Systematic comparison of the functional physico-chemical characteristics and biocidal activity of microbial derived biosurfactants on blood-derived and breast cancer cells

Hypothesis The cytotoxicity of biosurfactants on cell membranes may be influenced by composition of their hydrophilic head and hydrophobic tails. It is hypothesised that they form mixed micelles which exert a detergent-like effect that disrupts the plasma membrane. The functional physico-chemical and biocidal characteristics of four biosurfactants were concurrently investigated to determine which of their structural characteristics may be tuned for greater efficacy. Experiments Rhamnolipid-95, rhamnolipid-90, surfactin and sophorolipid were characterised using FTIR, LC-MS, HPLC, surface tension and critical micelle concentration. Their biocidal activity against HEK 293, MCF-7 and THP-1 cell lines were investigated by MTT assay, using doxorubicin as cytotoxic control. Growth curves were established for all cell lines using trypan blue (TB) and MTT assays, corresponding doubling time (DT) and growth rate were obtained and compared. Findings HEK 293 cell-line had the highest growth rate amongst the three cell lines. For TB assay, growth of HEK 293 > THP-1 and for MTT, HEK 293 > MCF-7 while the DT was in the order of THP-1 > MCF-7 > HEK 293. Sophorolipid showed anti-proliferative activity comparable to doxorubicin on THP-1 > MCF-7 > HEK 293. THP-1 showed high sensitivity to sophorolipid with IC50 of 10.50, 25.58 and 6.78 (μg/ml) after 24, 48 and 72 hr respectively. However, sophorolipid was cytotoxic from 24-72 hr on HEK 293 cell lines with IC50 of 21.53, 40.57 and 27.53 μg/ml respectively. Although, doxorubicin showed higher anti-proliferative activity than all biosurfactants, it had poorer selectivity index for the same time durations compared to the biosurfactants. This indicates that biosurfactants were more effective for slowing the growth of the tested cancer cell lines and hence may be potential candidates for use in human cancer therapy. Physico-chemical characteristics of the biosurfactants suggest that their mechanism of action may be due to activity on the cell membrane

Peptide based amphiphiles

Contains fulltext : 13849.pdf (publisher's version ) (Open Access

Importance of the long-chain fatty acid beta-hydroxylating cytochrome P450 enzyme YbdT for lipopeptide biosynthesis in Bacillus subtilis strain OKB105

Bacillus species produce extracellular, surface-active lipopeptides such as surfactin that have wide applications in industry and medicine. The steps involved in the synthesis of 3-hydroxyacyl-coenzyme A (CoA) substrates needed for surfactin biosynthesis are not understood. Cell-free extracts of Bacillus subtilis strain OKB105 synthesized lipopeptide biosurfactants in presence of L-amino acids, myristic acid, coenzyme A, ATP, and H2O2, which suggested that 3-hydroxylation occurs prior to CoA ligation of the long chain fatty acids (LCFAs). We hypothesized that YbdT, a cytochrome P450 enzyme known to beta-hydroxylate LCFAs, functions to form 3-hydroxy fatty acids for lipopeptide biosynthesis. An in-frame mutation of ybdT was constructed and the resulting mutant strain (NHY1) produced predominantly non-hydroxylated lipopeptide with diminished biosurfactant and beta-hemolytic activities. Mass spectrometry showed that 95.6% of the fatty acids in the NHY1 biosurfactant were non-hydroxylated compared to only ~61% in the OKB105 biosurfactant. Cell-free extracts of the NHY1 synthesized surfactin containing 3-hydroxymyristic acid from 3-hydroxymyristoyl-CoA at a specific activity similar to that of the wild type (17 ± 2 versus 17.4 ± 6 ng biosurfactant min−1·ng·protein−1, respectively). These results showed that the mutation did not affect any function needed to synthesize surfactin once the 3-hydroxyacyl-CoA substrate was formed and that YbdT functions to supply 3-hydroxy fatty acid for surfactin biosynthesis. The fact that YbdT is a peroxidase could explain why biosurfactant production is rarely observed in anaerobically grown Bacillus species. Manipulation of LCFA specificity of YbdT could provide a new route to produce biosurfactants with activities tailored to specific functionsPeer reviewedMicrobiology and Molecular Genetic

Lipopeptides: from self-assembly to bioactivity

This Feature Article discusses several classes of lipopeptide with important biomedical applications as antimicrobial and antifungal agents, in immune therapies and in personal care applications among others. Two main classes of lipopeptide are considered: (i) bacterially-expressed lipopeptides with a cyclic peptide headgroup and (ii) linear lipopeptides (with one or more lipid chains) based on bio-derived and bio-inspired amino acid sequences with current clinical applications. The applications are briefly summarized, and the biophysical characterization of the molecules is reviewed, with a particular focus on self-assembly. For several of these types of biomolecule, the formation of micelles above a critical micelle concentration has been observed while others form bilayer structures, depending on conditions of pH and temperature. As yet, there are few studies on the possible relationship between self-assembly into structures such as micelles and bioactivity of this class of molecule although this is likely to attract further attention

Diversity of Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Lipopeptide Biosurfactants

Lipopeptide biosurfactants (LPBSs) consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive) and Pseudomonas (Gram-negative) have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs). Production of active-form NRPSs requires not only transcriptional induction and translation but also post-translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed