Journal of Bioscience and Bioengineering

VOL. xx No. xx, 1e7, 2017

www.elsevier.com/locate/jbiosc

Metabolic dependent and independent pH-drop shuts down VirSR quorum

sensing in Clostridium perfringens

Keika Adachi,1 Kaori Ohtani,2, 3, x Michio Kawano,1 Ravindra Pal Singh,1, xx Basit Yousuf,1 Kenji Sonomoto,1
Tohru Shimizu,2, y and Jiro Nakayama1, *

Laboratory of Microbial Technology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan,1 Department of Bacteriology, Graduate School of Medical
Science, University of Kanazawa, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8203, Japan,2 and Miyarisan Pharmaceutical Co. Ltd., 1-10-3 Kaminakazato, Kita-ku, Tokyo 114-0016,
Japan3

Received 20 September 2017; accepted 20 December 2017

Available online xxx
Clostridium perfringens produces various exotoxins and enzymes that cause food poisoning and gas gangrene. The
genes involved in virulence are regulated by the agr-like quorum sensing (QS) system, which consists of a QS signal
synthesis system and a VirSR two-component regulatory system (VirSR TCS) which is a global regulatory system
composed of signal sensor kinase (VirS) and response regulator (VirR). We found that the perfringolysin O gene (pfoA)
was transiently expressed during mid-log phase of bacterial growth; its expression was rapidly shut down thereafter,
suggesting the existence of a self-quorum quenching (sQQ) system. The sQQ system was induced by the addition of
stationary phase culture supernatant (SPCS). Activity of the sQQ system was heat stable, and was present following
filtration through the ultrafiltration membrane, suggesting that small molecules acted as sQQ agents. In addition, sQQ
was also induced by pure acetic and butyric acids at concentrations equivalent to those in the stationary phase culture,
suggesting that organic acids produced by C. perfringens were involved in sQQ. In pH-controlled batch culture, sQQ was
greatly diminished; expression level of pfoA extended to late-log growth phase, and was eventually increased by one
order of magnitude. Furthermore, hydrochloric acid induced sQQ at the same pH as was used in organic acids. SPCS also
suppressed the expression of genes regulated by VirSR TCS. Overall, the expression of virulence factors of C. perfringens
was downregulated by the sQQ system, which was mediated by primary acidic metabolites and acidic environments. This
suggested the possibility of pH-controlled anti-virulence strategies.
Ó 2018, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Quorum sensing; Quorum quenching; Acidic pH; Autoinducing peptide; Clostridium perfringens; VirS/VirR two-component regulatory
system]

Clostridium perfringens is an anaerobic and spore-forming gram-
positive bacterium that inhabits animal intestinal tracts, and can
also reside in natural environments such as sewages and soil.
Certain C. perfringens strains produce exotoxins and enzymes that
can cause severe diseases such as gas gangrene and enteritis ne-
crosis (1). C. perfringens produces at least 16 types of pathogenic
toxins and extracellular enzymes (2), and are classified into 5 types
(type A-E) according to expressions of toxins produced: alpha
(CPA), beta (CPB), epsilon (ETX) and iota (ITX). The C. perfringens
type A strain is especially well-known as the major pathogen that
causes gas gangrene and food poisoning (3).
To effectively orchestrate the expression of a series of virulence
genes, some pathogenic bacteria employ the quorum sensing (QS)
system, which is a cell density-dependent regulatory system
mediated by diffusible chemical compounds collectively termed
* Corresponding author. Tel.: þ81 92 642 3020; fax: þ81 92 642 3021.
E-mail address: nakayama@agr.kyushu-u.ac.jp (J. Nakayama).
x
Present address: Department of Bacteriology and Bacterial Infection, Division of
Host Defense Mechanism, Tokai University School of Medicine, 143 Shimokasuya,
Isehara, Kanagawa 259-1193, Japan.
xx
Present address: Department of Biochemistry, John Innes Centre, Norwich
Research Park, Colney Ln, Norwich, NR4 7UH, United Kingdom.
y unnecessary metabolism. In addition, gram-negative bacteria
Deceased 25 February 2014.
autoinducers. Cyclic peptide-mediated QS is often found in low-GC
gram positive bacteria; the best-studied example of this is the
staphylococcal agr system (4e6). C. perfringens also employs an agr-
like QS system, in which the 5-amino acid residue thiolactone
peptide functions as an autoinducing peptide (AIPCp) (7). The agr-
like QS system is encoded by two operons; agrBDCp encodes the AIP
precursor and its processing enzyme, and virSR encodes the VirSR
two-component regulatory system (TCS) composed of an AIP
sensor kinase and its response regulator (8). The VirSR QS system is
involved in the control of multiple virulence genes, such as pfoA,
which encodes perfringolysin formely referred to as theta-toxin, as
well as plc, which encodes phospholipase C also called alpha-toxin.
In addition, it controls expressions of regulatory RNAs encoded by
virT, vrr, and virU. Notably, VR-RNA encoded by vrr acts as a key
global regulator that modulates the expression of 147 genes (9e21).
Previous studies have shown that some gram-negative bacteria
also possess a self-quorum quenching (sQQ) system that shuts
down their own QS. sQQ inducing factors can be classified into
three types: autoinducer-degrading enzymes, environmental or
metabolic factors, and starvation signals (22). Since QS can be en-
ergy consuming, sQQ system is a survival response that allows
rapid adaptation to the changing environment by shutting down

1389-1723/$ e see front matter Ó 2018, The Society for Biotechnology, Japan. All rights reserved.
https://doi.org/10.1016/j.jbiosc.2017.12.019

Please cite this article in press as: Adachi, K., et al., Metabolic dependent and independent pH-drop shuts down VirSR quorum sensing in
Clostridium perfringens, J. Biosci. Bioeng., (2017), https://doi.org/10.1016/j.jbiosc.2017.12.019
2 ADACHI ET AL.
degrade and assimilate extracellular autoinducers as nutrients
during starvation (22e28). However, little is known about sQQ in
gram-positive bacteria. Ohtani et al. (8,29) indicated that pfoA
transcripts are decreased from mid-log to stationary phase cultures,
suggesting the presence of sQQ-inducing agents. However, the
chemical nature of sQQ-inducing agents has not yet been clarified.
In this study, we elucidated the sQQ inducing factors and sQQ
mechanism of C. perfringens. The unveiled sQQ mechanism could be
a novel target for anti-infectious therapy.
MATERIALS AND METHODS
Bacterial strain and growth condition C. perfringens strain 13, a type A
strain, was used in this study. Cultivation of this strain was conducted at 37 C and
under anaerobic conditions, using an anaerobic gas generator, AnaeroPack-Anaero
(Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan). Cultures were generated by
streaking a glycerol stock of strain 13 onto 1.5 % (w/v) agar plates containing brain
heart infusion (BHI) medium and 5 % (v/v) defibrinated sheep blood. Following an
overnight incubation, colonies with hemolytic halos were inoculated into a liquid
medium of Gifu-anaerobic medium (GAM), and were grown as the pre-culture for
5 h until stationary growth phase was reached. To generate the main culture, the
pre-culture was inoculated into a liquid medium of tryptoneesoytoneefructose
(TSF) (40 g/L tryptone, 4.0 g/L soytone, and 5.0 g/L fructose, adjusted to pH 5.7 by
HCl) (30).
Reverse transcriptional quantitative PCR and northern blot
hybridization Transcriptional levels of genes listed in Supplementary Table S1
were quantified by reverse-transcriptional quantitative PCR (RT-qPCR). Briefly,
450 mL RNA protect Bacteria Reagent (QIAGEN, Germany) was added to 200 mL
culture broth immediately following sampling. The mixture was incubated for
5 min at room temperature, and cells were harvested via centrifugation at
15,300 g for 5 min. Total RNA was extracted and purified according to the
previously described protocol, using the hot-phenol method (31); 10 ng purified
RNA was used for the RT-qPCR assay. Reverse transcription was performed and
monitored with the One Step SYBR PrimeScript RT-PCR Kit-II (Perfect Real Time,
Takara Bio, Japan) in MxPro3000P (Stratagene Japan Co., Japan). The reverse
transcription and PCR amplification program was as follows: 1 cycle at 42 C for
5 min, 1 cycle at 95 C for 10 s, 40 cycles at 95 C for 5 s, 60 C, 58 C or 56 C for
20 s, and 72 C for 32 s. Melting curve analysis was then conducted by heating the
samples from 60 to 95 C to verify the identity of each targeted transcript. Primer
sequences and RT-qPCR conditions for each target gene are shown in Table S1.
Expression level of each transcript was normalized to that of 16S rRNA. Bacterial
cultures for each RT-qPCR assay were grown in triplicates, and RT-qPCR assays
from each culture were performed in duplicates.
Northern blot hybridization was performed as previously described (29), using
10 mg total RNA that was prepared as described above (13).
Time course of QS and sQQ Pre-culture of C. perfringens strain 13 (1 mL) was
inoculated into 100 mL TSF broth (pH 5.7) in a fermenter flask; cultures were stirred
and purged by filtered nitrogen gas for 2 min to maintain anaerobic conditions.
Growth was monitored by measuring the optical density of culture broth at 600 nm
(OD600) with a Biospec-1600 (Shimadzu Co., Tokyo, Japan). For pH-controlled
cultures, pH of culture broth was monitored with a pH meter (DKK-TOA Co.,
Japan) that was laid on the fermenter. pH values were adjusted to 5.7e5.8 by the
addition of 2.5 M NaOH using a pH controller (PHC-2201, Able, Japan) connected
to the pH meter. Sampling was performed every 30 min for 4 h and 6 h following
inoculation to quantify organic acids and pfoA transcripts. To quantify acetic acid
and butyric acid content, the culture supernatant (800 mL) was concentrated with
a Thermo Scientific Savant SpeedVac concentrator SPD1010 (Thermo Scientific,
Japan) without heating, and was dissolved in 200 mL MilliQ water. The solution
was then subjected to gas chromatography (GC, Agilent Technology, Japan) with a
15-m of MilliQ capillary column (Innowax; i.d. 0.53 mm, 19095N-121; Agilent
Technology, Germany), as previously described (32). Quantification of pfoA
transcripts was performed according to methods described above.
sQQ assay sQQ activity was assessed according to previously published
methods (31). To prepare QS-active cells, strain 13 was cultured in TSF medium (pH
5.7) up to mid-log growth phase, after which QS-active cells were harvested by
centrifugation at 15,300 g for 5 min. To examine QQ, cells were suspended with
samples to be tested, and were incubated at 37 C. Incubation time was fixed at
15 min with the exception of mode of action of sQQ. Following the incubation
period, total RNA was extracted and subjected to RT-qPCR for quantification of pfoA.
To prepare the stationary phase culture supernatant (SPCS), which has high QQ
activity, the pre-culture of C. perfringens strain 13 was inoculated into the TSF broth
to achieve a final concentration of 5 % (v/v). Cells were cultured for 6 h at 37 C, and
were removed via centrifugation at 15,300 g for 5 min.
To determine the heat tolerance of sQQ, SPCS was autoclaved at 121 C for
15 min. To estimate the molecular size of sQQ inducing agents, SPCS was subjected to
ultrafiltration with Amicon Ultra-Centrifugal filters (Ultracel-50K, 30K, 10K, 3K and
Please cite this article in press as: Adachi, K., et al., Metabolic dependent and independent pH-drop shuts down VirSR quorum sensing in
Clostridium perfringens, J. Biosci. Bioeng., (2017), https://doi.org/10.1016/j.jbiosc.2017.12.019
J. BIOSCI. BIOENG.,
1K, Merck EDM Millipore, Germany), and sQQ assays were performed on filtrated
fractions. Partial purification of the sQQ system was conducted with a strong anion
exchange column (Sep-pak accell QMA, Waters Co., USA). The columns were eluted
by stepwise increases in acetonitrile containing 0.1 % (v/v) trifluoroacetic acid; sQQ
activity of each fraction was monitored.
To examine the effect of organic acids, acetic acid and n-butyric acid, they were
diluted with sterilized MilliQ water to reach a final concentration of 1.0 g/L, and were
added to the indicator cell suspension. To examine the effect of pH on sQQ, hydro-
chloric acid was added to the TSF medium containing indicator cells. To examine the
mode of action of sQQ, rifampicin (200 mg/mL in 100% methanol), an antibiotic that
inhibits transcription in bacteria, and synthetic AIPCp, were added to the indicator
cell suspension. Synthetic AIPCp was prepared as previously described (31).
RESULTS
Time course of growth, primary metabolism, QS and sQQ of
C. perfringens strain 13 As shown in Fig. 1, in the anaerobic batch
culture, C. perfringens started to proliferate within 1 h following
inoculation, and entered log growth phase. According to the
growth curve, early-, mid- and late-log phases corresponded to
0e1 h, 1e2 h, and 2e3 h following inoculation. Acetic acid was
produced from early-log phase, whereas butyric acid production
was observed from late-log phase onwards. During organic acid
fermentation, culture pH dropped from 5.91 to 4.81. Transcription
of pfoA was highly activated during the mid-log phase and was
drastically downregulated from the late-log phase. These
observations suggested that the C. perfringens strain has a potent
sQQ system that intentionally downregulates pfoA transcription.
sQQ activity of SPCS in C. perfringens strain 13 We exam-
ined the effect of culture supernatants during log and stationary
phases on pfoA transcription. Similar to previous observations
(8,29), pfoA transcripts were broken down by the addition of SPCS
(Fig. 2A, B). This suggested the presence of sQQ-inducing agents
in SPCS.
To characterize the chemical nature of the sQQ-inducing agent,
SPCS was autoclaved and was subjected to the sQQ assay. Results
indicated that autoclaving did not abolish the sQQ activity of SPCS
(Fig. 2C). Subsequently, size fractionation of the sQQ-inducing
agent in SPCS was performed by ultrafiltration, using membranes
with different molecular weight cut-off sizes. The strongest sQQ
activity was detected in the <3 kDa fraction (Fig. 2D). The minor
activity was also detected in the other fractions, suggesting the
possibility that there are large molecules having sQQ activity in
culture supernatant. Furthermore, trypsin and chymotrypsin
treatments did not reduce sQQ activity (Supplementary Fig. S1).
sQQ activity was captured with an anion exchange cartridge col-
umn (Supplementary Fig. S2). These results suggested that the sQQ-
inducing agent is non-proteinaceous cationic compound(s). Since it
is known that C. perfringens produces acetic acid and butyric acid as
products of sugar fermentation, we examined sQQ activity of these
two organic acids at their natural concentrations in culture super-
natants during the late-log phase (Fig. 3A, B). It was found that both
acetic acid and butyric acid induce sQQ at concentrations equiva-
lent to those present in late-log to stationary phase cultures. Taken
together, we concluded that acetic and butyric acids produced by
C. perfringens act as QQ-inducing agents.
Acidic pH condition necessarily and sufficiently induces
sQQ The sQQ activity induced by organic acids disappeared
when pH was controlled at a neutral range (Fig. 3A, B). Interestingly,
not only organic acids, but inorganic acids such as hydrochloric acid
(HCl) also demonstrated obvious sQQ-inducing activities (Fig. 3C).
These results suggested that acidic condition is necessary and
sufficient for the induction of sQQ. To verify this hypothesis, pH-
controlled batch cultures of C. perfringens strain 13 was used
(Fig. 4). As compared with non pH-controlled cultures, pfoA
transcription was remarkably delayed in these cultures; its level
VOL. xx, 2017 pH-DEPENDENT QUORUM QUENCHING OF C. PERFRINGENS 3

FIG. 1. Time course of pfoA expression in C. perfringens strain 13. C. perfringens strain 13 was cultured anaerobically in TSF medium, and transcription levels of pfoA and 16S rRNA was
analyzed. Optical density (OD600), pH, and concentration of acetate and butyrate in the culture broth were monitored. Total RNA was extracted in triplicate. The transcriptional level
of pfoA and 16S rRNA was determined independently, and is expressed as mean  standard deviation.

was eventually elevated to approximately 10 times higher even late-log phase, and mostly disappeared during the stationary
sufficient concentration of organic acids were presence, indicating phase. This is likely attributed to general transcriptional arrest due
that pH-drop is necessary for sQQ at mid-log phase. However, pfoA to nutrient depletion but further studies are required to address
transcription in pH-controlled culture was diminished during the this phenomenon.

FIG. 2. QQ activity of culture supernatant, and properties of sQQ-inducing agent in the stationary-phase culture supernatant. (A) Northern blot hybridization of pfoA-extracted
C. perfringens strain 13 cells incubated in fresh TSF medium (lane 1), early-log phase culture supernatant (lane 2), and stationary-phase culture supernatant (lane 3). Under each
lane, bands of 23S rRNA and 16S rRNA in western blotting were indicated. (B) RT-qPCR of pfoA extracted C. perfringens strain 13 cells incubated in TSF medium (lane 1) and with
stationary-phase culture supernatant (lane 2). Experiments were performed in triplicates, expression levels are normalized to that of 16S rRNA, and are expressed as
mean  standard deviation. (C) Stability against autoclaving. Strain 13 cells from log-phase growing culture were incubated with fresh TSF medium (lane 1), the stationary-phase
culture supernatant (lane 2), and stationary-phase culture supernatant autoclaved at 121 C for 15 min (lane 3). Total RNA was extracted from the incubated cells, and was used to
assess QQ activity. Lane 1 is used as the negative control. (D) Size fractionation of QQ agent by ultrafiltration. The stationary-phase culture supernatant was sequentially subjected to
ultrafiltration membranes with molecular weight cut-off of 50 kDa, 30 kDa, 10 kDa and 3 kDa. Each filtrate was subjected to the QQ activity assay. Lane 1, fresh TSF medium; lane 2,
stationary-phase culture supernatant (positive control); lane 3, >50 kDa; lane 4, 30e50 kDa; lane 5, 10e30 kDa; lane 6, 3e10 kDa; lane 7, <3 kDa. The experiment was performed in
triplicate, and results are expressed as mean  standard deviation with the initial pH indicated by circles.

Please cite this article in press as: Adachi, K., et al., Metabolic dependent and independent pH-drop shuts down VirSR quorum sensing in
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4 ADACHI ET AL. J. BIOSCI. BIOENG.,

FIG. 3. QQ induction by acids. (A) QQ induction by acetic acid and its salt. (B) QQ induction by butyric acid and its salt. (C) QQ induction by inorganic acid. The cells at early-log phase
were incubated with various concentrations of acetic acid, butyric acid, sodium acetate, and sodium butyrate for 15 min at 37 C. The concentrations of chemicals used in the
experiments were 1.0, 0.5, 0.25, 0.125 g/L. Then, transcriptional level of pfoA and 16S rRNA was quantified by RT-qPCR. The experiment was performed in triplicate, and data are
expressed as mean  standard deviation, initial pH values are indicated by circles.

Mode of action of sQQ As shown in Fig. 5, when 1.0 mM of
synthetic AIPCp was added to C. perfringens cells at early-log
growth phase, pfoA transcription was rapidly induced. On the
other hand, when SPCS was added under the same conditions,
pfoA transcription was reduced to basal level within 10 min. This
rapid quenching of pfoA was similarly observed after addition of
rifampicin, which is an RNA polymerase inhibitor. These results
indicated that termination of nascent mRNA synthesis leads to
quenching of pfoA, and suggested that acidic conditions inhibit
transcription of pfoA even in the presence of AIPCp.

FIG. 4. QQ in pH-controlled and non pH-controlled culture of C. perfringens. C. perfringens strain 13 was grown in TSF medium controlled at pH 5.7e5.8 (left) or in TSF medium
without pH control (right). The concentration of organic acids was indicated lower side. Optical density at 600 nm (OD600), pH, concentrations of acetate and butyrate in culture
broth were monitored. Total RNA was extracted in triplicate. The transcriptional level of pfoA and 16S rRNA was determined independently, and is expressed as mean  standard
deviation.

Please cite this article in press as: Adachi, K., et al., Metabolic dependent and independent pH-drop shuts down VirSR quorum sensing in
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VOL. xx, 2017 pH-DEPENDENT QUORUM QUENCHING OF C. PERFRINGENS 5

DISCUSSION

Unlike the case of general QS, pfoA transcription under the

FIG. 5. Effect of stationary-phase culture supernatant and rifampicin on AIP-induced
pfoA expression. C. perfringens cells from early-log phase were suspended in fresh TSF
medium with 1.0 mM of AIPCp (circles), fresh TSF medium containing 200 mg/mL rifam-
picin (diamonds), or stationary-phase culture supernatant (triangles). As a control, data
of cells without AIP treatment was indicated (dashed line). Cells were incubated at 37 C
and collected sequentially. pfoA transcript levels were quantified by RT-qPCR, and
normalized to that of 16S rRNA. The experiment was done in triplicate and data are
expressed as mean  standard deviation; initial pH values are indicated by circles.
Effects of sQQ on expression of genes downstream of VirSR
TCS We examined the effect of sQQ on the transcription of a
series of genes downstream of VirSR TCS. Similar to what was
observed with pfoA, transcription of genes known to be directly
regulated by VirSR TCS was suppressed by the addition of SPCS
(Fig. 6). Especially, genes directly regulated by VirSR TCS were
strongly downregulated the transcription.
regulation of VirSR QS reached the peak at mid-log growth phase,
and was thereafter quenched. This phenomenon can be explained
by the association between sQQ and metabolic-linked pH drop. It is
also known that the agr system in Staphylococcus aureus is down-
regulated under acidic conditions (33e35). Similarly, acetoin
fermentation was shown to be regulated by QS and pH in Serratia
plymuthica (35).
Our results demonstrated that rifampicin reduced pfoA tran-
scripts by half in approximately 5 min, and resulted in complete
breakdown of pfoA in 10 min. This coincides with the general half-
lives of prokaryotic mRNA (36e39). Similarly, SPCS also broke down
pfoA transcripts even in the presence of AIPCp, suggesting that VirSR
TCS signaling is shut down under acidic conditions. It was observed
that inorganic acids also induced sQQ, suggesting that C. perfringens
cells are capable of sensing alterations in environmental pH, and
controls QS accordingly. Many studies have shown that QS is
quenched by environmental cues such as pH, nutrients and me-
tabolites (33e35,40).
It is known that VirR boxes, VirR-binding site which include two
imperfect repeating sequences, are located immediately upstream
of promoters for genes directly regulated by VirSR TCS (17,41). In
addition, two toxin genes under the control of VR-RNA, colA and plc,
were also down-regulated by the sQQ system. Regulatory gene
network under VirSR TCS shares several common target genes with
a small RNA named virX, other TCS orthologs (RevR), RNaseY and so
on (42,43). For example, Obana et al. (43) showed that RNaseY, a

FIG. 6. Effect of SPCS on the expression of genes downstream of VirSR TCS. C. perfringens cells from mid-log phase were incubated with 1 mM of AIPCp, and with or without SPCS for
15 min. Expression level of pfoA was quantitated by RT-qPCR and normalized to that of 16S rRNA. Fold changes as compared with control were determined for genes in the VirSR
regulatory networks (Known regulatory networks are indicated by gray arrows for positive regulation). Each experiment was performed in triplicate and results are expressed as
mean  standard deviation. Black arrows and squares next to black arrow mean genes and VirR boxes, respectively.

Please cite this article in press as: Adachi, K., et al., Metabolic dependent and independent pH-drop shuts down VirSR quorum sensing in
Clostridium perfringens, J. Biosci. Bioeng., (2017), https://doi.org/10.1016/j.jbiosc.2017.12.019
6 ADACHI ET AL.
major endoribonuclease, is involved in the post-transcriptional
cleavage and stability of toxin gene regulated by VirSR TCS. It is
known that RNaseY helps VR-RNA to directly regulate colA
expression through base pairing with colA mRNA in vivo; VR-RNA
stabilizes colA mRNA by cleaving the 50 -untranslated region (UTR)
(18,19). In addition to colA expression, VR-RNA regulates the
expression of plc (alpha toxin), cpd2 and several housekeeping
genes, suggesting that sRNA plays a critical role in the virulence and
metabolism of C. perfringens (20). Microarray analysis suggested
that VR-RNA regulates the expression of at least 147 genes, and is a
key global regulator (21).
This study showed that induction of a series of genes with VirR
box was strongly downregulated by SPCS and two genes which are
regulated indirectly by VirSR TCS, plc and colA, were slightly
downregulated by SPCS. However, link between pH-sensor
signaling and VirSR system is yet to be clarified. It is noted that
this regulation is rapid and solely triggered by exogenous acid,
suggesting involvement of direct external acid sensor such as his-
tidine kinase EvgS of Escherichia coli (44). It is known that Clos-
tridium acetobutyricum and Clostridium saccharoperacetobutyricum,
which are species closely related to C. perfringens, perform drastic
metabolic shift from acidogenesis to solventogenesis in response to
metabolic-linked pH drop (32,45). The VirSR QS system of
C. perfringens may cross-talk with global gene regulatory networks
associated with pH shift. Top-down transcriptomics may provide an
overview of these complex regulatory networks.
In conclusion, the virulence of C. perfringens is downregulated
by the sQQ system, which is mediated by primary metabolite
organic acids as well as acidic environmental conditions.
C. perfringens is an opportunistic pathogen that commonly inhabits
the gastro-intestinal tracts of humans and animals as well as
common food-borne pathogens. It should be further noted that
antibiotic-resistant strains are common among both clinical and
non-clinical isolates, as observed in other opportunistic pathogens
(46,47). There is a need to deviate from usage of antimicrobial
compounds, which promotes the emergence of drug resistant
bacterial strains (48). Instead, pH control can be exerted at sites
where C. perfringens contaminates, such as wound, intestine and
foods. This strategy can target pH-linked sQQ, and be utilized as a
novel anti-virulence approach in the future.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jbiosc.2017.12.019.
ACKNOWLEDGMENTS
We also thank Dr. Motomichi Takahashi and Dr. Kentaro Oka for
their helpful discussion. This work was supported by the Japan
Society for the Promotion of Science (JSPS, numbers KAKENHI
JP24380050, JP15H04480, and JP16F16101) for J. Nakayama.
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Clostridium perfringens, J. Biosci. Bioeng., (2017), https://doi.org/10.1016/j.jbiosc.2017.12.019
pH-DEPENDENT QUORUM QUENCHING OF C. PERFRINGENS 7
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