Bactericidal action of violacein revealed

In an article published in the journal Infectious diseases of SCA, Brazilian researchers describe the mechanism of bactericidal action of purplishin, a purple pigment produced by environmental bacteria, especially Chromobacterium violaceum.

According to the study's authors, the substance targets the cytoplasmic membrane of bacteria, mainly affecting gram-positive bacteria such as those of the genera. Streptococcus, enterococcus and Listeria. Its various biological activities include the ability to kill even bacteria that have become resistant to antibiotics.

The survey was conducted with the support of the São Paulo Research Foundation – FAPESP by research groups led by Frederico Gueiros-Filho of the Institute of Chemistry of the University of São Paulo ( IQ-USP) and Marcelo Brocchi of the Institute of Biology of the University of Campinas (IB-UNICAMP).

"The potent bactericidal properties of this pigment were discovered in 1945, but its mechanism of action has never been studied before.Many biologically active molecules are described in the literature, but if we want to use them for develop drugs, we must know how they work, "said Gueiros-Filho.

Attractive target

Purpletin is a natural pigment derived from the amino acid tryptophan. It is produced as a secondary metabolite by several phylogenetically distinct bacteria found in environments as diverse as oceans, glaciers, rivers and soils. C. violaceum is the first bacterium described as a producer of violacein and the most studied to date.

Purpleine has attracted attention because of its broad spectrum of biological activity. In addition to its potent activity against bacteria, including drug-resistant pathogens such as methicillin-resistant bacteria Staphylococcus aureus (MRSA), it has anti-fungal, antiprotozoal, antiviral, antitumor and antioxidant properties. Several studies describe these properties, but according to Gueiros-Filho, the target and mode of action of the violacein have never been accurately identified.

The first step in the study, he explained, was to treat bacteria of the species S. aureus and Bacillus subtilis with violacein. Using fluorescence microscopy and a set of indicator dyes, the group found that the pigment rapidly permeabilized bacterial cells. Cell permeabilization was accompanied by the appearance of visible discontinuities (holes or tears) in the cytoplasmic membranes, while the cell walls remained intact.

The researchers also demonstrated the permeabilization of membranes by measuring the leakage of ATP (adenosine triphosphate, a key energy transport and storage molecule) in treated cells.

Then, in collaboration with the group led by Professor Iol-I-USP Iolanda M. Cuccovia, they conducted in vitro experiments showing that violacein also disrupted the structure and permeability of liposomes, spherical vesicles with a water core and membrane created phospholipid test tube, the main components of organic cell membranes.

"With these experiments, we have shown that the phenomenon observed in cells could be attributed to a direct effect of violacein on the membrane," said Gueiros-Filho.

In addition, computer simulations of molecular dynamics have been used to explore how violacein interacts with lipid bilayers, such as those that form the cytoplasmic membrane.

Based on the results obtained, the authors of the study suggest that the presence of violacein between the phospholipid layers was sufficient to impair the organization of the membrane, thus increasing the distance between the phospholipid molecules and causing a loss of integrity of the membrane.

By damaging membranes, violacein is able to destroy dormant persistent bacteria as an antibiotic resistance strategy depending on the metabolic activity of the microorganism. These bacteria form biofilms to survive in hostile environments.

According to the researchers, the cytoplasmic membrane is an attractive and underused target for antimicrobials, for whom the discovery that purpurea is a membrane targeting compound should pave the way for future research into the utility of this natural product.

The research was supported by FAPESP via the projects "How do bacteria coordinate membrane biogenesis with cell growth and division?", "Antibacterial activity of the violacein against Staphylococcus aureus and "Pharmacokinetic Studies" and "Chemistry Interface: Drug, Peptide and Enzyme Interactions with Membrane Models".

The issue of the selectivity of violacein is at the heart of future research. The cytoplasmic membrane is similar and common to all living cells, so that violacein may also be able to disrupt eukaryotic cells such as those in humans. This property could be the reason why it displays activity against many types of pathogens, including fungi, protozoa as well as tumors, said Gueiros-Filho.

Preliminary results obtained by IQ-USP and IB-UNICAMP researchers suggest that purpurea is not highly selective for bacterial membranes and could become toxic to the host. If this were the case, the molecule would have to be chemically modified to make it more specific.

The spread of multidrug-resistant bacteria due to the overuse of antibiotics in human medicine and agriculture is one of the most important current threats to public health. The World Health Organization (WHO) warned of the imminence of a post-antibiotic era in which common infections and minor injuries that could be treated for decades would become deadly again.

The most commonly used antibiotics were discovered decades ago and are directed against a limited number of targets. It is therefore urgent to develop new antimicrobials with novel mechanisms of action.