Allicin causes fragmentation of the peptidoglycan coat in Staphylococcus aureus by effecting synthesis and aiding hydrolysis: a determination by MALDI-TOF mass spectrometry on whole cells.

PURPOSE: To determine the effect of allicin on Staphylococcus aureus cell wall peptidoglycans by the application of MALDI-TOF mass spectrometry on whole cells and to relate this to current knowledge of wall-processing enzymes. METHODOLOGY: Two different S. aureus strains were grown for 48 h after which period each culture was split into two, one part was then treated with sub-inhibitory levels of allicin while the other part left untreated as a control. After a further 24 h whole cells were recovered and analysed by MALDI-TOF mass spectrometry. RESULTS: Changes in the mass spectra between the treated and untreated cells revealed fragmented peptidoglycans identified by mass calculation only in the treated cells. These peptidoglycan fragments where identified as the products of specific peptidoglycan hydrolases. CONCLUSIONS: Allicin is known to target cysteine thiol groups. These are absent in peptidoglycan hydrolases and we might have expected identical results in both of the treated and untreated cells. Peptidoglycan synthesis enzymes such as the Fem family of enzymes do contain cysteines. Fem enzymes A, B and X all have a conserved conformation of 99 % for over 100 S. aureus strains and are therefore potential targets for allicin. Examination of FemA structure showed that cysteine102 is accessible from the surface. We propose that allicin has an inhibitory mechanism alongside others of targeting FemA and possibly other Fem enzymes by curtailing glycine bridging and leading to fragmentation. This study provided an insight into yet another antimicrobial mechanism of allicin.

Bioactivity and biocompatibility of a chitosan-tobermorite composite membrane for guided tissue regeneration.

A polymer-mineral composite membrane of the mucopolysaccharide derivative, chitosan, and calcium silicate hydrate phase, tobermorite, was prepared by solvent casting and characterised by scanning electron microscopy (SEM) and Fourier Transform infrared spectroscopy (FTIR). The bioactivity and biocompatibility of the chitosan-tobermorite composite were evaluated in vitro with respect to its potential for use as a biodegradable guided tissue regeneration (GTR) membrane. The in vitro bioactivity of the composite was confirmed by the formation of crystalline substituted hydroxyapatite on the surface of the embedded tobermorite particles in simulated body fluid. The presence of the composite membrane was found to enhance the growth of MG63 human osteosarcoma cells by up to 30%. The findings of this initial study have indicated that this novel chitosan-tobermorite composite may be a suitable material for GTR applications.

Induction of apoptosis in host cells: a survival mechanism for Leishmania parasites?

Leishmania parasites invade host macrophages, causing infections that are either limited to skin or spread to internal organs. In this study, 3 species causing cutaneous leishmaniasis, L. major, L. aethiopica and L. tropica, were tested for their ability to interfere with apoptosis in host macrophages in 2 different lines of human monocyte-derived macrophages (cell lines THP-1 and U937) and the results confirmed in peripheral blood mononuclear cells (PBMC). All 3 species induced early apoptosis 48 h after infection (expression of phosphatidyl serine on the outer membrane). There were significant increases in the percentage of apoptotic cells both for U937 and PBMC following infection with each of the 3 species. Early apoptotic events were confirmed by mitochondrial membrane permeabilization detection and caspase activation 48 and 72 h after infection. Moreover, the percentage of infected THP-1 and U937 macrophages increased significantly (up to 100%) following treatment with an apoptosis inducer. Since phosphatidyl serine externalization on apoptosing cells acts as a signal for engulfment by macrophages, induction of apoptosis in the parasitized cells could actively participate in spreading the infection. In summary, parasite-containing apoptotic bodies with intact membranes could be released and phagocytosed by uninfected macrophages.

The effect of cicerfuran, an arylbenzofuran from Cicer bijugum, and related benzofurans and stilbenes on Leishmania aethiopica, L. tropica and L. major.

The effect of 3 arylbenzofurans and 7 stilbenes on the growth of Leishmania parasites and human monocytes was evaluated. Promastigotes from cultures of L. aethiopica, L. major and L. tropica were tested in the exponential phase of growth. All compounds were active at concentrations of 100 microg/mL within 6 hours. The 2-hydroxylstibene showed activity at a concentration < 1 microg/mL, with an LD (50) of 3 - 5 microg/mL after 48 hours of incubation. The most active compounds: cicerfuran, 2-hydroxy-2'-methyl-4',5'-methylenedioxystilbene, 2-hydroxy-2'-methoxy-4',5'-methylenedioxystilbene and 2-hydroxystilbene had even stronger activity against the temperature-induced amastigotes of L. aethiopica, with the latter having the highest relative potency against all three species. Leishmanicidal activity seemed to be associated with the level of oxygen substitution in each compound. The ratio between leishmanicidal activity on promastigotes and toxicity to human cells suggested that the compounds could be considered as leishmanicidal drug leads.