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\begin{document}

\title{A Novel Water-Soluble Photosensitizer for Photodynamic Inactivation of
Gram-Positive Bacteria}



\author[1]{Zihuayuan Yang}%
\author[1]{Ying Qiao}%
\author[1]{Junying Li}%
\author[1]{Fu-Gen Wu}%
\author[1]{Fengming Lin}%
\affil[1]{Southeast University}%


\vspace{-1em}



  
  \date{June 13, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Antimicrobial photodynamic therapy (APDT) is a promising alternative to
traditional antibiotics for bacterial infections, which inactivates a
broad spectrum of bacteria. However, it has some disadvantages including
poor water solubility and easy aggregation of hydrophobic
photosensitizers (PS), and poor tissue penetration and cytotoxicity when
using UV as light source, leading to photodynamic therapy efficacy.
Herein, we develop a novel water-soluble natural PS (sorbicillinoids)
obtained by microbial fermentation using filamentous fungus Trichoderma
reesei (T. reesei). Sorbicillinoids could effectively generate singlet
oxygen (1O2) under UV light irradiation, and ultimately display
photoinactivation activity on Gram-positive bacteria, but not
Gram-negative ones. Staphylococcus aureus (S. aureus) treated with
sorbicillinoids and UV light displayed high levels of intracellular
reactive oxygen species (ROS), notable DNA photocleavage, and
compromised membrane permeability without overt cell membrane
disruption. Moreover, the dark toxicity, phototoxicity or hemolysis
activity of sorbicillinoids is negligible, showing its excellent
biocompatibility.%
\end{abstract}%



\sloppy


\textbf{1. INTRODUCTION}

Diseases caused by bacterial infections, such as pneumonia and sepsis,
threaten the lives of millions of people every year (Jones et al., 2008;
Theuretzbacher, 2013). The most widely used strategy for coping with
bacterial infections at present is antibiotics (Fischbach and Walsh,
2009). However, the abuse of antibiotics in recent decades has led to an
increase in the resistance of bacteria (Taubes, 2018), which causes the
emergence of multi-drug resistant (MDR) bacteria (Levy and Marshall,
2004; Tenover, 2006). Thus, it is urgent to develop new antibiotics and
efficient antibacterial therapies to fight MDR bacteria. Many novel
antibiotics have been explored to successfully combat MDR bacterial
infection, including carbon dots nanoparticles (NP) (Yang et al., 2016;
Ran et al., 2019) and cationic antibacterial peptides (Liu et al., 2009;
Lam et al., 2016). In addition, new efficient antibacterial therapies
have been also applied in treating bacterial infections, such as
photodynamic therapy (PDT) (Hamblin and Hasan, 2004; Liu et al., 2015)
and photothermal therapy (PTT) (Zhao et al., 2018; Wu et al., 2019).
Antimicrobial photodynamic therapy (APDT) has been widely reported as a
promising alternative to traditional antibiotics in the past few years
(Jia et al., 2017; Ravikumar et al., 2018). APDT generally involves
molecular oxygen, light source, and photosensitizer (PS). With light
irradiation of a suitable wavelength, PS converts molecular oxygen into
reactive oxygen species (ROS) (Liu et al., 2015), primarily singlet
oxygen (\textsuperscript{1}O\textsubscript{2}). The highly active ROS
can cause damage to important biomolecules such as lipids (Zhang et al.,
2019), proteins (Davies, 2003), and DNA (Alves et al., 2013), ultimately
leading to bacterial cell death. Unlike conventional antibiotics, APDT
only irradiates the lesion location and does not cause damage to other
parts (Hamblin and Hasan, 2004), thereby achieving selective
bacterium-killing. More importantly, APDT can inactivate a broad
spectrum of bacteria, opening new avenues for the development of new
antibacterial therapies (Liu et al., 2015).

Most currently available PSs for APDT have poor solubility in water,
showing a strong tendency to aggregate, ultimately leading to poor
photodynamic therapy efficacy (Almeida-Marrero et al., 2018). To improve
their hydrophilicity, a variety of modification methods were applied by
physical encapsulation or chemical covalent conjugation of PSs to
nanocarriers such as peptide nanoparticles (Han et al., 2015), polymer
nanoparticles (Parthasarathy et al., 2015), and liposomes (Malcher et
al., 2008). However, these surface modification methods are
time-consuming and usually bring some unexpected consequences such as
low biocompatibility, which may compromise the clinical application of
APDT. Another issue that plagues APDT was the need of UV light for some
PSs like metal oxide NPs zinc oxide (ZnO) NPs and titanium dioxide
(TiO\textsubscript{2}) NPs (Huh and Kwon, 2011), for the reason that UV
light is cytotoxic to human cells and has poor tissue penetration
(Wegener eet al., 2017). Therefore, it is urgent to find new PSs for the
APDT system to overcome these issues. Natural products from fungus and
plants have been reported as a promising alternative to conventional PSs
such as hypericin (Garc\selectlanguage{ngerman}ía et al., 2015), curcumin (Tonon et al., 2015),
hypocrellin (Zhenjun and Lown, 1990) and cationic riboflavin (Maisch et
al., 2014). The compounds are usually derived from secondary metabolism.
Natural products as PSs have the advantages of good biocompatibility,
wide source, and high \textsuperscript{1}O\textsubscript{2} yield
(Abrahamse and Hamblin, 2016).

Sorbicillinoids are hexaketide metabolites isolated from both marine and
terrestrial ascomycetes, such as \emph{Trichoderma} , \emph{Acremonium}
,\emph{Aspergillus} , \emph{Emericella} , \emph{Penicillium}
,\emph{Phaeoacremonium} , and \emph{Scytalidium} (Meng et al., 2016).
Most of these compounds have characteristic structures, including
bicyclic or tricyclic structures and C1'--C6' sorbyl sidechain.
Sorbicillinoids were divided into four classes based on their structure
(Harned and Volp, 2011): monomeric sorbicillinoids, bisorbicillinoids,
trisorbicillinoids, and hybrid sorbicillinoids. In recent years, various
potential applications of sorbicillinoids have been exploited, such as
antioxidants (Abe and Hirota, 2002; Kawahara et al., 2012), antibiotics
(Guo et al., 2015; Meng et al., 2018), and anticancer drugs (Lai et al.,
2019; Meng et al., 2019). However, research on sorbicillinoids as a PS
for the APDT system has not been reported.

In this study, we discovered a new type of natural material as a
water-soluble PS for APDT (Scheme 1). Sorbicillinoids produced
by\emph{Trichoderma} \emph{reesei} converts oxygen molecules
into\textsuperscript{1}O\textsubscript{2} effectively under mild
ultraviolet (UV) irradiation. As a result, sorbicillinoids exhibited
great APDT effect toward Gram-positive bacteria. The underlying
mechanism of the light-activated antibacterial activity of
sorbicillinoids was explored by monitoring intracellular ROS, DNA
photocleavage and cell membrane damage. Furthermore, the
biocompatibility of sorbicillinoids-mediated APDT was evaluated using
MTT assay and hemolysis assay.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Scheme 1} . Schematic Illustration of Photodynamic Inactivation
against S. aureus by Sorbicillinoids under UV Light Irradiation.

\textbf{2. MATERIALS AND METHODS}

\textbf{2.1 Materials}

Luria Bertani (LB) broth, LB agar and potato dextrose agar (PDA) were
purchased from Beijing Land Bridge. Singlet oxygen sensor green (SOSG)
kit was acquired from Molecular Probes Inc. (Eugene, Oregon, USA).
Propidium iodide (PI) was obtained from KeyGen Biotech (Nanjing, China).
Glutaraldehyde, 3-(4,5- dimethyl-2-thiazolyl)-2,5-diphenyl-2-\emph{H}
-tetrazolium bromide (MTT), methanol, ethanol, dimethyl sulfoxide
(DMSO), and triton X-100 were purchased from Aladdin Chemistry Co., Ltd.
(Shanghai, China). Reactive oxygen species assay kit was bought from
Beyotime Biotech Inc. (Shanghai, China). All other chemicals used in
this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). All
solutions were prepared with deionized water (18.2 M\selectlanguage{greek}Ω \selectlanguage{english}cm) purified by a
Milli-Q water purification system (Milli-Q, Millipore, USA).

\textbf{2.2 Sorbicillinoids production}

The conidia produced by \emph{Trichoderma reesei} strain ZC121 grown on
PDA plates for 7 days at 28 \selectlanguage{ngerman}°C, were inoculated into 10 mL sabouraud
dextrose broth (SDB) and incubated for 48 h with 200 rpm at 28 °C.
Pre-grown mycelia were inoculated with an inoculation ratio of 10\%
(v/v) into 50 mL \emph{Trichoderma} minimal media (Minty et al., 2013)
(TMM) with 2\% glucose, and then incubated for 120 h with 200 rpm at 28
°C. The suspension was centrifuged at 14000 rpm for 15 min at 4 °C to
remove \emph{Trichoderma reesei} cells and other solid materials, and
the supernatant was dried at 120 °C in an oven. The obtained powder was
dissolved in methanol to remove inorganic salt. Methanol was evaporated
at 60 °C under a nitrogen atmosphere to obtain sorbicillinoids. TMM
medium was composed of the following chemicals (all concentration unit
is g/L unless otherwise noted):
(NH\textsubscript{4})\textsubscript{2}SO\textsubscript{4}, 4.0;
KH\textsubscript{2}PO\textsubscript{4}, 6.5; Tween-80, 0.0186\% (v/v);
Yeast extract, 0.75; Tryptone, 0.25; Maleic acid, 11.6;
FeSO\textsubscript{4}·7H\textsubscript{2}O, 0.005;
MnSO\textsubscript{4}·H\textsubscript{2}O, 0.0016;
ZnSO\textsubscript{4}·7H\textsubscript{2}O, 0.0014;
CoCl\textsubscript{2}·6H\textsubscript{2}O, 0.002;
MgSO\textsubscript{4}, 0.60; CaCl\textsubscript{2}, 0.60; urea, 1.0.
Then the pH of TMM was adjusted to 5.8 by NaOH.

\textbf{2.3 Singlet oxygen generation assay}

The generation of \textsuperscript{1}O\textsubscript{2} was measured
with singlet oxygen sensor green (SOSG). 1 \selectlanguage{greek}µ\selectlanguage{english}L SOSG dissolved in methanol
was mixed with 2 mL 0.9\% NaCl and 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL sorbicillinoids in 0.9\%
NaCl, followed by UV light (2 mW/cm\textsuperscript{2}) or white light
irradiation (8 mW/cm\textsuperscript{2}). 0.9\% NaCl without any light
irradiation was set as control. Then, the fluorescence intensity of the
samples at 528 nm was measured by a spectrofluorophotometer (RF-5301PC,
Shimadzu, Japan).

\textbf{2.4 Antibacterial activity assay}

\emph{Escherichia} \emph{coli} (\emph{E} . \emph{coli} ),
and\emph{Staphylococcus} \emph{aureus} (\emph{S} . \emph{aureus} ) were
selected to present Gram-negative and Gram-positive bacteria,
respectively. \emph{E} . \emph{coli} or \emph{S} . \emph{aureus} were
cultivated overnight in LB medium at 37 \selectlanguage{ngerman}°C with 200 rpm. Bacteria were
obtained from bacteria suspension overnight by centrifugation at 8000
rpm for 5 min and then resuspended in 0.9\% NaCl. The optical density of
the resuspension at 600 nm (OD\textsubscript{600}) was measured using a
UV-vis spectrophotometer (UV-2600, Shimadzu, Japan). Then, the
resuspension was diluted to OD\textsubscript{600} = 0.5 with 0.9\% NaCl.
After the dilution, 100 \selectlanguage{greek}µ\selectlanguage{english}L bacteria suspension was mixed with 900 \selectlanguage{greek}µ\selectlanguage{english}L
sorbicillinoids dissolved in 0.9\% NaCl at different concentrations. The
final concentrations of sorbicillinoids were 0, 50, 100, 200, 250, and
300 \selectlanguage{greek}µ\selectlanguage{english}g/mL. for \emph{S} .\emph{aureus} (0, 200, 400, 600, 800, and 1000
\selectlanguage{greek}µ\selectlanguage{english}g/mL. for \emph{E} .\emph{coli} ). After irradiation under UV light (2
mW/cm\textsuperscript{2}) for 30 min, 100 \selectlanguage{greek}µ\selectlanguage{english}L solution was plated on LB
agar plates. The plates were placed in an incubator at 37 \selectlanguage{ngerman}°C for 24 h,
followed by colony counting. Antibacterial activity of sorbicillinoids
against Gram-negative bacterium \emph{Proteus vulgaris} (\emph{P}
.\emph{vulgaris} ), and Gram-positive bacteria \emph{Bacillussubtilis}
(\emph{B} . \emph{subtilis} ) and \emph{Micrococcus luteus} (\emph{M} .
\emph{luteus} ), were also tested in the same way.

\textbf{2.5 Intracellular ROS generation assay}

2,7-dichlorofluorescein diacetate (DCFH-DA) was applied to measure the
generation of intracellular ROS. 0.5 \selectlanguage{greek}µ\selectlanguage{english}L DCFH-DA was added into 500 \selectlanguage{greek}µ\selectlanguage{english}L
bacteria suspension (OD\textsubscript{600} =0.05), followed by
incubation at 37  for 10 min. After centrifugation at 8000 rpm for 5
min, bacteria were resuspended in 500 \selectlanguage{greek}µ\selectlanguage{english}L water, supplemented with 5 \selectlanguage{greek}µ\selectlanguage{english}L
sorbicillinoids (10 mg/mL for \emph{S} . \emph{aureus} and 40 mg/mL
for\emph{E} . \emph{coli} ) dissolved in water. After incubation at 37 
for 30 min, the suspension was irradiated under UV light (2
mW/cm\textsuperscript{2}) for 30 min. The fluorescence intensity was
measured by a flow cytometer (NovoCyte 2060, ACEA, USA). Channel used
for analyses was FITC with the excitation at 488 nm.

\textbf{2.6 Agarose gel electrophoresis of bacterial genomic DNA}

The genomic DNA of bacteria was extracted with the Bacterial DNA kit
which was bought from TIAGEN Biotech Co., Ltd. (Beijing, China). The
bacteria cell (1 \selectlanguage{ngerman}× 10\textsuperscript{6} CFU/mL) with different
treatments as indicated in the antibacterial activity assay were
collected by centrifugation at 8000 rpm for 5 min and then washed twice
with 0.9\% NaCl. The genomic DNA was extracted according to
manufacturer's instruction. Briefly, the bacterial cells were separately
resuspended in 200 \selectlanguage{greek}µ\selectlanguage{english}L GA buffer, 20 \selectlanguage{greek}µ\selectlanguage{english}L proteinase K solution and 220 \selectlanguage{greek}µ\selectlanguage{english}L
GB buffer. After incubation at 70  for 10 min, 220 \selectlanguage{greek}µ\selectlanguage{english}L ethanol was added
into each sample. Then the solution was transferred to a spin column CB3
and washed with GD buffer and PW washing solution. After that, the
genomic DNA was bound onto the spin column and then eluted with TE
buffer. The extracted DNA was analyzed by electrophoresis in Tris
Acetate-EDTA (TAE) buffer containing 0.8\% agarose (w/v) for 50 min at
100 V. The electrophoretic profiles were acquired with a Gel imaging
system (Tanon 3500R, Shanghai, China).

\textbf{2.7 PI staining}

The bacterial cells (1 \selectlanguage{ngerman}× 10\textsuperscript{6} CFU/mL) with different
treatments as indicated in the antibacterial activity assay were
incubated at 37 \selectlanguage{english} for 2 h and then collected by centrifugation at 8000
rpm for 5 min. After the centrifugation, the bacteria were stained with
red-fluorescent nucleic acid stain (PI) for 30 min. The bacteria samples
were imaged using a confocal microscope (TCS SP8, Leica, Germany).

\textbf{2.8 Morphological characterization of bacteria}

Morphological characterization of bacteria was carried out with a
scanning electron microscope (SEM, ULTRA Plus, Zeiss, Germany). The
bacterial cells (1 x 10\textsuperscript{6} CFU/mL) with different
treatments as indicated in the antibacterial activity assay were
collected by centrifugation at 8000 rpm for 5 min and then resuspended
in 0.9\% NaCl. After washing with 0.9\% NaCl three times, the bacteria
cells were mixed with 2.5\% (v/v) glutaraldehyde for 12 h to fix the
cell morphology, followed by dehydration using graded ethanol (30\%,
50\%, 70\%, 90\%, and 95\%). Then the bacterial cells were resuspended
in 100\% ethanol and dripped in a silicon slide for SEM imaging.

\textbf{2.9 Cytotoxicity evaluation assay}

The toxicity of sorbicillinoids towards AT-II (normal human lung cell)
by MTT assay. AT-II cells were cultivated in Dulbecco's modified eagle
medium (DMEM), containing 10\% fetal bovine serum, 100 U/mL of
penicillin, and 100 \selectlanguage{greek}µ\selectlanguage{english}g/mL streptomycin in an incubator with 5\%
CO\textsubscript{2} at 37 \selectlanguage{ngerman}°C. The cells were seeded in a 96-well plate
at a density of 5000 cells per well. After 24 h of culture, the cells
were mixed with sorbicillinoids dissolved in DMEM at the final
concentrations of 0 50, 100, 150 200, 250, 300, 350, 400 and 450 \selectlanguage{greek}µ\selectlanguage{english}g/mL,
followed by 24 h of culture. Then, 10 \selectlanguage{greek}µ\selectlanguage{english}L of MTT (5 mg/mL) was added into
each well. After incubation for 4 h, the solution was removed and 150 \selectlanguage{greek}µ\selectlanguage{english}L
DMSO was added into each cell. Then, the absorbance at 492 nm was
measured with a microplate photometer (Multiskan FC, Thermo Fisher
Scientific, USA).

\textbf{2.10 Hemolysis assay}

Fresh blood was collected from a healthy male mouse and restored in an
anticoagulation tube to avoid the blood coagulation. Red blood cells
(RBCs) were obtained from the blood by centrifugation at 2000 rpm for 5
min and resuspended in 0.9\% NaCl. The obtained RBCs were treated with
different concentrations of sorbicillinoids (50, 100, 200, 250, and 300
\selectlanguage{greek}µ\selectlanguage{english}g/mL) for 2 h at 37 \selectlanguage{ngerman}°C, and centrifuged at 2000 rpm for 5 min. Then the
resultant supernatants were transferred to a 96-well plate for the
absorbance measurement at 492 nm with a microplate photometer (Multiskan
FC, Thermo Fisher Scientific, USA). RBCs treated with phosphate-buffered
saline (PBS) and triton X-100 were set as the negative control and
positive control, respectively. The hemolysis percentage was calculated
by the following formula:

Hemolysis\%= {[}(absorbance of samples -- absorbance of negative
control) / (absorbance of positive control -- absorbance of negative
control){]} × 100\%.

\textbf{3.RESULTS AND DISCUSSION}

\textbf{3.1 Sorbicillinoids generated a high level
of\textsuperscript{1}O\textsubscript{2} under UV light irradiation}

Sorbicillinoids was produced from the fermentation of the
recombinant\emph{Trichoderma reesei} (\emph{T.} \emph{reesei} ) strain
ZC121 with an excellent sorbicillinoids production ability. The obtained
sorbicillinoids mainly included sorbicillinol, bisvertinolone and
oxosorbicillinol as identified by LC-MS (Li et al., 2018). Given that
sorbicillinoids has great absorbance at 370 nm (Derntl et al., 2017),
there is a possibility that sorbicillinoids can
generate\textsuperscript{1}O\textsubscript{2} under light irradiation.
To prove this, the \textsuperscript{1}O\textsubscript{2} generation
ability of sorbicillinoids under the irradiation of both UV and white
light was measured using a SOSG kit (Figure 1). Sorbicillinoids produced
significant \textsuperscript{1}O\textsubscript{2} when irradiated with
UV light. The produced \textsuperscript{1}O\textsubscript{2} was
increased noticeably with the irradiation time increasing, being 300\%
that of UV alone at 30 min. In contrast, when sorbicillinoids was
exposed to white light for 30 min,
insignificant\textsuperscript{1}O\textsubscript{2} production was
observed. No\textsuperscript{1}O\textsubscript{2} generation was
observed for sorbicillinoids alone. This result confirmed our hypothesis
that sorbicillinoids can synthesize
\textsuperscript{1}O\textsubscript{2} in the presence of UV light, but
not visible light. It is worth noting
that\textsuperscript{1}O\textsubscript{2} is highly toxic to the
microorganisms, serving as one major factor for APDT's antibacterial
effect (Jia et al., 2017; Mao et al., 2017). Therefore, sorbicillinoids
from \emph{T.} \emph{reesei} might be explored as a new PS for APDT to
kill microorganisms.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 1} . \textsuperscript{1}O\textsubscript{2} generation of
sorbicillinoids in 0.9\% NaCl as measured by the fluorescence intensity
changes of SOSG at 528 nm with the excitation wavelength of 504 nm.

\textbf{3.2 Sorbicillinoids showed photoinactivation against
Gram-positive bacteria under nontoxic dose of UV irradiation}

Inspired by their excellent
\textsuperscript{1}O\textsubscript{2}production ability under UV
irradiation, we investigated the APDT ability of sorbicillinoids.
\emph{S} . \emph{aureus} and \emph{E} .\emph{coli} representing
Gram-positive and Gram-negative bacteria respectively, were incubated
with different concentrations of sorbicillinoids in 1.5 mL sterilized
centrifuge tubes, followed by UV light irradiation for 30 min (Figure
2). When irradiated under UV light,\emph{S} . \emph{aureus} was killed
by 60.3\%, 68.5\%, 90.5\%, 98.1\%, and 99.5\% in the presence of 50,
100, 200, 250, and 300 \selectlanguage{greek}µ\selectlanguage{english}g/mL sorbicillinoids, respectively (Figure 2a
and 2b). Conversely, no antibacterial effect of sorbicillinoids was
observed on \emph{E. coli} , even when the administrated concentration
was increased up to 1000 \selectlanguage{greek}µ\selectlanguage{english}g/mL (Figure 2c and 2d). Sorbicillinoids or UV
alone was non-toxic to both\emph{S} . \emph{aureus} and \emph{E. coli}
(Figure 2). The undetected antibacterial effect of UV light in this
study is probably ascribed to the poor penetration ability of UV light
through the plastic centrifuge tube (Ran et al., 2012). It appears that
while the plastic is mitigating the intensity of incident UV light to
the point where there is no cell death in the negative control, there is
still enough incident UV light to sensitize sorbicillinoids to generate
drastic \textsuperscript{1}O\textsubscript{2}, leading to the killing of
\emph{S} . \emph{aureus} .

Moreover, the UV-mediated APDT ability of sorbicillinoids was tested on
another two Gram-positive bacteria \emph{B} . \emph{subtilis}
and\emph{M} . \emph{luteus} , and another Gram-negative bacterium
\emph{P} .\emph{vulgaris} . In the presence of 25, 50, 75, 100, 200 and
300 \selectlanguage{greek}µ\selectlanguage{english}g/mL sorbicillinoids and under UV light irradiation, \emph{B}
.\emph{subtilis} was killed by 36.7\%, 40.4\%, 61.4\%, 89.3\% and 98.5\%
and 98.9\%, respectively, whereas \emph{M} . \emph{luteus} was killed by
29.5\%, 80.8\%, 98.0\%, 99.7\% and 99.9\%, respectively in the presence
of 50, 100, 200, 250 and 300 \selectlanguage{greek}µ\selectlanguage{english}g/mL sorbicillinoids (Figure S1). By
contrast, no antibacterial ability was observed against \emph{P}
.\emph{vulgaris} (Figure S2) as it is in the case of \emph{E}
.\emph{coli} . Obviously, sorbicillinoids with the irradiation of
nontoxic UV light can successfully kill Gram-positive bacteria, but not
Gram-negative ones. This Gram-selective antibacterial ability of
sorbicillinoids may be attributed to the different cell wall structures
of Gram-positive bacteria and Gram-negative bacteria. It is well-known
that compared to Gram-positive bacteria, Gram-negative bacteria have an
outer membrane that lies outside of the plasma membrane, serving as a
penetration barrier and protecting Gram-negative bacteria from
antibiotics and PSs (Lam et al., 2016; Lien et al., 1968). This outer
membrane may prevent sorbicillinoids from entering Gram-negative cells
to realize APDT. The localization of PSs largely determines the
efficiency of PDT, as the half-life
of\textsuperscript{1}O\textsubscript{2} is very short (\textless{} 0.04
\selectlanguage{greek}µ\selectlanguage{english}s) and its action radius is only shorter than 0.02 \selectlanguage{greek}µ\selectlanguage{english}m in the biological
system (Moan and Berg, 1991). Therefore, despite it could produce
abundant \textsuperscript{1}O\textsubscript{2} in the solution under UV
light irradiation, the extracellular sorbicillinoids exhibited no
bactericidal activity against Gram-negative bacteria. For Gram-positive
bacteria without the outer membrane, the water-soluble sorbicillinoids
with small molecular weight might readily diffuse through cell surface
into the cytosol to exert APDT.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 2} . The APDT ability of sorbicillinoids against \emph{S}
.\emph{aureus} (a, b) and \emph{E} . \emph{coli} (c, d). Agar plate
photographs of \emph{S} . \emph{aureus} (a) and \emph{E} . \emph{coli}
(c) treated with sorbicillinoids under (no) UV light irradiation
conditions. The corresponding dependence of bacterial survival fraction
on the concentration of sorbicillinoids was measured (b, d).

\textbf{3.3 Abnormally high level of intracellular ROS was found in S.
aureus treated with sorbicillinoids and UV irradiation, leading to DNA
damage}

To evidence our speculation that sorbicillinoids enters Gram-positive
bacteria to realize APDT, the generation of intracellular ROS was tested
employing 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe.
DCFH-DA freely passes through the cell membrane into cells where it is
hydrolyzed by intracellular esterase to form
2',7'-dichlorodihydrofluorescein (DCFH) that cannot penetrate the cell
membrane and stay inside cells. DCFH is subsequently oxidized by the
intracellular ROS into 2',7'-dichlorofluorescein (DCF) that has
fluorescence. Neither DCFH-DA nor DCFH has fluorescence. Therefore, the
level of ROS in the cells can be measured by detecting the fluorescence
of DCF. After treated with sorbicillinoids and irradiated under UV
light, the fluorescence intensity of \emph{S} . \emph{aureus} increased
significantly by 5 times as compared to the untreated \emph{S}
.\emph{aureus} , whereas no increasing fluorescence intensity was
observed for \emph{E} . \emph{coli} (Figure 3a). This suggested that the
treatment of sorbicillinoids with the irradiation of UV induced \emph{S}
.\emph{aureus} to generate large sums of intracellular ROS, which might
further damage biomacromolecules like proteins, DNA and lipids. This
result matched well with the selective photoinactivation of
sorbicillinoids against Gram-positive bacteria as we observed above.

Inspired by its ultra-high intracellular ROS level, DNA photocleavage
of\emph{S} . \emph{aureus} with the treatment of sorbicillinoids and UV
irradiation was detected by agarose gel electrophoresis (Figure 3b).
There was a marked reduction of DNA for \emph{S} . \emph{aureus} treated
with 100 \selectlanguage{greek}µ\selectlanguage{english}g/mL sorbicillinoids under UV light irradiation, indicating
nucleic acid photocleavage took place in the treated bacterial
cells.\emph{S} . \emph{aureus} treated with sorbicillinoids or UV light
alone did not display a reduction of DNA. On the contrary,
sorbicillinoids did not result in observable DNA reduction in \emph{E} .
\emph{coli} with or without UV light. Obviously, the unusually high
intracellular ROS induced by sorbicillinoids upon UV irradiation caused
cellular DNA damage in \emph{S. aureus} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3} . a) Generation of intracellular ROS in \emph{S}
.\emph{aureus} and \emph{E} . \emph{coli} . b) Electrophoretic profiles
of DNA extracted from \emph{S} . \emph{aureus} and \emph{E} .
\emph{coli} . 1: no treatment; 2: UV light; 3: sorbicillinoids; 4:
sorbicillinoids and UV light; M: molecular weight marker.

\textbf{3.4 Sorbicillinoids with UV irradiation compromised cell
semi-permeability of S. aureus without overt cell membrane disruption}

Both DNA and cytoplasmic membrane are two crucial cellular components
targeted by ROS in APDT (Cieplik et al., 2018). Therefore, the effect of
sorbicillinoids on the cell membrane of \emph{S} . \emph{aureus}
and\emph{E} . \emph{coli} was investigated using both propidium iodide
(PI) staining and SEM imaging (Figure 4 and Figure S3). PI cannot
penetrate into viable bacteria cells with complete membrane. Yet it can
enter bacterial cells with damaged membrane to intercalate between the
base pairs of the accessible double-stranded DNA and emit red
fluorescence with the excitation at 580 nm. \emph{S} . \emph{aureus}
were dyed red by PI after the treatment of sorbicillinoids and UV
irradiation (Figure 4a), demonstrating their membrane permeability was
compromised. Nevertheless, no red fluorescence was observed in the
irradiated\emph{E} . \emph{coli} cells in the presence of
sorbicillinoids, showing their cell membrane was not affected (Figure
S3a). This finding was consistent with that sorbicillinoids exhibited no
APDT activity against\emph{E. coli} . Similar to most classical PSs
(Cieplik et al., 2018), sorbicillinoids induces oxidative damage to the
cell membrane of\emph{S} . \emph{aureus} , contributing to its overall
antimicrobial activity together with the high intracellular ROS level
and the notable DNA photocleavage.

Furthermore, SEM experiments were carried out to monitor the impact of
sorbicillinoids on the morphology of bacterial surface. No noticeable
morphology change was found in the SEM pictures of the
irradiated\emph{S. aureus} (Figure 4b) and\emph{E. coli} (Figure S3b),
including wrinkles, holes or leaked intracellular substances as reported
previously for some photodynamic antibacterial materials (Jia et al.,
2017; Mao et al., 2019). Sorbicillinoids killed \emph{S. aureus} without
overt cell membrane disruption, as in the case of other PSs like EPS-RB
NPs (Li et al., 2018). Taken together, sorbicillinoids can readily
diffuse through the cell wall of Gram-positive bacteria into the cells.
When irradiated under UV light, it produced large sums of intracellular
ROS, thereby targeting and destroying both cytoplasmic membrane and DNA
to achieve APDT effect. This does not happen to Gram-negative bacteria
due to their distinctive outer membrane that is an extra effective
penetration barrier to block sorbicillinoids out of cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 4} . a) Confocal fluorescence images of \emph{S}
.\emph{aureus} that was incubated with PI after different treatments as
indicated in the figure. Bacteria with damaged cell membrane would be
stained by PI, showing red fluorescence. (scale bar = 10 \selectlanguage{greek}µ\selectlanguage{english}m). b) the
corresponding SEM images were also taken.

\textbf{3.5 Sorbicillinoids-mediated APDT exhibited good
biocompatibility}

Last but not least, the biocompatibility of sorbicillinoids was assessed
by the cytotoxicity measurement and hemolysis assay (Figure 5).
Sorbicillinoids showed a little cytotoxicity to AT-II cells at 400 \selectlanguage{greek}µ\selectlanguage{english}g/mL
(Figure 5a) that was higher than its bactericidal concentrations
for\emph{S} . \emph{aureus} (Figure 4a), \emph{B} . \emph{subtilis}
(Figure S1a) and \emph{M} . \emph{luteus} (Figure S1b). When the
concentration of sorbicillinoids was increased to 450 \selectlanguage{greek}µ\selectlanguage{english}g/mL, more than
85\% of AT-II cells remained alive, demonstrating a high safety of
sorbicillinoids (Figure 5a). More importantly, even under the
irradiation of UV light, only a slight decrease of the cell viability
was found with \textgreater{} 75.66\% AT-II cells alive when the
concentration of sorbicillinoids was increased up to 450 \selectlanguage{greek}µ\selectlanguage{english}g/mL. Thus,
sorbicillinoids did not induce marked dark toxicity or phototoxicity to
AT-II cells. On the other hand, only 0.89\% hemolysis was observed with
the treatment of 300 \selectlanguage{greek}µ\selectlanguage{english}g/mL sorbicillinoids (Figure 5b and Figure S4).
Sorbicillinoids show excellent biocompatibility, which is of benefit to
their future practical applications in clinic.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 5} . Biocompatibility evaluation of sorbicillinoids. a)
Viabilities of AT-II cells in the presence of different concentrations
of sorbicillinoids with and without UV light. b) Hemolysis rates of RBCs
after incubation with various concentrations of sorbicillinoids. RBCs in
Triton X-100 and PBS were set as the positive and negative controls,
respectively.

\textbf{4. CONCLUSIONS}

We discovered a novel water-soluble PS (sorbicillinoids) from
filamentous fungus \emph{Trichoderma} \emph{reesei} by microbial
fermentation. The obtained sorbicillinoids could transform oxygen
molecules to \textsuperscript{1}O\textsubscript{2} effectively under
nontoxic dose of UV irradiation. As a result, the irradiated
sorbicillinoids by nontoxic dose of UV light displayed photoinactivation
activity against Gram-positive bacteria including \emph{S}
.\emph{aureus} , \emph{B} . \emph{subtilis and M} . \emph{luteus} .
\emph{S} .\emph{aureus} treated with sorbicillinoids and UV light
irradiation exhibited high level production of intracellular ROS, marked
DNA reduction, and damaged cell membrane without observable cell
morphology change, leading to the cell death of Gram-positive bacteria.
Sorbicillinoids can be obtained on a large scale through microbial
fermentation by \emph{T} . \emph{reesei} or other microorganisms, which
is sustainable, environmentally-friendly and low cost. This
newly-discovered PS has good water solubility. Furthermore, they
displayed good biocompatibility. Therefore, sorbicillinoids-based APDT
presents a potential platform for combating pathogen-related diseases.

\textbf{ACKNOWLEDGMENTS}

This work was supported by grants from the National Natural Science
Foundation of China (31700040), and the Fundamental Research Funds for
the Central Universities.

\textbf{CONFLICT OF INTERESTS}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

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