The influence of pH on the transcription of
cpb2 of Clostridium perfringens.
Drs. J.E. Duval
Supervisors:
Dr. A.J.A.M. van Asten
Drs. J.G. Allaart
Prof. Dr. A. Gröne
Department of Pathobiology
Division of Strategic Infection Biology
Faculty of Veterinary Medicine
Utrecht University, the Netherlands
January 2010
TABLE OF CONTENTS
Abstract
page 3
Introduction
page 4
Materials and methods
page 6
Results
page 8
Discussion
page 10
Acknowledgements
page 11
References
page 12
Appendices
page 14
2
ABSTRACT
Research has shown that the correlation of cpb2 is strong in cases of porcine enteritis
(85%), especially from cases of neonatal porcine enteritis (91.8%). This suggests that
the β2-toxin is involved in the pathogenesis of enteritis by C. perfringens type A. In
other animal species a correlation exists, but it is not that strong.
The toxin production occurs only under certain circumstances, but the environmental
conditions that lead to changes in the expression of cpb2 are not clarified. In Western
Blot analysis differences in the amount of β2-toxin production were measured after
growing the bacteria under different pH conditions. Our hypothesis is that the
transcription of cpb2 is influenced by the pH in the environment of the bacteria. The
aim of this study was to verify this hypothesis using the Quantitative-PCR technique,
in order to measure the relative transcription of cpb2 compared to the gene encoding
for gyrase (reference gene). Our provisional results demonstrate that the relative
transcription of cpb2 is influenced by the pH; the transcription of cpb2 is higher at pH
7 compared to that at pH 5. This suggests that the transcription of cpb2 is pH
dependent.
3
INTRODUCTION
Clostridium perfringens is a Gram-positive, spore-forming, anaerobic bacterium and
might be the most important clostridial enteric pathogen of domestic animals. The
bacterium is widespread in the environment and is commonly found in the gastrointestinal tract of animals, including humans. The bacterium does not invade healthy
cells, but under certain circumstances it can produce toxins in the gastro-intestinal
tract. There are at least 15 different toxins known, but each individual C. perfringens
strain produces a selection of these toxins. Based upon the production of the four
major toxins, alpha, beta, epsilon and iota, the species are classified in five types of C.
perfringens (type A-E). Pathogenicity and lesions are correlated with the major toxins
produced, thus typing of the bacterium has diagnostic and epidemiological
significance. All enteric toxins of C. perfringens share two common features: they are
all single polypeptides of modest size (~25–50 kDa), although lacking sequence
homology and they act in general by pore-forming activity or by forming channels in
plasma membranes of host cells.1, 8, 10, 11
In 1997 the nucleotide sequence of the gene (cpb2) encoding for a formerly unknown
toxin was identified. The C. perfringens strain harbouring cpb2 was isolated from a
piglet that suffered from necrotic enteritis.3 Cpb2 showed no significant homology
with the sequence encoding the β-toxin (15% nucleotide similarity) or at amino-acid
level with any other known protein sequence, but the toxin was termed β2 because of
its similar biological activity as the β-toxin. The β2-toxin disrupts the cell membrane,
but it’s exact mechanism is still unknown.9, 14
Since its first description cpb2 harbouring C. perfringens has been shown to occur in
many other animal species, including horses, cattle, poultry and humans.1, 9 Research
shows that the correlation of cpb2 is strong in cases of porcine enteritis (85%),
especially from cases of neonatal porcine enteritis (91.8%). This suggests that the β2toxin is involved in the pathogenesis of enteritis by C. perfringens type A. The
correlation in other animal species is not that strong.2 It is hypothised that cpb2 is of
more importance than cpb in the pathogenesis of diarrhea or enteritis in pigs, since the
cpb2 showed to be more apparent in cases of diarrhea in piglets than the cpb.5 The
recently discovered correlation of enteritis in pigs with the production of the β2-toxin
has also implications for the use of vaccination to prevent disease in pigs as a result of
a C. perfringens infection. The commercially available vaccines for pigs registered in
the Netherlands are at the moment toxoïd vaccines containing the β-toxin toxoid. In
order to offer complete prevention against disease when the β2-toxin is involved the
vaccines should also contain the β2-toxin toxoïd.4, 7, 14
The cpb2 gene resides on plasmids and there is evidence that the expression of cpb2 is
regulated at the level of transcription, by the VirR/ VirS two component bacterial
regulatory system.7, 9, 13 However, the conditions that lead to changes in the expression
of the toxin are still not clarified. In certain C. perfringens strains, originating from
horses, the expression of cpb2 was induced by antibiotic therapy with gentamicin or
streptomycin. This was probably due to frameshifting.12
In a Quantitative Polymerase Chain Reaction (Q-PCR) the amplified product can be
detected and measured during the reaction. In this study SYBR Green was used as
4
binding dye, it binds to double stranded DNA and its fluorescence increases after this
binding. The amount of fluorescence will double after each PCR cycle, until a plateau
phase starts when the reaction components are consumed and become the limiting
factor. When enough amplified products accumulate a reliable fluorescent signal can
be detected, the cycle number at which this occurs is the threshold cycle (Ct). The Ct
of a reaction is mainly determined by the amount of template at the start of the
amplification reaction. The change in transcription of the gene of interest is compared
to the transcription of a reference gene. A reference gene should have a constant
transcription level across all the test samples and the transcription should not be
affected by the experimental treatment under study. As a reference gene we used gyr
A, the gene encoding for DNA-gyrase, an enzyme that is essential in the replication of
DNA.6
It is hypothised that production of the β2-toxin is influenced by environmental
conditions, such as the pH of the environment in which the bacteria are growing
(unpublished data). The purpose of this study was to clarify the role of the pH on the
transcription of cpb2. Using the Q-PCR, we investigated whether the differences in
toxin production after growth under different pH circumstances, as measured by
Western Blot, were due to changes in transcription or translation of cpb2.
5
MATERIALS AND METHODS
Bacterial strains and growth conditions
The bacterial strains were grown overnight under anaerobic conditions at 37 ºC, in
MRS broth. The cultures were transferred in equal amount to 12 tubes and centrifuged
for 10 minutes at 3.880 x g. Then for each timepoint sets of 4 pellets were
resuspended in tubes containing 3 ml MRS broth at a pH5, 6, or 7. The suspensions
were incubated under anaerobic conditions for 1, 2, 3 or 4 hours at 37 ºC. After
incubation the pH of the growing medium was measured and serial dilutions of the
bacterial suspensions were made and spread on LB plates in order to determine the
number of viable bacteria (colony forming units (CFU)).
Tubes were centrifuged for 10 minutes at 3.880 x g. And both supernatant and pellets
were stored at -20 ºC. The supernatants were used for Western Blot analysis, the
pellets for RNA isolation.
Prior to RNA isolation the pellets were divided in 3, to prevent repetitively thawing
and freezing of the pellets, since this could affect the quality of the samples. The
pellets were, after thawing, dissolved in 2.5 ml PBS (4 C) and distributed over 3
fresh eppendorf tubes. Centrifuged at 4 C, for 5 minutes at 16.100 x g. The
supernatant was discarded and the pellets stored at -20 C.
RNA isolation
Total RNA was isolated using the TRIzol Reagent kit (Invitrogen) according to the
manufacturer’s protocol, with some minor modifications (See appendix I). After RNA
isolation the RNA concentration and purity was measured with the Nanodrop
spectrophotometer. Electrophoresis through a 1% agarose gel, containing 0.5 g/ ml
ethidium bromide, was used to check for possible degradation of the RNA and the
presence of ribosomal bands.
Preparation DNA-free RNA prior to RT-PCR and cDNA synthesis
In order to remove the genomic DNA, the RNA samples were treated with DNase I,
RNase-free (Fermentas) prior to cDNA synthesis. The input of total-RNA for this
reaction being 1 g (See appendix II). The effectiveness of the reaction was checked
by performing a PCR on the samples using, Taq polymerase (Fermentas) and the
beta2totalF2 and beta2totalR primers (Table 1). The PCR was followed by
electrophoresis through a 1% agarose gel containing 0.5 g/ ml ethidium bromide, to
identify a possible PCR product by UV transillumination (See Appendix III)
For the cDNA synthesis the RevertAid First Strand cDNA Synthesis Kit (Fermentas)
was used, with the random hexamer primer (see Appendix IV).
6
Quantitative PCR
A quantitative PCR was performed to measure the transcription of cpb2, relative to
the transcription of the (housekeeping) gene gyr A (See Appendix V). Per reaction
12.5 l IQ SYBR Green Supermix (BioRAD), 3 l primermix 1.5 M (Table 1) and
4.5 l aquadest was used. The input of cDNA, with a concentration of 5 ng/l, was 5
l. By standardising this concentration the conditions in the wells were approximately
the same for all the samples. Amplification of the genes was performed using the
following protocol: 3 minutes at 95 C, followed by 40 cycles consisting of 30
seconds at 95 C, 30 seconds at 48 C and 30 seconds at 72 C. And two final steps,
both one cycle, 1 minute at 95 C, followed by 1 minute at 65 C. The amplification
was followed by the formation of a melt curve with the following protocol: 60 cycles,
with a start temperature of 65 C, +0.5 C every 10 seconds. The Ct values of the
samples were determined in triplicates for cpb2 as well as for gyr A
As a control we used electrophoresis through a 1% agarose gel containing 0.5 g/ ml
ethidium bromide, to check the samples by UV transillumination for by-products
other then the expected PCR products of cpb2 or gyr A.
After performing the quantitative PCR, the average and the standard deviation of the
Ct values of the triplicates were calculated. This was followed by the calculation of
∆Ctcpb2-Ctgyr A and the relative transcription of cpb2. These values were put in a
graph.
Gene
Primer code
Primer sequence(5’-3’)
Annealing
temperature
Expected length
PCR product
cpb2
beta2totalF2
beta2totalR
5’-AAATATGATCCTAACCAAMAA-3’
5’-CCAAATACTYTAATYGATGC-3’
48 C
525 bp
gyr A
gyrasAclosF
gyrasAclosR
5’-AAGAATAATAAGTTTGAGTGTG-3’
5’-CCCTTGATAATATTGATGATGT-3’
48 C
512 bp
Table 1: primers
M = A or C
Y = C or T
7
RESULTS
RNA isolation
The precipitation of RNA with isopropanol in the RNA isolation protocol according
to the manufacturers’ description is as followed: 10 minutes at room temperature.
Precipitation of RNA from identical C. perfringens samples, for 15 minutes at -20 C,
doubled the final concentration of RNA at the end of the procedure. All other steps in
the protocol were identical in both cases.
Quantitative PCR assay
By measuring the fluorescence of the amplicons during the PCR reaction we were
able to compare the relative transcription of cpb2 to the transcription of gyr A. The
mean Ct values of cbp2 and gyr A were calculated and used to calculate ∆Ctcpb2Ctgyr A. An example of these provisional results is given in figure 1. It became
apparent that the relative transcription of cpb2 increases at a certain pH, especially at
pH 7 compared to a lower pH.
relative transcription cpb2 to gyr A
Cp 15
relative transcription cpb2
1,00
0,80
0,60
0,40
0,20
0,00
5.0 5.1 5.2 5.3 5.4 6.0 6.1 6.2 6.3 6.4 7.0 7.1 7.2 7.3 7.4
sample: pH and time
Figure 1 Relative transcription cpb2 of Cp 15.
8
The melt curve shows a clear peak around 80 C (Figure 2). UV transillumation, after
electrophoresis, did not show other products then bands at the height that is an
indication for the PCR products of cpb2 or gyr A.
Meltcurve q-PCR Cp15
Figure 2: Meltcurve q-PCR Cp15
9
DISCUSSION
The production of the β2-toxin by C. perfringens is probably influenced by the
environmental conditions in which the bacteria are growing, for example the pH.
Western blot experiments showed that the amount of β2-toxin produced, differs when
growing the bacteria at different pH. This leads to that hypothesis that the production
of the β2-toxin depends on the pH in the environment of C. perfringens. The purpose
of this study was to determine the role of the pH on the transcription of cpb2. By
using the q-PCR technique we were able to distinguish changes in the transcription of
cpb2 when growing C. perfringens under various pH.
Performing the experiments we were faced with some technical problems leading to
inconsistent q-PCR results. The first problem was the low output of RNA after the
RNA-isolation. By performing the RNA precipitation step at -20 C for 15 minutes,
the final concentration of RNA almost doubled. According to the protocol the
incubation should by at 15-30 C for 10 minutes. All other steps during the procedure
were performed identical in both cases. Secondly, the inactivation of DNase I
(Fermentas) by adding 1 l 25mM EDTA and heating the samples at 65 C for 10
minutes, according to the manufacturers’ description, proved to be insufficient to stop
degradation of DNA. This was the case using DNase I from Bovine pancreas as well
as with recombinant DNase I (Fermentas). In our experiment the next step would be
the preparation of cDNA. If there is still some DNase activity left this could lead to
the degradation of the cDNA as well. And this might influence the results obtained
further along in the experiments, like the results of the Q-PCR. However, in the
preparation of cDNA there are several heating steps that might inactivate the DNase.
This is the reason why we have decided to continue with the Fermentas DNase I-kit.
We have also performed inactivation experiments with different concentrations of
EDTA and by raising the concentration of EDTA to 0.2M we showed that the DNase
activity was stopped. Further research can be done to determine what the minimum
concentration of EDTA is to inactivate the DNase.
With the Q-PCR technique we were able to identify the changes in transcription of
cpb2 when growing C. perfringens under various pH circumstances. After calculating
the ∆Ctcpb2-CtgyrA- value, the relative expression of cbp2 compared to gyr A under
different pH circumstances, was visualised in a graph. The provisional Q-PCR results
show that the pH of the growing medium does effect the transcription of cpb2
compared to gyr A in C. perfringens strain 15. At pH 7 the relative transcription of
cpb2 increases, compared to the relative transcription at pH 5. The fact that there are
these differences underlines the hypothesis that the β2-toxin production by Cl.
perfringens is regulated at the level of transcription. And it shows that the pH in the
environment is at least one of the factors that is involved in the regulation of the
production of the β2-toxin and thus in the development of disease. However, further
optimizing the q-PCR assay is essential, as it was not possible to have detectable Ctvalues for cpb2 in other strains than Cp15. This could be due to difference between
strains itself, as they manifest differences in growing curves. Since the Ct-values have
been very high (Ct>30) for cbp2, an option could be to raise the input of cDNA in the
q-PCR assays, this might lower the Ct-values to a detectable level.
10
ACKNOWLEDGEMENTS
I would like to thank everyone in the SIB group for their help and guidance in my first
steps in the wonder world of research. And I would like to thank Peter Cornelissen for
his help with the Q-PCR technique.
11
REFERENCES
1. van Asten, A.J.A.M., Georgios, N.N., Gröne, A., (2008) The occurrence of
cpb2-toxigenic Clostridium perfringens and the possible role of the -2 toxin
in enteric disease of domestic animals, wild animals and humans. The
Veterinary Journal.
2. Bueschel, D.M., Jost, B.H., Billington, S.J., Trinh, H.T., Songer, G., (2003)
Prevalence of cpb2, encoding beta2-toxin, in Clostridium perfringens field
isolates: correlation of genotype wih phenotype, Veterinary Microbiology 94:
121-129.
3. Gibert, M., Jolivet-Renaud, C., Popoff, M.R., (1997) Beta2 toxin, a novel
toxin produced by Clostridium perfringens, Gene 203: 65-73.
4. Hendriksen, S.W.M, van Leengoed, L.A.M.G, Roes, H.I.J., van Nes, A.,
(2006) Neonatale diarree bij biggen: α- en β2-toxine producerende
Clostridium perfringens, Tijdschr Diergeneeskd 131: 910-913.
5. Klaassen, H.L.B.M, Molkenboer, M.J.C.H., Bakker, J., Miserez, R.,Häni, H.,
Frey, J., Popo¡, M.R., van den Bosch, J.F., (1999) Detection of the 2 toxin
gene of Clostridium perfringens in diarrhoeic piglets in The Netherlands and
Switzerland, FEMS Immunology and Medical Microbiology 24: 325-332.
6. Livak, K.J., Schmittgen, T.D., (2001) Analysis of Relative Gene Expression
Data Using Real-Time Quantitative PCR and the 2-∆∆Ct Method, METHODS
25, 402-408.
7. Ohtani, K., Kawsar, H.I, Okumura, K., Hayashi, H., Shimizu, T, (2003) The
VirR/ VirS regulatory cascade a¡ects transcription of plasmid-encoded
putative virulence genes in Clostridium perfringens strain 13. FEMS
Microbiology Lett. 222:137-141.
8. Petit, L., Gibert, M., Popoff, M.R., (1999) Clostridium perfringens: toxinotype
and genotype. Trends in Microbiology 104 Vol. 7 No. 3.
9. Schotte, U., Truyen, U., Neubauer, H., (2004) Significance of b2-Toxigenic
Clostridium perfringens Infections in Animals and Their Predisposing Factors
– A Review, J. Vet. Med. B 51, 423–426.
10. Smedley, J. G., Fisher, D.J., Sayeed, S., Chakrabarti, G., McClane, B.A.,
(2004) The enteric toxins of Clostridium perfringens. Rev Physiol Biochem
Pharmacol 152:183–204.
11. Songer, G.J., Uzal, F.A., (2005) Clostridial enteric infections in pigs, J Vet
Diagn Invest 17:528–536.
12. Vilei, E.M., Schlatter, Y., Perreten, V., Straub, R., Popoff, M.R., Gibert, M.,
Gröne, A. Frey, J., (2005) Antibiotic-induced expression of a cryptic cpb2
12
gene in equine b2-toxigenic Clostridium perfringens, Molecular Microbiology
57(6), 1570–1581.
13. Waters, M., Raju, D., Garmory, H.S., Popoff, M.R., Sarker, M.R., (2005)
Regulated Expression of the Beta-2 Toxin Gene (cpb2) in Clostridium
perfringens Type A Isolates from Horses with Gastrointestinal Diseases.
Journal of Clinical Microbiology, Aug. 4002-4009.
14. Waters, M., Savoie, A. , Garmory, H.S., Bueschel, D., Popoff, M.R., Songer,
J.G, Titball, R.W., McClane, B.A., Sarker, M.R., (2003) Genotyping and
Phenotyping of Beta2-Toxigenic Clostridium perfringens Fecal Isolates
Associated with Gastrointestinal Diseases in Piglets, Journal of clinical
microbiology, Vol. 41 No. 8.
13
APPENDICES
Appendix I: RNA isolation
1. Homogenization:
Lyse the cells in TRIzol Reagent (Invitrogen) by repetitive pipetting. Use 1 ml
TRIzol per 1 x 10^6 bacterial cells.
2. Phase separation:
a. Incubate the homogenized samples for 5 minutes at 15 to 30C
b. Add 0.2 ml of chloroform per 1 ml TRIzol Reagent. Cap sample tubes
securely and shake the tubes vigorously by hand for 15 seconds.
c. Incubate the samples for 2-3 minutes at 15-30C.
d. Centrifuge the samples at 12.000 x g for 15 minutes at 2 to 8C.
After centrifugation, the mixture separates into a lower red, phenol-chloroform
phase, an interphase, and a colourless upper aqueous phase. RNA remains in
the aqueous phase.
3. RNA precipitation:
a. Transfer the upper aqueous phase, containing the RNA, to a fresh
eppendorf tube.
b. Mix with 0.5 ml isopropanol per 1 ml TRIzol Reagent used for initial
homogenization in order to precipitate the RNA.
c. Incubate the samples for 15 minutes at -20 C.
d. Centrifuge at 12.000 x g for 10 minutes at 2 to 8C.
After centrifugation the RNA precipitate forms a gel-like pellet on the side and
bottom of the tube.
4. RNA wash:
a. Remove the supernatant.
b. Wash the pellet with 70% ethanol, adding at least 1 ml of 70% ethanol per
1 ml TRIzol Reagent used for initial homogenization.
c. Mix the sample.
d. Centrifuge at 16.100 x g for 10 minutes at 2 to 8C.
Repeat the washing step once again.
5. Redissolving the RNA:
a. Remove the supernatant.
b. Air-dry the RNA pellet.
c. Redissolve the pellet in 100 l aquadest by passing the solution a few
times through a pipette tip.
d. Incubate for 10 minutes at 55 to 60C.
14
Appendix II: protocol for the preparation of DNA-free RNA prior to RT-PCR
1. Add to a RNase-free tube:
- Total RNA: 1 g
- 10X reaction buffer with MgCl2: 1 l
- Aquadest: to 9 l
- DNase I, RNase-free: 1l (1u/ l)
2. Incubate at 37C for 30 minutes
3. Add 1l 25 mM EDTA and incubate for 65C for 10 minutes.
4. Use the prepared RNA as a template for reverse transcriptase.
15
Appendix III: RT-PCR
1. Prepare mixture for PCR using Taq polymerase (Fermentas) containing:
- Sample
1.0 l
- Forward primer 100ng/µl
1.0 l
- Reverse primer 100ng/µl
1.0 l
- dNTPs 10mM
1.0 l
- Taq polymerase 5U/µl
0.2 l
- Buffer + KCl 10X
5.0 l
- MgCl2 25mM
3.0 l
- Aquadest
37.8 l
Total volume
50.0 l
2. Mix the samples gently and spin down.
3. PCR protocol
1 cycle:
Initial denaturation
35 cycles:
Denaturation
Annealing
Elongation
1 cycle
Final elongation
5. After PCR the sample can be used for gel electrophoresis.
95C, 2 minutes
95C, 30 seconds
48C, 30 seconds
72 C, 30 seconds
72C, 7 minutes
16
Appendix IV: First strand cDNA synthesis
1. RevertAid First strand cDNA Synthesis Kit (Fermentas). Thaw, mix and
briefly centrifuge the components of the kit. Store on ice.
2. Add the following reagents into a sterile, nuclease-free tube on ice in the
indicated order.
- Total RNA
0.1-0.5 g
- Random hexamer primer 100µM
1 l
- DEPC-treated water
to 12 l
Total volume
12 l
3. Mix gently, centrifuge briefly and incubate at 65 for 5 minutes. Chill on ice ,
spin down and place the vial back on ice.
4. Add the following components in the indicated order:
- reaction buffer 5X
4 l
- RiboLock RNase Inhibitor (20 u/ l)
1 l
- dNTP mix10 mM
2 l
- RevertAid M-MuLV Reverse Transcriptase (200 u/ l)
1 l
Total volume
20 l
5. Mix gently and centrifuge.
6. Incubate for 5 minutes at 25C, followed by 60 minutes at 42C.
7. Terminate the reaction by heating at 70C for 5 minutes.
17
Appendix V: Quantitative PCR
1. Dilute the cDNA with aquadest to a concentration of 5ng/ l cDNA.
2. Pipette per sample 5 l of the diluted cDNA in the well of the 96-wells
PCR plate.
3. Add to each well 20 l of the mastermix. Prepare a mastermix for the
number of samples and negative control in triplicates.
Components mix per reaction:
- IQ SYBR Green Supermix (BioRAD)
- Primermix 1.5 M
- Aquadest
Total volume mix
Sample
Total volume per reaction
12.5 l
3.0 l
4.5 l
20 l
5 l
25 l
4. Cover the PCR plate with the adhesive seal.
5. Enter the 96-well plate in the Q-PCR machine, close the lid and enter the
q-PCR protocol.
Q-PCR protocol
- 1 cycle
Initial denaturation
- 40 cycles
Denaturation
Annealing
Elongation
- 1 cycle
- 1cycle
95C, 3 minutes
95C, 30 seconds
48C, 30 seconds
72C, 30 seconds
95C, 1 minute
65C, 1 minute
Melt curve: 60 cycles, 10 seconds, +0.5C
6. Select or edit the plate setup.
7. Fill in the total volume per reaction and start the run.
18