein gD level of expression is not affected by the bacteriocin, an altered pattern of gD intracellular localization was detected by immunofluorescence assay in subtilosin treated culture. On the other hand, at high concentrations subtilosin displays virucidal action.Fil: Quintana, Verónica Mara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Torres, Nicolás. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Wachsman, Mónica B.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Sinko, Patrick J.. State University of New Jersey; Estados UnidosFil: Castilla, Viviana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Chikindas, Michael. State University of New Jersey; Estados Unido

Mode of action of lactocin 160, a bacteriocin from vaginal Lactobacillus rhamnosus.

OBJECTIVES: To determine the mechanism of antimicrobial action of lactocin 160, a bacteriocin produced by the healthy vaginal strain of Lactobacillus rhamnosus, using an established model, with Micrococcus luteus ATCC 10420 as a test organism. METHODS: Sensitivity of M. luteus to lactocin 160 was determined by the diffusion assay. Loss of cellular ATP in the lactocin-treated cells was elucidated using a commercially available ATP determination kit (luciferin-luciferase bioluminescence assay). Luminescence intensity as a reflection of ATP quantity was determined using a luminometer. Dissipation of membrane potential (Deltapsi) was studied using fluorophore DiSC3(5) with the fluorescence spectrum sensitive to changes in Deltapsi. RESULTS: Lactocin 160 inhibited growth of M. luteus ATCC 10420 at a concentration of 5 microg/ml. There were no significant changes in the intracellular ATP level of M. luteus upon the addition of 20 microg/ml of lactocin 160. However, the extracellular ATP level increased significantly. This means that the treatment of cells with lactocin 160 resulted in an efflux of ATP from inside the cells. Therefore, a partially purified lactocin 160 preparation (16 microg /ml of the bacteriocin in the sample) killed sensitive cells and dissipated 3.12 +/- 0.36% of Deltapsi. CONCLUSION: Lactocin 160 has a mode of action typical for bacteriocins. It disturbs the cellular membrane (Deltapsi dissipation) and induces ATP efflux, most likely because of the pore formation, which is a common mechanism of action for many bacteriocins

Exploration and Understanding of Beneficial Properties of Lactic Acid Bacteria: 10 years of experience in Applied Food Biotechnology

The scientific community is currently facing a more than exponential increase of knowledge in all areas and disciplines. In the last 10 years, the contribution of the journal Applied Food Biotechnology was eminent in distributing that knowledge by providing a tribune for researchers from different countries all over the world to share their ideas, observations, and hypotheses. With a focus on different aspects of applied and fundamental biological sciences, the journal Applied Food Biotechnology was established as a reference of the Iranian scientific community. In the last 10 years, the journal has been covering several topics related to exploring the beneficial properties of lactic acid bacteria (LAB) and their role in modern food technologies. LAB are already proven as a realization of Hippocrates' vision for the potential role of food in human and other animals' health. However, what will be the next frontier? What are the challenges in understanding the interactions between microbiota and host microorganisms? What will be the novel analytical tools, facilitating a new era of probiotic research? These were only a few research topics presented and discussed in Applied Food Biotechnology in the last decade. This editorial overview aims to celebrate the scientific contribution of Applied Food Biotechnology in the area of research associated with the beneficial properties of LAB, summarizing some of the studies published in the journal

Safety Study of an Antimicrobial Peptide Lactocin 160, Produced by the Vaginal Lactobacillus rhamnosus

Objective. To evaluate the safety of the antimicrobial peptide, lactocin 160. Methods. Lactocin 160, a product of vaginal probiotic Lactobacillus rhamnosus 160 was evaluated for toxicity and irritation. An in vitro human organotypic vaginal-ectocervical tissue model (EpiVaginal) was employed for the safety testing by determining the exposure time to reduce tissue viability to 50% (ET-50). Hemolytic activity of lactocin160 was tested using 8% of human erythrocyte suspension. Susceptibility of lactobacilli to lactocin160 was also studied. Rabbit vaginal irritation (RVI) model was used for an in vivo safety evaluation. Results. The ET-50 value was 17.5 hours for lactocin 160 (4.9 hours for nonoxynol 9, N9). Hemolytic activity of lactocin 160 was 8.2% (N9 caused total hemolysis). Lactobacilli resisted to high concentrations of peptide preparation. The RVI model revealed slight vaginal irritation. An average irritation index grade was evaluated as “none.” Conclusions. Lactocin 160 showed minimal irritation and has a good potential for intravaginal application

Heterologous Processing and Export of the Bacteriocins Pediocin PA-1 and Lactococcin A in Lactococcus Lactis:A Study with Leader Exchange

The bacteriocins pediocin PA-1 and lactococcin A are synthesized as precursors carrying N-terminal extensions with a conserved cleavage site preceded by two glycine residues in positions -2 and -1. Each bacteriocin is translocated through the cytoplasmic membrane by an integral membrane protein of the ABC cassette superfamily which, in the case of pediocin PA-1, has been shown to possess peptidase activity responsible for proteolytic cleavage of the pre-bacteriocin. In each case, another integral membrane protein is essential for bacteriocin production. In this study, a two-step PCR approach was used to permutate the leaders of pediocin PA-1 and lactococcin A. Wild-type and chimeric pre-bacteriocins were assayed for maturation by the processing/export machinery of pediocin PA-1 and lactococcin A. The results show that pediocin PA-1 can be efficiently exported by the lactococcin machinery whether it carries the lactococcin or the pediocin leader. It can also compete with wild-type lactococcin A for the lactococcin machinery. Pediocin PA-1 carrying the lactococcin A leader or lactococcin A carrying that of pediocin PA-1 was poorly secreted when complemented with the pediocin PA-1 machinery, showing that the pediocin machinery is more specific for its bacteriocin substrate. Wild-type pre-pediocin and chimeric pre-pediocin were shown to be processed by the lactococcin machinery at or near the double-glycine cleavage site. These results show the potential of the lactococcin LcnC/LcnD machinery as a maturation system for peptides carrying double-glycine-type amino-terminal leaders

Spermicidal Activity of the Safe Natural Antimicrobial Peptide Subtilosin

Bacterial vaginosis (BV), a condition affecting millions of women each year, is primarily caused by the gram-variable organism Gardnerella vaginalis. A number of organisms associated with BV cases have been reported to develop multidrug resistance, leading to the need for alternative therapies. Previously, we reported the antimicrobial peptide subtilosin has proven antimicrobial activity against G. vaginalis, but not against the tested healthy vaginal microbiota of lactobacilli. After conducting tissue sensitivity assays using an ectocervical tissue model, we determined that human cells remained viable after prolonged exposures to partially-purified subtilosin, indicating the compound is safe for human use. Subtilosin was shown to eliminate the motility and forward progression of human spermatozoa in a dose-dependent manner, and can therefore be considered a general spermicidal agent. These results suggest subtilosin would be a valuable component in topical personal care products aimed at contraception and BV prophylaxis and treatment

The Role of Phonological Awareness and Phonetic Radical Awareness in Acquiring Chinese Literacy Skills in Learners of Chinese as a Second Language

There is much research into the roles of phonological awareness and phonetic radical awareness in the development of Chinese character reading and writing skills in native-speaking children, but there is comparatively little work on the relationship between such metalinguistic skills and character literacy skills in adult learners of Chinese a second language (CSL). In this study, we explored this issue with 83 Arabic and English CSL learners who had studied Chinese in their home country. Their knowledge of phonological awareness, phonetic radical awareness, and Chinese character reading and writing was measured. There were two main findings. Firstly, the learners’ phonological awareness, but not their phonetic radical awareness, predicted the acquisition of character reading and writing skills directly or indirectly. Secondly, phonetic radical awareness did not mediate the effect of phonological awareness on character reading and writing skills. The results point to the different roles that phonological awareness and phonetic radical awareness play in the development of character literacy skills, and the still unclear relationship between phonological awareness and phonetic radical awareness. These findings are important for understanding the contribution of phonological awareness and phonetic radical awareness to the acquisition of character literacy skills for CSL learners