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Potential biocide roles of violacein
Ignacio Rivero Berti
1
, Melisa E. Gantner
2
, Santiago Rodriguez
2
,
German A. Islan
1
, Wagner J. Fávaro
3
, Alan Talevi
2
,
Guillermo R. Castro
4
* and Nelson Durán
4
,
5
*
1
Laboratorio de Nanobiomateriales, Centro de Investigación y Desarrollo en Fermentaciones Industriales
(CINDEFI), Departamento de Química, Facultad de Ciencias Exactas, CONICET-UNLP (CCT La Plata), La
Plata, Argentina,
2
Laboratorio de Investigación y Desarrollo de Bioactivos (LIDeB), Departamento de
Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), La Plata,
Argentina,
3
Laboratory of Basic and Applied Bacteriology, Department of Microbiology, Center of
Biological Sciences, Londrina State University, Londrina, Brazil,
4
Nanomedicine Research Unit (Nanomed),
Center for Natural and Human Sciences, Federal University of ABC (UFABC), São Paulo, Brazil,
5
Laboratory
of Urogenital Carcinogenesis and Immunotherapy, Department of Structural and Functional Biology,
Institute of Biology, University of Campinas, Campinas, Brazil
Violacein is a pigment produced by Gram-negative bacteria, which has shown
several beneficial biological activities. The most relevant activities of violacein
include the interference in the physiological activities of biological membranes,
inhibition of cell proliferation, antioxidant, and anti-inflammatory activities.
Moreover, the antiviral activities of violacein against some enveloped and non-
enveloped viruses have also been reported. Violacein showed a wide spectrum of
protease inhibition, both experimentally and in silico. Other in silico studies have
suggested that violacein binds to the SARS-CoV-2 spike. Empirical
physicochemical studies indicate that violacein (or, occasionally, its derivatives)
may be administered orally to treat different disorders. In addition, different
alternatives to product violacein, and molecular devices for delivery of this
pigment are reviewed.
KEYWORDS
violacein, COVID-19, membrane interaction, antioxidant activity, anti-inflammatory
activity, protease inhibition, violacein antiviral activities
1 Introduction
In the last five decades, viral infections have negatively affected the world population,
mainly because of accelerated globalization and rapid urbanization, which are associated
with strong changes in social habits. Many viral infections have spread worldwide through
different vectors, such as dengue (DENV), Ebola, Hepatitis B virus, herpes virus, HIV/AIDS,
Middle East respiratory syndrome, rotaviruses, and Zika. Recently, the global population has
been in peril with the SARS-CoV-2 pandemic. The world death toll of SARS-CoV-
2 overpassed 6.8 million people worldwide, and long-term deleterious consequences
have been described in some of the 755 billion infected people based on the World
Health Organization (WHO) website (WHO, 2023). Different therapeutic strategies have
been developed worldwide for the treatment and prevention of SARS-CoV-2 infection.
Many old and traditional antiviral drugs were on the battlefront trying to stop the viral
pandemic but with poor or disappointing results (Das et al., 2020). The processes to develop
SARS-CoV-2 vaccines, get the corresponding approval, and deliver them worldwide took
approximately six to 12 months. Although large-scale worldwide vaccination has
substantially reduced the death toll, new viral variants still challenge the effectiveness of
marketed vaccines making their production more complex. Effective drug treatments are,
however, still needed to complement vaccine development, especially for unvaccinated
OPEN ACCESS
EDITED BY
Raj Kumar,
University of Nebraska Medical Center,
United States
REVIEWED BY
Rahul K. Suryawanshi,
Gladstone Institutes, United States
Anisha Dsouza,
Massachusetts Eye and Ear Infirmary and
Harvard Medical School, United States
*CORRESPONDENCE
Guillermo R. Castro,
grcastro@gmail.com
Nelson Durán,
nduran@unicamp.br
RECEIVED 14 March 2023
ACCEPTED 16 June 2023
PUBLISHED 21 July 2023
CITATION
Rivero Berti I, Gantner ME, Rodriguez S,
Islan GA, Fávaro WJ, Talevi A, Castro GR
and Durán N (2023), Potential biocide
roles of violacein.
Front. Nanotechnol. 5:1186386.
doi: 10.3389/fnano.2023.1186386
COPYRIGHT
© 2023 Rivero Berti, Gantner, Rodriguez,
Islan, Fávaro, Talevi, Castro and Durán.
This is an open-access article distributed
under the terms of the Creative
Commons Attribution License (CC BY).
The use, distribution or reproduction in
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original author(s) and the copyright
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comply with these terms.
Frontiers in Nanotechnology frontiersin.org01
TYPE Review
PUBLISHED 21 July 2023
DOI 10.3389/fnano.2023.1186386
people or groups that may not appropriately respond to vaccination
(such as immunocompromised patients), and to cover viral
mutations not covered by available vaccines (Mei and Tan, 2021).
Nature provides an enormous number of secondary metabolites
with a wide variety of molecular scaffolds produced from different
biological kingdoms. Among secondary metabolites, pigments have
been used in many applications since the beginning of human
society and are presently employed in many industrial areas,
such as cosmetics, textiles, foods, and healthcare (Kulandaisamy
et al., 2020). Microorganisms are one of the relevant pigment
producers because they can be cultured under controlled
conditions, have simple nutritional requirements, fast and
reproducible growth, are easy to scale up, and provide well-
established purification methods that are available in the market;
they are also environmentally friendly (Mumtaz et al., 2019). In silico
screening is among the modern approaches to find novel drug
candidates; in particular, structure-based virtual screening departs
from experimental or in silico predicted protein structures to select
from large public chemical databases or relatively small in-house
libraries, those compounds that are more likely to bind to a known
or putative binding site (Rahman et al., 2022). Virtual screening has
been extensively applied to find potential therapeutics against
COVID-19. For example, using this approach 26 synthetic
derivatives of coumarins and quinolines were analyzed by
molecular docking and molecular dynamics; among them, six
compounds were predicted to possess high binding capacity
against SARS CoV-2 main protease (M
pro
)(Yañez et al., 2021).
Several microbial secondary metabolites are currently marketed
for therapeutic applications that started at the beginning of the last
century with penicillin, diverse anthracyclines, mitomycins, etc., and
have recently been reviewed (Abdelghani et al., 2021). Since the
sanitary SARS-CoV-2 emergency, antiviral applications of many
molecules and pigments, and their biological mechanisms have
recently been revisited (Azman et al., 2018; Ma et al., 2020; Selim
et al., 2021).
Violacein has recently attracted the attention of researchers
owing to its wide variety of biological activities. During the last
two decades, several reports have described numerous biological
activities of this pigment, including immunomodulatory,
antimicrobial, antiparasitic, antifungal, anticancer, and antiviral
activities (Duran et al., 2021a;Duran et al., 2021b;Duran et al.,
2022). The relevance of the antioxidant properties of violacein must
be analyzed in the context of COVID-19 pathology, as acute
infections of SARS-CoV-2 can produce cell death and long-term
neurological pathologies. In general, most viral infections are related
to the reduction of antioxidant physiological pathways mainly by the
inhibition of Nuclear Factor Erythroid 2 (NRF2), a transcription
factor (i.e., leucine zipper), which triggers antioxidant proteins and
hampers the NLRP3 inflammasome, which mediates the release of
many cytokines (Zhu et al., 2021). Additionally, SARS-CoV-
2 induces activation of Nuclear Factor kappa B (NF-κB),
promoting inflammation and oxidative stress. The direct
consequence of SARS-CoV-2 is the development of elevated
levels of inflammation and generalized oxidative processes. These
processes are developed by the activation of pro-inflammatory
cytokines (i.e., Tumor Necrosis Factor-alpha o TNF-α, IL-1β, and
IL-6) produced by macrophages and monocytes, high recruitment of
immune and endothelial cells, and platelets (Tay et al., 2020). The
NF-κB activation induces high activities of cyclooxygenase 2
(COX2) and NOX2 (NADPH oxidase) responsible for ROS
production and mitochondrial oxidative stress. Some of the
postulated molecular mechanisms of SARS-CoV-2 infection have
recently been reviewed (Chernyak et al., 2020). Besides, COVID-19
is not only considered a respiratory viral disease, but is also
associated with endotheliopathy, triggering many molecular
markers such as angiopoietin 2 plasminogen activator inhibitor 1
(PAI-1) and Willebrand factor (vWF), among others, and compared
to healthy individuals. These factors can be associated not only with
fatigue and circulatory diseases (i.e., immune thrombosis and
myocardial infarction) but also with neuropsychiatric pathologies
(i.e., cognitive disorders and stroke) in acute COVID-19 infections
and long-term COVID (Laforge et al., 2020;Fodor et al., 2021;Li
et al., 2021). A recent study conducted in Nigeria showed that the
levels of glutathione; vitamins A, C, and E; enzymes with antioxidant
activities such as superoxide dismutase, catalase, and glutathione
peroxidase; and the concentrations of Cu, Mg, Se, and Zn are lower
in patients with COVID-19 than in healthy people (Muhammad
et al., 2021). Intravenous administration of vitamin C, a well-known
antioxidant compound, at high concentrations ameliorates
inflammation and oxidative stress in patients with severe
COVID-19 (Vollbracht and Kraft, 2022). COVID-19 pathology
mediated by inflammation associated with the oxidative stress
cascade unquestionably contributes to disease severity. Several
trials of potential therapeutic antioxidant molecules to treat
SARS-Co-2 infection are in progress in the US (https://
clinicaltrials.gov/).
It is of particular interest to analyze the antiviral activities of
violacein against viruses since the emergence of SARS-CoV-19 is
correlated with the scarce activity of many therapeutic molecules.
The present work endeavors to describe the potential
mechanisms and properties of violacein as a potential antiviral
agent against SARS-CoV-2. Analyses of the effects of violacein
on cellular membranes, and antioxidant, anti-inflammatory, and
antiviral activities are overviewed (Figure 1). Studies on protease
inhibition and in silico studies have also been summarized. Finally,
alternatives for the production and delivery of violacein are
discussed.
2 Violacein properties and biological
activities
2.1 Violacein general properties
Violacein is a low molecular weight (MW = 433.41 g/mol)
bacterial purple pigment produced mainly by Gram (−) bacteria
and has been recently reported (Duran et al., 2021a).
Chromobacterium spp. and Janthinobacterium spp. are the most
common violacein producers. (Duran et al., 2021b).
The IUPAC formula for violacein is 3-[2-hydroxy-5-(5-hydroxy-
1H-indol-3-yl)-1H-pyrrol-3-yl]indol-2-one (C
20
H
13
N
3
O
3
). From a
structural point of view, violacein is a bisindole molecule composed
of three heterocycles and a 2-pyrrolidone molecule that links two
indoles, an oxindole, and 5-hydroxyindole (Figure 2).
Violacein is a very hydrophobic molecule characterized by a P
of 2.7 (PubChem) and Log Po/w = 3.34 (Choi et al., 2020).
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Rivero Berti et al. 10.3389/fnano.2023.1186386
Violacein is insoluble in aqueous media but is partially soluble in
organic solvents such as acetone and dioxane, less soluble in ethanol
and propanol, and soluble in ethyl acetate, DMSO, and methanol.
The UV-Visible spectra of violacein showed two maximum
peaks at λmax = 577–585 nm depending on the organic solvent,
and a UV peak at λ
max
= 260.3 nm in methanol (Abboud and
Arment, 2013).
Violacein biosynthesis involves four genes arranged in a gene
cluster vioABCD of 8 kb. The synthesis of violacein involves the
condensation of two L-tryptophan molecules at the oxindole
position, followed by intramolecular reorganization at the 5-
hydroxy-indole ring (1→2 shift) (Duran et al., 2021a).
The physicochemical properties of violacein were analyzed in
relation to some typical empirical rules, namely: Lipinski and Veber
(which predict whether a drug like compound is likely to be orally
bioavailable via passive absorption) (Lipinski et al., 2001;Veber
et al., 2002) and Pfizer 3/75 rule (which suggests that a drug
candidates in the chemical space of low calculated log P and high
topological surface area are not likely to cause significant
toxicological effects at total plasma concentrations below 10 μM)
(Hughes et al., 2008). Violacein fulfills all of them, thus being a good
candidate for oral administration with likely low toxicity
(Supplementary Table S1).
2.2 Violacein antioxidant activity
The antioxidant activity of violacein was reported for the first
time in 1998 and confirmed by several extensive studies by Duran’s
research group during the 2000s and recently reviewed (Duran et al.,
2021a;Durban et al., 2021b). The presence of several conjugated
FIGURE 1
Potential antiviral violacein activities. Example: SARS-Cov-2. Abbreviations: IL-6 (Interleukin 6), CXCL-1 (chemokine ligand 1), TNFR1 (Tumor
necrosis factor receptor 1), TLR8 (Toll-like receptor 8), HSV-1 (Herpes simplex virus 1), RV-SA11 (Simian rotavirus SA11), PV-2 (Poliovirus type 2), MHV
(Murine hepatitis virus), Huh7 (human hepatocellular carcinoma cell line, which contains complete genome replicon of Hepatitis C virus), RBD:ACE2
(receptor-binding domain to angiotensin converting enzyme 2 [ACE2]), HIV-1-RT (human immunodeficiency virus -1 reverse transcriptase), MMP-2
and MMP-9 (matrix metalloprotease 2 and 9 respectively). Coronavirus COVID-19 structure illustration was created at the Centers for Disease Control
and Prevention (CDC). Modified from the CDC, Alissa Eckert, MS; Dan Higgins, MAM (Public Domain).
FIGURE 2
ChemicalStructure of deoxyviolacein(A ) and(B) violacein,adapted
with permission from Nelson Durán et al., licensed under CC BY-SA 4.0.
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Rivero Berti et al. 10.3389/fnano.2023.1186386
double bonds and two peaks in the UV-visible spectra suggests a
protective effect against visible and UV radiation. The experimental
antioxidant activity of violacein has been confirmed by theoretical
studies based on an electron density model and spectroscopic
analysis (Cao et al., 2007;Jehlička et al., 2015). The estimated
ionization potential (IP) of violacein is 146.88 kcal/mol which is
about 5% lower than a well-known antioxidant a-tocopherol (IP =
154.90 kcal/mol). The antioxidant activity of violacein has been
attributed to the N7-H7 band of 5-hydroxyindole (Cao et al., 2007).
In 2006, violacein antioxidant activity against lipid peroxidation
was experimentally evaluated in three models of lipid membranes,
including egg and soybean phosphatidylcholine liposomes, and also
in rat liver microsomes. Protection of lipid membranes by violacein,
either in solution or reconstituted within the liposomes, was
evaluated against nitric oxide, 1,1-diphenyl-2-picryl-hydrazyl
(DPPH), and ascorbyl radicals in the presence or absence of the
biodye showing IC
50
values of 21, 30, and 125 μM, respectively
(Konzen et al., 2006). It was also observed that reconstitution of
violacein into the liposomes enhanced its antioxidant activities.
Liposomes in the absence of violacein were used as control. A
more recent in vitro study on violacein antioxidant properties
evaluated violacein partially purified from the isolate
Chromobacterium vaccinii CV5 against common radicals. The
IC50 of violacein against DPPH, superoxide (produced from
phenazine methosulphate), nitric oxide (produced from sodium
nitroprusside), hydrogen peroxide, and hydroxyl (produced from
ferrous ammonium sulfate) were 0.87 µM, 0.91 µM, 1.19 µM,
0.86 µM, and 0.85 µM, respectively (Vishnu and Palaniswamy,
2018). The lower in vitro IC
50
values reported in this study could
be attributed to the partial purification of violacein. In any case, all
these results suggested a protective effect mediated by violacein at
micromolar or sub-micromolar concentrations against peroxidation
produced by radical species.
In another study, the antioxidant activity of violacein produced
by C. violaceum wild-type and mutants less- and non-biodye
producers (i.e., CV9, CV13, and CV14, respectively) was
analyzed (Abboud and Arment, 2013). All C. violaceum strains
were UV-irradiated at λ= 253.7 nm with 6,000 μWs
-1
cm
-2
. Non-
violacein-pigmented mutants did not grow, whereas the viability of
the wild-type strain and violacein hyper-producer mutants was
reduced by UV irradiation for 48 h. These results suggest the
potential protective effects of Violacein against DNA UV-induced
damage. Additionally, catalase activity in non-violacein-producing
strains was enhanced compared with the enzymatic activity of
violacein hyper-producer C. violaceum strains. This experiment
strongly suggested an active scavenging role of violacein against
reactive oxidative species (ROS). Violacein has been added to
sunscreens to increase its protective effect against potential UV
damage (Suryawanshi et al., 2015).
2.3 Violacein anti-inflammatory activity
The analgesic, antipyretic, and immunomodulatory effects of
violacein produced by the newly isolated C. violaceum
ESBV4400 were evaluated in Wistar albino rats and mice
(Antonisamy and Ignacimuthu, 2010). The effects of violacein
against non-inflammatory and inflammatory pain, anaphylactic
reactions and fever were assayed by injecting with ovalbumin,
acetic acid, formalin, sheep red cells, and Saccharomyces
cerevisiae yeast to create traumatic trials. The harmful effects in
the treated animals were countered by using indomethacin,
dexamethasone, disodium cromoglycate, naloxone, and morphine
as control drugs. In the delayed hypersensitivity and provoked paw
anaphylaxis tests employing red blood sheep cells and ovalbumin.
Similar results were observed with 10 mg kg
-1
dexamethasone paired
with 5 mg kg
-1
disodium cromoglycate and 40 mg kg
-1
violacein,
with less than 2% difference. In the case of severe pain induced by
acetic acid, the results showed a 93.9% and 78% reduction in
writhing using 5 mg kg
-1
and 10 mg kg
-1
morphine and
indomethacin. Similar response was elicited by 40 mg kg
-1
violacein. Comparable results were obtained using the formalin
assay. Hyperthermia induced by the yeast S. cerevisiae injection
in rodents showed normalization of temperature with 150 mg kg
-1
paracetamol after 60 min, and a similar response was observed with
20 mg kg
-1
, and 40 mg kg
-1
violacein after 120 min and 60 min,
respectively. The authors concluded that violacein exerts an
immunosuppressive effect on inflammatory physiological
responses through T Cells, antiallergic activity to the anaphylactic
Ig E-mediated response, and antipyretic response by inhibiting
prostaglandin synthesis.
In another report, the effect of violacein on acute and chronic
inflammation in mice (C57BL/6) produced by intraperitoneal
injection of 1.0 µg LPS from Escherichia coli 0111:B4 was
analyzed (Verinaud et al., 2015). Dendritic cells and their
CD80 and CD86 markers treated with LPS and violacein did not
show variations compared to control cells. Comparable results were
obtained for B- and T-cells, which did not show signs of toxicity. In
addition, mice treated with LPS-violacein exhibited a significant
decrease in neutrophil infiltration in the peritoneal cavity compared
to control mice. In addition, cytokines IL-6 and CXCL-1 were
reduced, whereas IL-10 was increased in the serum. An
Experimental Autoimmune Encephalomyelitis mouse model was
developed by induction with myelin oligodendrocyte glycoprotein
(MOG35–55). The authors reported an increase in scurfin protein
levels (FoxP3, considered a primary regulator of T regulatory cells
(Tregs) in mice treated with violacein.
A cellular study using immune lineages of human peripheral
blood mononuclear cells (PBMCs) and two murine macrophage
cells (ANA-1 and Raw 264.7, established by retroviral infections)
treated with violacein was carried out (Venegas et al., 2019).
Macrophages exposed to violacein displayed elevated levels of
pro-inflammatory TNFαconcomitant with TNF receptor 1
(TNFR1) activation, which induces cellular apoptosis. In
addition, the authors suggested that Toll-like receptors (TLRs)
could be activated by violacein in murine cells, which are
strongly associated with PAMPs (i.e., pathogen-associated
molecular patterns). Particularly, human TLR8 was activated by
violacein in the transfected HEK-293 cell line, and an in silico
docking proposed a binding mode of violacein to the receptor
similar to the antiviral drug imidazoquinolinone (Venegas et al.,
2019). Members of the imidazoquinolines and imidazoles, which
resemble the heterocycle structure of violacein, are known to activate
TLR7/8, triggering cytokine production by the immune system
associated with the clearance of virally infected cells and
treatment of skin cancer. The activation of TLR7/8 by
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Rivero Berti et al. 10.3389/fnano.2023.1186386
imidazoquinolines and imidazoles depends on the electronic
configuration of the heterocycles (Kaushik et al., 2021).
Briefly, the major anti-inflammatory effects of violacein were
associated to the reduction of pro-inflammatory cytokines such as
TNF-α, TGF-ß, IL-1β, and IL-6, and chemokines CXCL1 and
CXCL12 involved in the cell recruitment but also increase the
expression of IL-4 and IL-10 (Antonisamy and Ignacimuthu,
2010;Platt et al., 2014;Verinaud et al., 2015).
2.4 Violacein antiviral activities
In a pioneering study, May et al. (1989),May et al. (1991)
reported the in vitro antiviral activity of violacein/deoxyviolacein
(90/10) in HeLa cells infected with poliovirus and herpes simplex
virus (HSV-1). The IC
50
value of violacein against HSV-1 infected
cells was estimated to be 0.577 µM. A decade later, another study
challenged the in vitro antiviral activity of violacein against HSV-1,
Simian rotavirus SA11 (RV-SA11), and Poliovirus type 2 (PV-2)
strains with pigment concentrations approximately 1/3–1/2 below
the cytotoxicity levels on the tested cell lines (i.e., HEp-2, FRhK-4,
MA104, or Vero cell cultures). The antiviral activity of 1.25 μM
violacein against the tested virus by the MTT assay ranged from
24.3% to 8.5%, depending on the virus and strain (Andrighetti-
Fröhner et al., 2003).
The differences in violacein effectiveness against the tested
viruses entail analyzing the main characteristics of the viral
surface to underpin the potential antiviral mechanism of the
pigment. Rotaviruses are non-enveloped viruses that contain a
glycoprotein, VP7, attached to the cell surface. Glycoprotein
VP7 is composed of 326 amino acids, with two hydrophobic
domains in the amino-terminal group (Poruchynsky et al., 1985).
In addition, polioviruses are non-enveloped viruses with three main
proteins, VP1, VP2, and VP3, on the capsid surface, that are folded
in a hydrophobic ß-barrel (He et al., 2000). On the other hand,
members of the herpes virus family are enveloped, having a lipid
membrane structure obtained from the host covering the virion.
Similarly, viral structures can be observed in coronaviruses and
human immunodeficiency virus (HIV). Despite the different surface
characteristics of the three tested viruses, all shared hydrophobic
moieties on their surfaces that could interact with water-insoluble
molecules, such as violacein. Since the interaction of violacein with
molecules with hydrophobic motifs (i.e., non-ionic surfactants,
cyclodextrins, aromatic ionic liquids, lipid carriers) were
previously reported (de Azevedo et al., 2000;Rivero Berti et al.,
2019;Rivero Berti et al., 2020;Rivero Berti et al., 2022). It is expected
that hydrophobic interactions appear to be the unspecific major
mechanism of interaction between the virus and violacein.
Murine hepatitis virus (MHV), like SARS-CoV-2, belongs to the
genus Betacoronavirus. Both are enveloped, with glycoproteins on
the surface and a genome composed of positive-sense single-
stranded RNA within a nucleocapsid. The inhibition of MHV-3
in L929 (mouse fibroblasts, ATCC CCL-1) infected cells by 20 µM
violacein was 42% after incubation at 37°C for 1 h (Gonçalves et al.,
2023).
In a recent physicochemical study, the in vitro inhibitory effects
of purified violacein and deoxyviolacein on CoV-2 spike RBD:
ACE2 and HIV-1 Reverse transcriptase proteins were analyzed
(Supplementary Table S2). These results indicated that
deoxyviolacein had a low inhibitory effect on both proteins at
millimolar concentrations. Meanwhile, 1 mM violacein inhibited
94.3% of HIV-1 Reverse transcriptase, and CoV-2 spike RBD:
ACE2 was inhibited by 53% at 2 mM violacein (Dogancıet al.,
2022). The concentration of the pigment needed to inhibit the
activity of these proteins is approximately one thousand times
higher than the high levels required to induce cytotoxicity in
most of the reported cellular cell lines (de Sousa Leal et al., 2015;
Duran et al., 2021a).
The low antiviral activity of violacein against HSV-1, Simian
rotavirus 219 SA11, and Poliovirus type 2 reported previously
(Andrighetti-Fröhner et al., 2003) could be attributed to its
extremely poor solubility of the biodye under physiological
environmental conditions, which may be a major obstacle to
replicate the conditions of in vitro assays (e.g., the concentration
of the dye) in cell assays or in vivo. The insolubility of violacein in
aqueous media can be attributed to the lack of polar groups and the
presence of aromatic motifs. The interaction of indole aromatic
residues of different molecules that can pile on each other, which is
attributed to π-πstacking, reduces violacein interactions with other
molecules, including water. Consequently, molecular aggregates of
violacein display reduced biological activities, similar to some low-
solubility marketed drugs containing aromatic rings (Islan et al.,
2012). It would be of most interest to study whether it is possible to
generate violacein derivatives that retain or even potentiate the
molecular interactions responsible for its antiviral effects
(increasing potency) and, simultaneously, include structural
modifications that diminish their π-πstacking potential, reducing
their tendency to aggregate. It is important to bear in mind, however,
that π-πstacking can be exploited in the design of last-generation
drug delivery systems, including high purified carbon- or graphene-
based nanomaterials, nanocomposites, targeted delivery systems,
and prodrug delivery systems such as self-assembled polyprodrug
amphiphiles (Zhuang et al., 2019). The key question appears to be:
Can violacein molecular structure be modified in a way that the
derivatives retain the ability to interact with viral constituents
(which, as explained later, also requires π-πstacking) and load
the dye into state-of-the-art drug delivery systems, and at the same
time display enhanced aqueous solubility? Are these mutually
exclusive properties? In any case, fine-tuning would likely be
required at the molecular optimization step.
Besides that, the antiviral mechanisms of violacein were not
reported yet, based on the biophysical properties of the biodye it
could be speculate that the hydrophobic interaction between the
virus moieties and violacein will be the major driven force for virus
inactivation.
2.5 Violacein and membrane interactions
Enveloped viruses are causative agents of diverse infections.
Enveloped viruses have a lipid membrane covering the viral
structure (i.e., the capsid) that originates from the infected host.
Among the advantages of encapsulated viruses, the fusion of the
viral membrane with the cell membrane is considered to be one of
the most effective strategies for infecting cells. In particular, many
coronaviruses have a bilayer of lipids and proteins, forming an
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Rivero Berti et al. 10.3389/fnano.2023.1186386
envelope around their capsids. This envelope is derived from host
membranes during virion formation but also plays a crucial role
during infection when virus particles adhere to the host cell
membrane (Nardacci et al., 2021). Therefore, the role of lipids in
SARS-CoV-2 infection and pathogenesis cannot be underestimated.
In a previous study, the existence of pockets of high hydrophobicity
in the SARS-CoV-2 protein S (spike), which firmly bind linoleic
acid, was described. This binding seemingly locks the S protein in a
state that decreases its association with the ACE2 receptor (Toelzer
et al., 2020). These types of hydrophobic pockets have previously
been used to develop drugs to treat rhinoviruses (Casanova et al.,
2018).
Additionally, lipid membranes play a key role in the replication
of all RNA (+) viruses, including coronaviruses. These viruses
manipulate the host membranes to form viral replication
organelles. In addition to sequestering the cellular machinery
necessary for viral multiplication, these organelles may also play
a role in evading the immune response. It is known that
coronaviruses alter the cellular metabolism of lipids, favoring the
synthesis of sterols and fatty acids that are propitious to them in an
analogous way to that in which tumor cells reprogram lipid
metabolism to ensure their survival (Borella et al., 2022;
Rudiansyah et al., 2022). Furthermore, coronaviruses interfere
with exosome formation, autophagy, and lipid rafts (Casari et al.,
2021). This reorganization of lipid metabolism and membrane
mechanisms ensures or promotes viral multiplication and is,
therefore, a potential target for new drugs or therapeutic strategies.
Therefore, studies of violacein-related lipids and membrane
systems are of interest. While violacein does not have a typical
amphiphilic head-tail structure, it is hydrophobic and has some
polar groups; therefore, direct interaction with cell or viral
membranes is highly probable (de Souza et al., 2017). Cauz et al.
(2019) described some interactions between violacein and
membranes, initially, to explain its antimicrobial activity.
However, these findings may be useful in explaining its antiviral
activity. In vitro experiments have shown that the addition of
violacein to large unilamellar vesicles (LUVs) disrupts these
vesicles and produces leaks, thereby affecting their permeability.
Additionally, because of the low concentration of violacein required
to produce these effects, they hypothesized that violacein does not
act as a tensioactive, like many antimicrobials, but rather produces
defects and discontinuities in phospholipid organization that alter
membrane permeability (Cauz et al., 2019). This partially contrasts
with previous results; de Souza et al. (2017) explored violacein
interactions with phospholipid thin films to model eukaryotic
plasma membranes and found that violacein effectively interferes
with the correct ordering of phospholipids, affecting their
compressibility and viscoelasticity, and therefore fluidity, but does
not produce changes in permeability in the tested models (de Souza
et al., 2017). In a recent study, the effect of violacein on synthetic
monolayer lipid membranes composed of 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-
ethylphosphocholine chloride salt (DSEPC), and 1,2-dipalmitoyl-
sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG) was
studied using the Langmuir-Blodgett technique. The isotherms of
violacein in the cationic and zwitterionic lipid monolayers showed a
decrease in lipid molecular area, suggesting compaction. However,
compaction was not observed in negatively charged lipid
monolayers. Further analyses with X-rays indicate that violacein
decreases the lipid tilt angle, which consequently induces the
thickening of lipid monolayers (Gupta et al., 2021;Gupta and
Ghosh, 2023). These results indicate the interaction of the
aromatic electrons of violacein with the positive residues of lipid-
like π-σnon-specific molecular interactions.
A recent study showed that the release of violacein from the cells
in one of the main bacterial pigment producers, Chromobacterium
violaceum (ATCC 12472), occurred through approximately 100 nm
extracellular membrane vesicles. The study revealed that 79.5% of
the violacein encapsulated in the membrane vesicles remained in
solution, and the membrane vesicles provided an increase of
violacein estimated in 1740 folds (Choi et al., 2020). The strategy
of violacein release in C. violaceum possesses two relevant
characteristics: first, the pigment will not be attached to the cell
surface because the bacteria grow in aqueous media and violacein is
a very hydrophobic molecule; and second, the released membrane
vesicles can be attached to the hydrophobic surfaces of several
molecules and structures, such as the VP7 glycoprotein of
rotaviruses and the ß-barrel structure of VP1-VP3 of poliovirus.
The interaction of viral glycoproteins present on the virus surface
handles virus-cell interactions, leading to causative infections
(Cosset and Lavillette, 2011). Consequently, membrane vesicles
containing violacein can easily merge with other membranous
lipid structures of enveloped viruses (HSV-1, SARS-CoV-2,
HIV, etc.).
The main cellular target of violacein are biological membranes
and molecules because of violacein hydrophobicity (i.e., low
solubility in watery environments). Additionally, the recent
finding of violacein which is released from the producer cells in
the form of membranous structures will favor the interaction within
mammalian cell membranes and also facilitates the entrance of
violacein to the cytoplasm.
2.6 Violacein inhibition activity on proteases
The traditional mechanism of SARS-CoV-2 infection involves
the binding of the spike (i.e., S glycoprotein) to ACE2 (i.e.,
angiotensin-converting enzyme type 2) to produce a membrane
fusion by a serine protease (i.e., TMPRSS-2) or by the cysteine
protease pathways (i.e., cathepsins). In recent studies, the spike in
SARS-CoV-2 variants was found with a wider spectrum of action by
activating the transmembrane serine protease 13 (i.e., TMPRSS-13)
and the matrix metalloproteases 2 and 9 (i.e., MMP-2 and MMP-9).
High MMP levels were found in patients with serious SARS-CoV-
2 infections. Supporting the role of proteases in viral infection,
protease inhibitors can reduce the infection of the SARS-CoV-2αin
the human fibrosarcoma HT1080 cell line (Benlarbi et al., 2022).
In previous work, the inhibition of matrix metalloproteases
(MMPs) by violacein was reported (Platt et al., 2014). The
authors reported that violacein inhibited MMP-2 in the MCF-7
cell line, and the metalloprotease played a relevant role in the
secretion of CXCL12, an inflammatory cytokine. Additionally,
violacein inhibited MMP-9, which downregulates the
a-chemokine receptor (CXCR4) specific for stromal-derived
factor-1, a chemotactic molecule for the recruiting lymphocytes
(Platt et al., 2014).
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Recently it was suggested that violacein presumably acted as a
protease inhibitor against the ACE2 receptor, and as an
immunomodulator against COVID-19 (Durán et al., 2022). M
pro
,
also called 3CL
pro
, and also the papain-like protease (PL
pro
)is
responsible for viral polyprotein disruption, a process important
for the virus to survive and replicate. M
pro
is a decisive coronavirus
enzyme that plays essential roles in modulating two key processes:
viral transcription and replication, which makes it an attractive
pharmacological target against SARS-CoV-2.
Then, one strategy could be protease inhibition as a target to M
pro
,
once it was shown that inhibition of many proteases was discovered by
researching the cytotoxic actions of violacein. Decease caused in
CD34+/c-Kit+/P-glycoprotein+/MRP1+ TF1 leukemia progenitor
cells was moderated by calpain (calcium-dependent protease-cysteine
protease) suppression and by decease-associated protein kinase 1
(DAPK1). Comparative analysis showed that violacein also induced
several protein kinases activities, such as protein kinase A (PKA),
pyruvate dehydrogenase kinase (PDK), and protein kinase B (AKT),
that were monitored by structural modifications induced by
endoplasmic reticulum stress and Golgi apparatus collapse, as led to
cell decease (Queiroz et al., 2012). It is attainable concluding some
chemical structural comparison between violacein activity and that of
protease suppressors such as Ebselen (Figure 3).
Following the suppression data, the HIV-1 RT suppression rate
of violacein (1 mM) from the Janthinobacterium sp. GK strain was
higher than 90%, and the SARS-CoV-2 spike RBD:
ACE2 suppression rate of violacein (2 mM) was higher than 50%.
In silico studies were performed to explore the potential interactions
among deoxyviolacein and violacein and three reference compounds
with the target proteins: ACE2, SARS-CoV-2 spike RBD (i.e.,
receptor binding domain), and HIV-1 RT (i.e., reverse
transcriptase). Violacein seems to bind strongly to the three
receptors as showed by their docking binding energies:
−9.94 kcal/mol for ACE2, −9.32 kcal/mol for HIV-1 RT,
and −9.32 kcal/mol for SARS-CoV-2 spike RBD. Comparable
results were obtained for deoxyviolacein: −10.38 kcal/mol for
ACE2, -9.50 kcal/mol for HIV-1 RT, and −8.06 kcal/mol for
SARS-CoV-2 spike RBD. Following these outcomes,
deoxyviolacein and violacein seem to bind to all the receptors
with high efficiency. HIV-1-RT and SARS-CoV-2 spike protein
suppression searches with deoxyviolacein and violacein were
reported for the first time in the literature (Doganci et al., 2022).
The inhibition of diverse proteases involved in the viral infection
process by violacein observed experimentally and by in silico
molecular docking is a relevant advantage from therapeutic point
of view because it is additional feature to the many biological
activities of violacein which will reduce the chance of drug multi-
resistance displayed by many molecules available in the market.
2.7 Violacein cytotoxicity
In vitro cell toxicity of violacein was evaluated in four
mammalian cell lines (i.e., Vero, MA104, FRhK-4, and HEp-2)
using cell morphology by light microscopy, cell viability by
Trypan blue and by the MTT assays (Andrighetti-Fröhner et al.,
2003). The violacein cytotoxic concentration 50% was in the range of
2.07 a 3.55 µM, which depends on the used technique and the cell
line. While the concentrations of violacein showed the best antiviral
activity at 1.25 µM against HSV-1 (i.e., strains KOS, VR-733, RV-
SA11) with inhibition percentages in the range of 17.75%–24.27%
and compared with acyclovir with inhibition percentages close to
100% Besides, the antiviral violacein concentration assayed was
35.5 to 8.9 times lower compared to the acyclovir (Andrighetti-
Fröhner et al., 2003). In general, violacein cytotoxicity in non-
tumoral cell lines is in the range of 5 µM–10 μM, meanwhile
tumoral cell lines are more sensitive to the biodye with a
cytotoxic range of 0.71 µM to about 5 µM (de Sousa Leal et al.,
2015;Rivero Berti et al., 2020). Several mechanisms of violacein
cytotoxicity including disturbance of mammalian cell membrane
and polarization of mitochondrial membrane, disruption of actin
function, p44/42 mitogen activated protein kinase (MAPK),
apoptosis mediated by caspase 3, etc. were reviewed recently
(Duran et al., 2016;Durban et al., 2021a). Besides, it is currently
accepted that violacein displays low toxicity in-vitro against non-
tumoral cell lines.
2.8 Violacein in silico binding studies
In an attempt to further study the potential binding site of
violacein within the SARS-CoV-2 Spike protein, five potential
binding sites reported in the literature were explored through
molecular docking using AutoDock4 (Morris et al., 2009). The
binding sites analyzed in this study are shown in Figure 4. Two
sites are located within the S2 subunit: site 1 refers to the arbidol site
previously published (Shuster et al., 2021) and site 2 corresponds to
the fusion peptide binding site described in a recent report (Hu et al.,
2021). The other sites are located within the S1 subunit: site 3 is a
potential allosteric site reported by Wang et al. (2022), site
4 corresponds to the experimentally determined biliverdin site in
the N-terminal domain (NTD) (Rosa et al., 2021), and site
5 addresses two possible reported sites in the RBD subunit,
which are the Spike: ACE2 protein-protein interaction (PPI)
interface (site 5α)(Bojadzic et al., 2021) and the site reported by
Dogancıet al. (2022) for violacein from the blind docking study on
the isolated RBD subunit (site 5β). In addition, the entire RBD
subunit was considered for the docking calculations to explore other
possible binding sites within such subunit (site 5γ). The biliverdin
site has not been considered an inhibitor site, because biliverdin
binding helps Spike evade antibody immunity (Rosa et al., 2021).
Nevertheless, it was decided to include it in the present study
FIGURE 3
Ebselen molecular structure, reprinted from Benjah-bmm 27,
Public Domain, via Wikimedia Commons
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Rivero Berti et al. 10.3389/fnano.2023.1186386
because it is an experimentally validated binding site for small
molecules.
The docking results obtained are shown in Supplementary Table
S3. The most favorable score considering the three protomers was
obtained for the 5γsite in the RBD subunit. While site 5γis likely to
be the most probable binding site, site 2 and site 3 are also probable
candidates.
Considering site 5γin protomer A, violacein was located in an
environment defined by residues Phe338A, Tyr365A, Tyr369A,
Ala372A, Ser373A, Phe374A, Asp405C, Glu406C, Arg408C,
Gln409C, Thr415C, Gly416C, Lys417C (Figure 5A), finding a
parallel-displaced π-stacking with Tyr365A, a T-shaped π-
stacking with Tyr369A, hydrogen bonds with Tyr369A, Ser373A,
and Thr415C, and a π-cation interaction with Arg408C (Figure 5B).
To further study the different interactions between violacein and
the 5γbinding site in the protomer A, 50 nanoseconds of molecular
dynamics (MD) simulation was carried out using GROMACS
2020.4 Molecular Dynamics engine (Abraham et al., 2015).
Figure 5C shows the root-mean-square deviation (RMSD) for
violacein as a function of the simulation time. The average RMSD
value was 0.645 Å with a standard deviation of 0.152 Å, indicating high
ligand stability at the 5γsite. By exploring the interactions that stabilize
the complex during the MD simulation through the measurement of
the distances between violacein atoms and the key residues of the
protein defined by docking (Supplementary Figures S1–S5), it can be
concluded that the stabilization of violacein at the 5γsite appears to be
mediated mainly by π-stacking and π-cation interactions, with a
coordinated contribution from hydrogen bonding. The docking and
MD procedure are described in the Supplementary Material together
with a detailed analysis of the interactions observed in this study.
Finally, due to the novelty of this site, the SARS-CoV-2 Spike
protein was further analyzed through the DoGSiteScorer web server,
a grid-based method that uses a Difference of Gaussian filter to the
binding pocket prediction, characterization, and druggability
estimation (Volkamer et al., 2012). This analysis ranked the 5γ
site in third place out of more than 100 potential sites found in the
protein, with a druggability score of 0.85 (0.86 being the highest
druggability score obtained).
2.9 Violacein production
The large-scale production of microbial pigments is seriously
limited by the production-purification costs and regulations in
dominant pharmaceutical countries. In 2018, the world market
for organic dyes was valued at U$D 3.5 billion, with an estimated
growth of about 37% by 2024 (Cassarini et al., 2021). The most
representative studies on violacein production are briefly described
below, including the use of recombinant microorganisms and the
new trend of recycling waste in the frame of the circular economy.
Violacein production was initially studied in the 2000s. The first
work on violacein production was conducted in C. violaceum and
Janthinobacterium lividum in synthetic liquid media (Mendes et al.,
2001;Nakamura et al., 2003). The main guidelines for the
production of violacein from C. violaceum can be found in the
websites of the American Type Culture Collection (ATCC,
United States, https://www.atcc.org/products/12472), and the
National Collection of Type Cultures (NCTC, United Kingdom,
https://www.culturecollections.org.uk/products/bacteria/detail.jsp?
collection=nctcandrefId=NCTC+9757).
FIGURE 4
3D structure of the SARS-CoV-2 Spike protein, where all potential violacein binding sites studied by molecular docking are highlighted on protomer
A. Protomer A is colored light blue while the corresponding RBD subunit is cornflower blue. Site 1 (navy blue) refers to the arbidol site located within the
S2 subunit, site 2 (magenta) corresponds to a fusion peptide site, site 3 (salmon) is a potential allosteric site within the S1 subunit, site 4 (lime green)
corresponds to the biliverdin site in the NTD, site 5α(red) refers to the Spike: ACE2 PPI interface, site 5β(yellow) corresponds to the site reported by
Dogancıet al. (2022) for violacein from a blind docking study on the RBD subunit, and site 5γ(purple) emerged by considering the entire RBD subun it for
docking calculations; to the best of our knowledge, this is the first report on this putative binding site.
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In general, it is currently accepted that carbon sources play a
crucial role in pigment synthesis; in particular, glycerol increases
while glucose represses its production (Pantanella et al., 2007;Duran
et al., 2021). These results indicate shared pathways for simple
carbon sources, as well as the presence of catabolic repression by
glucose. A systematic statistical study of violacein production by C.
violaceum CCT 3496 cultured in a 50 mL flask containing glucose-
rich media supplemented with tryptophan was conducted using a
fractional factorial design followed by a central composite design.
The violacein production increased from 7.5 g L
−1
dry cell mass and
0.17 g L
−1
crude violacein concentration to 21 g L
−1
and 0.43 g L
−1
,
respectively (Mendes et al., 2001). The ratios of dry cell mass/crude
violacein concentration were approximately 44 and 49 for initial and
optimized production, respectively. These results indicate that
coupled violacein production is related to bacterial cell mass.
The violacein producer Duganella sp. B2, isolated from a glacier
in China, was statistically optimized using a two-level Placket-
Burman, Box-Behnken, and surface response for biodye
production. The experiments were carried out in 250 mL stirred
flasks containing minimal saline medium and starch as a carbon
source supplemented with tryptophan. The highest violacein
concentration produced in the optimized medium was 1.62 g L-1
after 32 h of culture (Wang et al., 2009).
The production of violacein from a wild-type isolated
psychotropic bacterium RT102, close to J. lividum, cultivated in a
3-L fermenter containing rich media (i.e., glucose, casein, and yeast
extract) under physicochemical controlled parameters resulted in a
maximum violacein concentration and productivity of 3.7 g L
-1
and
0.12 g L
-1
h-1 at 40 h culture (Nakamura et al., 2003).
Several efforts have been made to obtain recombinant bacterial
strains that are capable of producing high concentrations of
violacein. Most studies have focused on E. coli (Rodrigues et al.,
2012;Rodrigues et al., 2013;Fang et al., 2015). The E. coli
TOP10 strain transformed with the plasmids pJP1000 and pPSX-
Vio + which are harboring the violacein operon pBvioABCDE from
C. violaceum showed low levels of violacein production ranging
from 0.006 to 0.025 g L
-1
, which was comparable to the biodye
production by J. lividum DSM 1522 under the same experimental
conditions (Rodrigues et al., 2012). In another study by the same
research group, different E. coli strains were transformed with
plasmids and the integrated gene responsible for violacein
production. The mutant E. coli dVio-1 and dVio-6 expressing the
vioABCDE gene cluster accumulate 0.18 g L-1 and 320 mg L-1 of
deoxyviolacein intracellularly, respectively. Integration of the vioD
gene into the E. coli genome, E. coli Vio-4, produced 0.71 g L
-1
of
intracellular violacein by fed-batch fermentation (Rodrigues et al.,
FIGURE 5
Binding site 5γin protomer A predicted for violacein by molecular docking and characterized by the DoGSiteScorer web server (A). Pose prediction
for violacein. Some relevant interactions are shown with black dashed lines (B). Violacein’s RMSD (Å) vs. simulation time plot (C).
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2013). In future work, different recombinant E. coli BL21 strains
were used as a chassis for the expression of violacein genes inserted
in a plasmid associated with the enhanced production of tryptophan.
Flask culture in glucose saline medium of the recombinant strain
E. coli B2/pED + pVio produces 0.60 g L
-1
violacein at a
concentration of 48 h. Scale-up of violacein production in a
fermenter with an optimized glucose medium showed a titer of
1.75 g L
−1
associated with 36 mg L−1h
−1
biodye productivity (Fang
et al., 2015).
In another microbial recombinant strategy, Citrobacter freundii
containing the violacein gene cluster in a plasmid (i.e., pCom10vio)
was cultured in a 2 L minimal salt medium supplemented with
glycerol and tryptophan in a 5-L fermenter using a fed-batch
technique. The optimized yield parameters were 3.34 g L-1 dry
cell mass, maximum violacein concentration of 4.13 g L
-1
with
violacein productivity of 0.083 mg L
−1
h
−1
(Yang et al., 2011).
However, some issues must be taken into account for potential
commercial applications of violacein in pharmaceutical applications
because the high production and/or productivity of violacein in
recombinant strains depends on the strain stability during scale-up
and successive microbial cultures, violacein purification complexity
(i.e., steps and procedures) because the biodye is located
intracellularly in most recombinant microorganisms, and the
presence of violacein precursors and/or cometabolites (i.e.,
protoviolaceinic acid, deoxyviolacein, etc.). Alternatively, the use
of agricultural food waste can be an alternative to reduce violacein-
producing costs as well as co-production with other metabolites of
industrial interest. Pioneering investigations reported in the
literature for violacein production were developed by Ahmad’s
research group at the University Technical of Malaysia. A wild-
type strain of C. violaceum isolated from a waste plant has been
extensively studied for the production of violacein in liquid media
supplemented with local agricultural wastes and/or cheap substrates,
such as brown sugar, molasses, solid pineapple waste, sugarcane
bagasse, and commercial-rich media. Violacein production in the
rich medium was very low or negligible. While the high biodye
productions were 0.19 g L
-1
and 0.82 g L
-1
obtained in a medium
containing 1% and 3% sugarcane bagasse supplemented with
tryptophan, a pigment precursor, respectively (Ahmad et al., 2012).
Violacein production in C. violaceum UTM5 was studied by
combining static and stirred growth conditions, and violacein
production was between 0.17 g L
-1
to 0.26 g L
-1
. However, high
production was observed when the microorganism was first
cultivated under static conditions followed by stirring. After
environmental optimized culture conditions, the microorganism
produced 16.26 ± 0.44 g L
-1
of violacein in a 50 L fermenter
containing 45 L of the medium of filtered pineapple waste
supplemented tryptophan at 30°C for 24 h (Aruldass et al., 2015).
Microparticulate wheat bran, a low-cost substrate with high
lignocellulose content, was used to supplement the Luria-Bertani
medium for violacein production using C. vaccinii in batch culture
(Cassarini et al., 2021). The optimal violacein production was
0.208 g L
-1
after 73 h of culture.
In a recent study, the simultaneous production of poly
hydroxybutyrate (PHB) and violacein was reported (Kumar et al.,
2021). The synthesis of PHB and violacein by wild-type Iodobacter
sp. PCH194 was optimized in a 22-L fermenter containing glucose
and tryptone in a saline medium. The maximum production of PHB
was 11.0 ± 1.0 g L
-1
, and a mixture of violacein (50%–60%) and
deoxyviolacein (40%–50%) was obtained. The highest concentration
of violacein was 1.5 g L
-1
at 48 h of cultivation.
The main data on violacein production using different
microorganisms, methodologies, and brief culture conditions are
displayed in Supplementary Table S4. Another factor to be considered
is related to the obtention of violacein from the culture media. Briefly, at
the late stationary phase, the bacterial cells were centrifuged, and the
violacein was extracted from the pellet with ethanol (Mendes et al., 2001).
Among the advantages of violacein production are the standard
conditions of production, short times for production, the facile
bacterial cultivation in fermenters, and scale up with traditional
strains, the low nutritional requirements of the microorganism for
the biodye production which could include wastes (e.g. with the
advantage of the circular economy), easy recovery of the pigment
and purification because of high hydrophobicity.
2.10 Development of violacein devices
Violacein attached to different materials has two main
applications: as a filter barrier with antiviral activity or loaded
into particulate systems for drug delivery (Khaksar et al., 2021;
Fu et al., 2023).
Because the human coronavirus is mainly transmitted by aerosols
caused by coughing, sneezing, breathing, or speaking, there is a need to
establish a physical barrier for the dispersion of these aerosols. However,
the infectivity over time of the coronavirus in a droplet of aerosol
depends greatly on the surface where it is deposited, being able to
remain active for several hours in some cases (Fu et al., 2023). This is
why a functional material must not only retain droplets but also
inactivate the virus.
Strategies have been developed to use violacein as a biodye in the
development of functional fabrics, taking advantage of its antiviral
properties. Three different methods for dyeing polyamide fabrics
with violacein have been tested (Kanelli et al., 2018). Direct
fermentation of J. lividum on the fabric, exposure of the fabric to
the culture after fermentation, and exposure of the fabric to an
acellular extract of the culture. Improved color retention and
antimicrobial and biofilm inhibition activities were obtained
using the first method (Kanelli et al., 2018). With the same goal
of producing functional fabrics, Gao et al. (2019) developed a silk-
based composite that takes advantage of the antimicrobial synergy
between violacein and silver nanoparticles (Gao et al., 2019).
Electrospinning techniques have also been tested to design fabrics
based on violacein-containing synthetic polymers, such as Nylon-66
and Polyvinyl-alcohol/polyvinyl-pyrrolidone (Osman and Setu,
2018;Rosli and Setu, 2018). Also, by using electrospinning, Lee
et al. (2022) have recently taken this concept further; developing
prototype respiratory masks from polyacrylonitrile nanofibers with
violacein, demonstrating aerosol retention from 0.8 to 3.4 µm
(human-produced aerosols being between 0.74 and 2.12 µm),
antimicrobial activity against S. aureus and antiviral activity
against human influenza A and human coronavirus. Violacein
also provides selective UV protection to the fabric (Lee et al.,
2022). In particular, these violacein-loaded materials retain the
inactivating activity of coronaviruses and influenza and could
constitute products with real applications, such as surgical masks.
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In another study, viscose fabric was incubated in the presence of
violacein, followed by the incorporation of silver and titanium
dioxide nanoparticles by sonication. The antimicrobial activity of
the fabric containing violacein and nanoparticles showed a 3- to 6-
log reduction in the growth of S. aureus,B. cereus, and E. coli,
indicating the effectiveness of the fabric derivatized with violacein
and nanoparticles (Khaksar et al., 2021).
Another potential application of violacein is to include biodye in 3D
matrices for molecular delivery.In2000,violaceinwasaddedtoaß-CD
solution and precipitated. The violacein complexed in cyclodextrin
showed the same biocide activity against E. coli, but the cytotoxicity in
lung fibroblasts of Chinese hamster V-79 cell cultures was reduced, and
lipid peroxidation was fully inhibited at 500 µM of the violacein-ß-
cyclodextrin complex (). Later, a proof of concept was developed by the
encapsulation of violacein in nanoparticles of poly-(D, L-lactic-co-
glycolic acid) for assaying antimicrobial activity. PLGA nanoparticles
with 116 and 139 nm diameter containing violacein efficiently inhibit
five strains of S. aureus, including antibiotic-resistant (MRSA) strains, at
micromolar concentrations three to five times lower than free violacein
(Martins et al., 2011). In a subsequent study by Durán et al.,violacein
was encapsulated in nanoparticles made of poly (ε-caprolactone)
covered with chitosan to produce a positively charged device and
increase mucoadhesiveness (Berni et al., 2013). Violacein
nanoparticles of 201–320 nm were tested to prevent bovine mastitis
against S. aureus MRSA with a low minimal inhibitory concentration
compared to free biodye. Additionally, violacein encapsulation
diminished ecotoxicity by approximately 10 to 5 times using
Daphnia similis according to the OCDE guidelines.
Another strategy for violacein encapsulation is the use of Arabic
gum as a matrix (Venil et al., 2015). Violacein microparticles can be
used to provide color to foods, such as jellies and yogurts. Violacein
encapsulated in Arabic gum was spray-dried and displayed high
stability in the range of 25°C–60°C for 30 days.
Similarly, based on the low violacein solubility in aqueous media,
thebiodyewasfirst suspended in a polyoxyethylene sorbitan
monolaurate solution and showed high stability at room
temperature for 6 days. Later, the violacein emulsion was
successfully encapsulated in gelatin-pectin coacervate and tested
against HCT-116 colon cancer (Rivero Berti et al., 2019). More
recently, violacein was encapsulated in a nanostructured lipid carrier
with an active release by lipase and 3D printed using a hydroxypropyl
cellulose-chitosan matrix. The formulation was tested against the
A549 and HCT-116 cancer cell lines (Rivero Berti et al., 2022).
Other strategies have been developed to increase the biocidal
activity of violacein by using metallic nanoparticles. Silver
nanoparticles with violacein adsorbed in the surface displayed
high antibacterial activities against multiresistant bacterial strains
of P. aeruginosa, E. coli, and S. aureus, also tested against Aspergillus
tamari, Aspergillus tubingensis, and Fusarium proliferatum, and also
algicidal activities against Dictyosphaerium sp. strains DHM1 and
DHM 2, and Pectinodesmus sp. strain PHM 3. In all cases, the
biocide activity of AgNPs capped with violacein against bacteria,
fungi, and algae were enhanced by the presence of the biodye (Arif
et al., 2017). Similar results were obtained for silk coated with AgNPs
and violacein, as previously described (Gao et al., 2019).
A novel strategy for repositioning old antibiotics is to develop a
combination of antibiotics and violacein. The authors developed a 1:
1 individual mixture of 20 antibiotics with violacein and tested these
mixtures against Salmonella typhi,Vibrio cholerae,P. aeruginosa,K.
pneumoniae, and S. aureus. In most cases, the biocidal activity of
violacein is synergistic and/or additive concerning the antibiotic
(Subramaniam et al., 2014). This strategy offers several advantages.
First, the antibiotic-violacein mixtures can increase the biocide
activity against pathogens because of different molecular targets,
decreasing the amount of the antibiotic concomitantly with a
decrease in potential toxicities, and increasing the bioavailability
of the components.
Additionally, the interaction of ten imidazole ionic liquids with
violacein was analyzed. Imidazole ionic liquids and violacein form
stable micellar solutions that are encapsulated in solid lipid
nanoparticles. The anticancer activity of the violacein-imidazole
ionic liquids formulation against A549, HeLa, and HCT116 cells was
tested. These results indicated that the imidazole group of the ionic
liquids associated with violacein displayed strong anticancer activity
(Rivero Berti et al., 2020). Further computational studies have
demonstrated the effectiveness of imidazole derivatives against
SARS-CoV-2 (Belhassan et al., 2020). Data from both studies
showed promising results for the use of violacein–imidazole ionic
liquid formulations for SARS-CoVid-2 therapy.
Taking into consideration what is stated in this section, not only
should the biological activity of violacein be considered, but also its
ability to form functional materials. The late one is a relevant
challenge that will result in a improve biocide and antiviral activities.
3 Conclusion
Many biological activities of violacein were extensively described in
theliterature.Thepreviousfocuswastouseviolaceininthetreatmentof
different types of cancer. However, recently found violacein properties
suchasitseffectoncellularmembranes,andanti-inflammatory,
antioxidant, and antiviral activities suggest that another application
is possible. Other detailed studies of violacein activities revealed a wide
spectrum of protease inhibition. Additionally, in silico studies through
molecular docking confirmed the binding activities of violacein within
the SARS-CoV-2 spike in several sites. However, in some cases, the
antiviral titers of violacein are not enough to be considered as a potential
virucide agent. These results could be attributed to two factors, first the
presence of other molecules in the violacein extracts because of a lack of
appropriate purification steps and quality controls, and second because
of violacein low biodisponibility due to its very low solubility in
physiological media. Besides, the production of violacein from
different strains and procedures is in the state of the art,butitis
essential to standardize pigment purification procedures. Also, the
potential oral administration of violacein will require the
development of novel strategies for molecular delivery systems to
obtain more efficient biocide activities.
Author contributions
IR, writing, drawing, reviewing. MG, docking and molecular
studies, writing. SR, docking and molecular studies, writing. GI,
editing, reviewing. WF, editing, reviewing. AT, critical review, and
text edition, discussion, and manuscript revision. ND, writing, text
edition, and discussion. GC, organization, writing, editing, critical
Frontiers in Nanotechnology frontiersin.org11
Rivero Berti et al. 10.3389/fnano.2023.1186386
review, discussion, and manuscript revision. All authors contributed
to the article and approved the submitted version.
Funding
Financial support by UNLP (X815), CONICET (PIP 0034), and
ANPCyT (PICT 2016-4597; PICT 2017-0359) is gratefully acknowledged.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fnano.2023.1186386/
full#supplementary-material
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