SUPPLEMENTARY MATERIALAntibacterial mode of action of 1,
8-dihydroxy-anthraquinone from Porphyra haitanensis Against
Staphylococcus aureusYuxi Wei 1*, Qi Liu2, Jia Yu1, Qiang Feng1,3,
Ling Zhao2, Huiping Song1 and Wenxiu Wang1As one kind of
anthraquinone dihydroxy derivatives, 1, 8-dihydroxy -anthraquinone
(Dan) with strong antibacterial activity against Staphylococcus
aureus was first isolated from Porphyra haitanensis. Here we report
on the investigation of the antibacterial mode of action of Dan on
the gram-positive bacterium Staphylococcus aureus. The results show
that Dan strongly inhibited cell growth at logarithmic phase. In
present study, the Dans antibacterial activity was analysed by
using phosphorus standard solutionPSS, p-nitrophenyl phosphate
(pNPP)o-nitrophenyl--D-galactopyanoside (ONPG), scanning electron
microscopy (SEM) and transmission electron microscopy (TEM). The
results suggested that Dans antibacterial activity is due to its
interaction with the cell wall and cell membrane, in which it
increases cell envelope permeability and leads to the leakage of
cytoplasm and the deconstruction of cell. The present study
indicates that Dan as a natural product in seaweeds deserves
further investigation for applications as an antibacterial
bioactive substance in food safety control and drugs.Key
wordsPorphyra haitanensis; 1, 8-dihydroxy-anthraquinone;
antimicrobial action; Staphylococcus aureus1College of Life
Sciences, Qingdao University, Qingdao 266071, PR China; 2Yellow Sea
Fisheries Research Institute, Chinese Academy of Fishery Sciences,
Qingdao 266071, PR China; 3Center for Disease Control and
Prevention of Jining City, Jining 272000, PR China*Corresponding
author. E-mail address: [email protected] Experimental
Materials
Porphyra haitanensis sample collected from the Jinjiang
coastline (Fujian, China) in February 2013 was authenticated by
Institute of Oceanology, Chinese Academy of Sciences. The voucher
specimen (QD-SW-20130068) was deposited in the Biochemistry Lab of
Qingdao University, Qingdao, China. 1, 8-dihydroxy- anthraquinone
(Dan) was isolated from the sample by the reference method (Feng et
al., 2013). Staphylococcus aureus (CAU0519) was supplied by
Institute of Microbiology, Chinese Academy of Sciences. Bacterial
cells were cultured and activated in fresh tryptone soybean broth
(TSB) at 28 for 12 h.
Effect of Dan on the Growth Curve of S. aureus The experiment
was carried out according to reference (Ai et al. 2007) with some
modifications. BrieflyS. aureus culture was inoculated in nutrient
broth medium with an inoculation amount of 106 CFU/mL and cultured
at 37 with the air bath and homothermal vibrator at 150 rpm.
Optical density (OD) at 550 nm was monitored with spectrophotometer
every hour during a course of 13 hours as control group. The
procedure for experimental group was the same as above except that
bacterial suspension contained 80 g/mL Dan.
Effect of Dan on the phosphorus metabolism of S. aureus The
experiment was carried out according to reference (Hancock &
Rozek 2002, Qian et al. 2010, Zhai et al. 2006) with some
improvements. S. aureus culture was inoculated in 5 mL of nutrient
broth with an inoculation amount of 106 CFU/mL. Then 5 mL of
glucose standard solution (1 mg/mL), 2 mL of KH2PO4 standard
solution (0.17 mg/mL) were added. The mixture was cultured at 37
with the air bath and homothermal vibrator at 150 rpm. For each
hour, the culture solution was centrifuged at 5000 rpm for 10 min.
The supernatant (0.5 mL) was mixed with 4 mL CCl3COOH-FeSO4
solution (containing 10mg/mL thiourea, 100.0 mg/mL CCl3COOH and 0.6
mg/mL FeSO4) and layed up for 10 min. After each centrifugation,
the OD value of the supernatant was determined by ammonium
molybdate spectrophotometry at 630 nm as control group. The
procedure for experimental group was the same as above except that
bacterial suspension contained 80 g/mL of Dan.
Effect of Dan on the cell wall permeability of S. aureus
determination of alkaline phosphatase activityMethods for bacterial
culture and centrifugation of culture solution were the same as
above. The experiment was performed as previously described (Wang
et al. 2006). After centrifugation for each hour, the supernatant
(2 mL) containing 80 g/mL Dan was mixed with 5 mL p-nitrophenyl
phosphate (pNPPthe Sigma Chemical Co. St. Louis, MO, USA) substrate
buffer. The mixture was heated at 40 for 5 min. A 2.0-mL portion of
0.5 mol/L Na2CO3 was added to stop reaction, and then absorbance
reading was taken at 410 nm using a spectrophotometer. OD410 value
was used to indicate the activity of alkaline phosphatase as
experimental group. The procedure for control group was the same as
above except that there was no Dan added.
Effect of Dan on the membrane permeability of S. aureus
Protein content determination of culture media Methods for
bacterial culture and centrifugation of culture solution were the
same as above. After centrifugation for each hour, the content of
proteins in culture of both groups was assayed by Coomassie
brilliant blue method at 595nm (Bradford 1976). -galactosidase
activity assay of culture media. Methods for bacterial culture and
centrifugation of culture solution were the same as above for both
groups. The experiment was carried out according to references (Li
et al., 2012; Ibrahim et al. 2000). After centrifugation for each
hour, 4 mL o-nitrophenyl--D-galactopyanoside (ONPGthe Sigma
Chemical Co. St. Louis, MO, USA) substrate buffer was mixed with
1.0 mL supernatant of culture solution obtained above (containing
80g/mL Dan) and the mixture was heated at 50 for 30 min. A 2.0-mL
portion of 1.0 mol/L Na2CO3 was added to stop reaction, and then
absorbance reading was taken at 420 nm using a spectrophotometer.
OD420 value was used to indicate the activity of -galactosidase.
The procedure for control group was the same as above except that
there was no Dan added.
Sample Preparation of S. aureus and Observation by SEMThe
experiment was carried out according to reference (Tang et al.
2010) with some improvements. S. aureus pure culture was inoculated
with an inoculation amount of 106 CFU/mL, in nutrient broth medium
(control group) or nutrient broth medium containing 80 g/mL Dan
(experimental group) respectively. After being allotted into three
tubes and cultured at 37 with the air bath and homothermal vibrator
for 3, 5 and 7 h respectively, the culture solution was centrifuged
at 5000 rpm for 10 min to collect bacteria cells. The pellets were
prefixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH7.3),
postfixed in phosphate buffer (pH7.4), and dehydrated in graded
ethanol solutions (30%, 50%, 70%, 80%, 90% and 100%). Pellets were
then dried at critical point, coated with gold palladium by
direct-current sputtering and examined with a HITACHI S-450
scanning electron microscope. Fig.S6 The morphology of S. aureus at
incubation time of 0, 3, 5 and 7 h. Fig.S7 The morphology of S.
aureus after incubation with Dan for 3, 5 and 7 h
Sample Preparation of S. aureus and Observation by TEM The
experiment was carried out according to reference (Xing et al.
2009) with some improvements. The culturing, collecting, washing,
fixing and dehydration procedures of S. aureus sample treated with
Dan were the same as those used for preparing the sample observed
by SEM. Pellets were embedded in Epon 812, fixed at 37, 45 and 65
and then cut using an ultramicrotome (UltracutE). Changes of
ultrastructure of S. aureus treated by Dan were observed under
transmission electron microscope (JEM-1200EX) after the thin
sections were stained by uranyl acetate and lead nitrate. The same
suspension was incubated in nutrient broth medium with distilled
water instead of Dan as control group and the sample preparation
for TEM observation was the same as that for experimental group.
Fig.S8 The ultrastructure of S. aureus at incubation time of 0, 3,
5 and 7 h.
Fig.S9 The ultrastructure of S. aureus after incubation with Dan
for 3, 5 and 7 h Statistical analysis All data were analysed by
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