Violacein Targets the Cytoplasmic Membrane of Bacteria

Abstract

Violacein is a tryptophan-derived purple pigment produced by environmental bacteria, which displays multiple biological activities, including strong inhibition of Gram-positive pathogens. Here, we applied a combination of experimental approaches to identify the mechanism by which violacein kills Gram-positive bacteria. Fluorescence microscopy showed that violacein quickly and dramatically permeabilizes B. subtilis and S. aureus cells. Cell permeabilization was accompanied by the appearance of visible discontinuities or rips in the cytoplasmic membrane, but it did not affect the cell wall. Using in vitro experiments, we showed that violacein binds directly to liposomes made with commercial and bacterial phospholipids and perturbs their structure and permeability. Furthermore, molecular dynamics simulations were employed to reveal how violacein inserts itself into lipid bilayers. Thus, our combined results demonstrate that the cytoplasmic membrane is the primary target of violacein in bacteria. The implications of this finding for the development of violacein as a therapeutic agent are discussed.

Death in a sphere: Chromobacterium violaceum secretes outer membrane vesicles filled with antibiotics

“…Chromobacterium violaceum forms smooth violet colonies on common laboratory media, which is due to the production of the secondary metabolite violacein, a blue‐purple pigment (Cauz et al ., ). Other bacteria like Collimonas sp., Pseudoalteromonas , Alteromonas , Duganella sp., and Janthinobacterium also produce violacein, suggesting that violacein producers are phylogenetically distinct and can be found in diverse environments, as for example in the rhizosphere of olive groves or forest soils, oceans, glaciers, rivers, or associated with the surfaces of sea sponges (Choi et al ., ).…”

“…This allows researchers to use violacein production as a visual readout for functionality of quorum sensing, as for instance when studying quorum sensing inhibitors (Choi et al ., ). Biosynthesis of violacein is catalysed by gene products of a cluster of five genes and involves the oxidative condensation of two tryptophan molecules giving rise to violacein, which is a highly hydrophobic compound (Cauz et al ., ). It is thought that C. violaceum produces violacein as a mechanism to fight off competing bacteria in an ecological niche and so far, violacein was found to exhibit antifungal, antiprotozoal, antiviral, antitumoral and antioxidant activity.…”

“…This is due to its mechanism of action as is inserts itself into lipid bilayers, as shown with the quick permeabilization of artificial membranes as well as Gram‐positive bacterial cells. This was visualized as initial holes in the membranes of Bacillus subtilis cells, which later dissolved (Cauz et al ., ). Since Gram‐negative bacteria are resistant to violacein, it is thought that the outer membrane of Gram‐negative bacteria works as a protection to keep violacein away from the inner membrane, its main target (Cauz et al ., ).…”

“…This was visualized as initial holes in the membranes of Bacillus subtilis cells, which later dissolved (Cauz et al ., ). Since Gram‐negative bacteria are resistant to violacein, it is thought that the outer membrane of Gram‐negative bacteria works as a protection to keep violacein away from the inner membrane, its main target (Cauz et al ., ). Interestingly, violacein is also a potential cancer therapeutic, as it showed cytotoxicity against various cancer cell lines and promoted gastroprotective effects in a gastric ulcer rat model (Choi et al ., ).…”

“…After accumulation of violacein in the periplasm, it would be of interest to establish how OMV secretion is regulated and how violacein is prevented from damaging the cytoplasmic membrane from the periplasmic side. Since violacein disrupts the cytoplasmic membrane (Cauz et al ., ), one could speculate that periplasmic transport and secretion of violacein must be tightly regulated processes to avoid cell damage.…”