Determination of Lytic Abilities in a Clostridium Septicum Alpha Toxin Mutant

 

 

 

 

 

 

 

 

 

Submitted to State Oklahoma Junior Academy of Science

April 1-2 2004

Alva High School

Elizabeth Rathgeber

Abstract

            Clostridium septicum is a gram-positive, anaerobic organism located widely throughout the soil and in animals, such as humans and horses. Clostridium septicum alpha toxin is a lethal protoxin which vacuolates victim cells, resulting in death through cell lysis within less than 24 hours. The purpose of this research project was to determine if the loop region in domain two of C. septicum alpha toxin is necessary for pore formation. By changing the serines, on the nucleotides 220 and 269, to cystines by using multiple Polymerase Chain Reactions (PCR), an alpha toxin mutant (S220C/S269C) was generated. By doing this, a disulfide bond (a reversible covalent bond) would be created. The disulfide bond was formed in hopes of locking down the Trans Membrane Domain, located in Domain two, which was believed to be the cause of lysis. Alpha toxin mutant proteins that were structurally sound were grown up to a large scale of 10 L for purification. The purified protein was run through gels to determine if it would oligomerize on victim cell membranes. The mutant was added with red blood cells and ran in a hemolytic assay to determine the lysing abilities of this mutant. Dithiothreitol (DTT) was also added to the mutant, in hopes to reduce the disulfide bond. The mutant (S220C/S269C) was found to obtain no lysing abilities, but when DTT was added to the mutant, it regained all of the lysing abilities. In conclusion, DTT was found to reduce the disulfide bond that was generated, and that the S220C/S269C mutant possessed no lysing abilities.

 

 

 

Introduction

 

            Clostridium septicum is an anaerobic, motile, gram-positive organism that produces spore-forming rods and powerful exotoxins (1). C. septicum is ubiquitous in soil, and other places where it is found include the intestinal tracts and feces of humans, and also iatrogenic infections (which are unfavorable responses to therapy, mostly found in horses) (1, 2). C. septicum can cause gas gangrene, which is a common gas producing CO2/H2 (1). The incubation period for gas gangrene infection takes several days, and follows direct contamination of a traumatic wound, such as castration or docking (amputation of the base of a horse’s tail), genital infections, and failed delivery (2). The infection spreads along the fascial (skin tissue) planes, and includes symptoms such as bloody exudates, edema, muscle liquefaction, hemorrhage, and necrosis (1, 2). Lesions develop, which soon become crepitant (sensation occurring when possessing gas gangrene), cold with a loss of feeling, followed by fever, anorexia, finally ending in death within less than 24 hours through cell lysis (1, 2).

            Twenty percent of C. septicum is an alpha toxin, which is often used as a research tool for studying lysing and vacuolating characteristics of other toxins (such as Toxoplasma gondii GPI tachyzoites), and finding a possible alpha-toxoid vaccine in guinea pigs (1, 3-5). Alpha toxin is a lethal protoxin, which generally is hemolytic, a pore former, and is able to necrotize and vacuolate (2, 3). Alpha toxin binds to receptors via amino acids in Domain 1 through the Receptor Binding Domain by a proteolytic cleavage, which then releases a 45 amino acid fragment, which is believed to be the Trans Membrane Domain (TMD) (6). Alpha toxin then oligomerizes into a membrane-bound pre-pore complex and forms pores at temperatures ≥ 25°C (7). A relative to C. septicum, C. perfringens beta toxin displays neurological effects, while C. septicum alpha toxin produces a rapid-shock like effect (8). Both toxins, however, show effects that copy characteristics of both human and animal diseases (8). Alpha toxin is also able to increase capillary permeability and myonecrosis (which encourages the spread of infection) (2). Alpha toxin therapy is commonly ineffective, while an immunization with bacterial toxoids produces a lifelong immunity (2).

            The purpose of this research project was to determine if the movement of the loop region in Domain 2 of Clostridium septicum alpha toxin was necessary for the pore formation of victim cells. The working hypothesis was that the movement of the loop region in Domain 2 in C. septicum alpha toxin was required for pore formation in victim cells.

 

Procedure

 

To determine if the movement of the loop region of Domain 2 was necessary for pore formation, and to attempt to prevent cell lysis in victim cells, the following procedure was conducted:

Bacterial strains, plasmids, cell lines, and chemicals: The gene for alpha toxin (AT) was cloned into the pET-22(b)+ expression vector (Novagen) (designated pBRS10) and placed into either E. coli XL1-Blue, for cloning purposes, or BLR-DE3 cells for high-level expression. All chemicals were obtained from Sigma Chemical Company (St. Louis, MO) and all enzymes form Gibco BRL (Rockville, MD) unless otherwise specified.

Generation of S220C/S269C: The point mutations S220C/S269C were produced using Quik-Change PCR mutagenesis according to manufacturer’s instructions (Stratagene). A PCR reaction containing 50 ng template DNA (pBRS10), 5 µl 10x reaction buffer, 1 µl dNTPs (final concentration of each nucleotide at 10 mM), 10 ng mutagenic primer 1 (list primer), 10 ng mutagenic primer 2 (list primer), 1 µl PfuTurbo DNA Polymerase (Stratagene), and water for a final volume of 20 µl, was placed into a PCR machine and run through 15 rounds of the following cycles: 95şC denaturing (30 sec), 55şC primer annealing (1 min), 68şC extension (9 min). Following PCR, 1 µl Dpn1 was added to all reactions and incubated at 37şC for 1 hour. Ten µl of each reaction was then mixed with 5 µl sample buffer and all samples were run on a 1% agarose gel for visualization of product DNA. One µl from a PCR reaction containing visible product was used to transform supercompetent E. coli XXL1-Blue cells. Transformant cells were individually grown-up and harvested for DNA purification with Wizard Mini-Prep purification kits (Promega), and the DNA was used for sequencing or transformation of E. coli Tuner DE3 cells.

Mini-Inductions of AT: To monitor for proper expression and stability of S220C/S269C, individual transformed Tuner DE3 colonies were grown in roller tubes containing 10 ml sterile terrific brothe (TB) broth and 200 µg/ml Ampicillin at 37şC until an OD of 1.0 was reached. The cells were then induced for expression of the AT gene with the addition of 0.2 mM isopropyl-#946; -D-thiogalactopyranoside (IPTG) for 2 hours at 37şC. The cells were then harvested and resuspended in 1 ml phosphate buffered saline (PBS). 30 µl of the cell suspension was mixed with 6µl SDS-Sample buffer (6X), boiled for 2 min at 95şC, and separated on a 10% SDS-PAGE gel. The proteins were transferred to nitrocellulose paper and the blot was incubated with affinity purified anti-AT antibody. After 1 hr, unbound primary antibody was removed by washing the blot three times in

blot wash (10 mM Tris-HC1, 150 mM NaCl, .05% Tween 20, pH 8.0) and then secondary antibody conjugated to horseradish peroxidase was added to the blot and incubated for an additional 45 min. The blot was again washed three times in blot was to remove unbound antibody. Colorimetric development of the bands recognized by the antibody was accomplished by developing the blot with the color development solution 4-chloro-1-naphthol (4CN) according to manufacturer’s instructions (Bio-Rad).

Expression and Purification of AT: For large scale purification of S220C/S269C, E. coli Tuner DE3 harvesting pBRS10 with the S220C/S269C mutations was initiated by inoculating 10 L sterile TB containing 200 µg/ml ampicillin with a 1:33 inoculum of an overnight culture. The culture was incubated at 37şC with constant sterile aeration until an OD of 1.0 was obtained. The culture was induced for expression of the AT gene with 0.2 mM IPTG for 2 hours. Cell pellets were harvested by centrifugation and stored overnight at -80şC. The cell pellets were resuspended in 150 ml of buffer A (10 mM MES {2-[N-morpholino] ethanesulphonic acid} {Research Organics, Cleveland, OH}, 150 mM NaCl, pH 6.5). Lysis of cells was carried out in an EmulsiFlex-C5 high pressure homogenizer (Avestin, Ottawa, ON, Canada) at 15,000 psi. Cell debris was pelleted by centrifugation at 21,000 x g for 10 min. The AT-containing supernatant was loaded onto a column of chelating Sepharose Fast Flow (Pharmacia), preloaded with Co˛+. The column was washed with 130 ml buffer A containing 5 mM imidazole. AT-containing fractions were pooled and loaded onto a cation-exchange column packed with SP Sepharose HP (Pharmacia) equilibriated in 10 mM MES, 1 mM EDTA, pH 6.5. The desired protein was eluted form the column with a 315 ml linear gradient from 0.15-0.50 M NaCl in the same buffer. AT-containing fractions eluted from the cation-exchange column were combined in a Micro-ProDiCon System (Spectrum, Gardena, CA) with a 10,000 MWCO Micro-ProDiCon membrane for simulation dialysis and concentration. Samples were dialyzed against 10 mM MES, 500 mM NaCl, 1 mM EDTA, pH 6.5 (buffer B) overnight at 4şC. For cystine-substituted proteins, 1 mM dithiothreitol was included in the dialysis buffer. 10% glycerol was added to the concentrated toxin before storage at -80şC. Protein concentration was determined by absorbance at 280 nm using a molar extinction coefficient of 63,000 Mˉą cmˉą (unpublished data).

Expression and Purification of AT cont.: The growth and harvesting of E. coli BLR-DE3 carrying expressing native AT and the various AT derivates was done according to Sellman et al. (Sellman et al., 1997). The cell pellets were resuspended in 150 ml of buffer A (10 mM MES {2-[N-morpholino] ethanesulphonic acid} {Research Organics, Cleveland, OH}, 150 mM NaCl, pH 6.5). Lysis of cells was carried out in an EmulsiFlex-C5 high-pressure homogenizer (Avestin, Ottawa, ON, Canada) at 15,000 psi. Cell debris was pelleted by centrifugation at 21,000 x g for 10 min. Purification of AT from the supernatant over a cobalt-chelating column and cation-exchange column was done as previously described (Sellman et al., 1997). AT-containing fractions eluted from the cation-exchange column were combined in a Micro-ProDiCon System (Spectrum, Gardena, CA) with a 10,000 MWCO Micro-ProDiCon membrane for simultaneous dialysis and concentration. Samples were dialyzed against 10 mM MES, 500 mM NaCl, 1 mM EDTA, pH 6.5 (buffer B) overnight at 4şC. For cystine-substituted proteins, 1 mM dithiothreitol was included in the dialysis buffer. 10% glycerol was added to the concentrated toxin before storage at -80şC. Protein concentration was determined by absorbance at 280 nm using a molar extinction coefficient of 63,000 M-1 cm-1 (unpublished data).

Activation and Oligomerization of S220C/S269C on SupT1 Membranes: Wild type and mutation protoxin (ATpro) (5 µg) was activated using trypsin at a ratio of 1:1000 trypsin: toxin (w/w). The mixture was incubated at 37şC for 30 min and the trypsin was inhibited by the addition of a 30-fold molar excess of the protease inhibitor TLCK (tosyllysine chloromethyl ketone). SupT1 membranes (10 µl) were added to the activated toxin and the volume was adjusted to 30 µl with PBS and incubated at 37şC for 2 hr. SDS sample buffer (8 µl 6X) and 7 µl of 10% SDS were added to the samples, boiled at 90şC for 2 min and separated on a 4-15% gradient gel. The proteins were transferred to nitrocellulose paper and the blot was incubated with affinity purified anti-AT antibody. After 1 hr, unbound primary antibody was removed by washing the blot three times in buffer D (10 mM Tris-HC1, 150 mM NaCl, .05% Tween 20, pH 8.0) and then secondary antibody conjugated to horseradish peroxidase was added to the blot and incubated for an additional 45 min. The blot was again washed three times in buffer D to remove unbound antibody. Colormetric development of the bands recognized by the antibody was accomplished by developing the blot with the color development solution 4-chloro-1-napthol (4CN) according to manufacturer’s instructions (Bio-Rad, Hercules, CA).

Hemolytic Assay: 10 ml of 0.1% trypsin-PBS was made by adding 1 µl trypsin (1 mg/ml) to 10 ml 1X PBS, then 50 µl was filled in the appropriate wells of a round-bottom 96-well plate. 2 µg of toxin plus 1 X PBS was added to the first well in a row equaling 50 µl with the entire well equaling a total volume of 100 µl. 2X serial dilution was carried out in wells 1-12 by taking 50 µl out of well #1, mixing it with well #2, then taking 50 µl out of well #2, mixing it with well #3, etc. through remaining wells. The remaining 50 µl was discarded after well #12. Fifty µl of 5% blood was added to all wells, covered, and then incubated at 37şC for 1 hr.

HD50  Determination: The Hemolytic Assay plate was spun at 6000 X g for 5 minutes to pellet any unlysed erythrocytes. 25 µl supernatant was removed from each well and placed into the wells of a different flat-bottomed 96-well plate containing 75 µl 1X PBS in all wells, then the plate was placed into a spectrophotometric plate reader and read at A540 to quantify hemoglobin release. A540 values were plotted (y-axis) against well number (x-axis) and produced a linear graph. A540 was determined at 50% by dividing the highest A540 value for wild-type toxin in half, then the A540 was found at 50% for all toxins and the value was determined on the x-axis at the same point.

Statistical Procedure 1:     HD50= (2well#) (1/x) (2µg).

Statistical Procedure 2:     Percent hemolysis= (HD50 wild type ÷ HD50

toxin tested) (100).

 

 

 

 

 

 

Equation 1

 

            Equation 1 shows the t-test that was used to determine whether a statistical difference existed between the amount of lysed red bloods cells without DTT added and the amount of lysed red blood cells with DTT added. In the equation X represented the wells (letters A-F of a 96 well plate) that did not contain any DTT, while Y represented the wells that did contain DTT, and n represented the sample size. Three different tests were run with PBS, wild type alpha toxin, and the S220C/S269C mutant, each with two rows of wells containing either no DTT, or 5mM of DTT. Negative values were calculated as zero, since it is impossible to have negative lysed red blood cells.

Results

Data Table 1: A540 readings from hemolytic assay plate

 

1

2

3

4

5

6

7

8

9

10

11

12

A

Blank

Blank

Blank

-0.012

-0.012

-0.014

-0.007

-0.012

-0.014

-0.021

-0.019

-0.015

B

0.263

0.258

0.222

0.222

0.168

0.103

0.055

-0.025

-0.025

-0.029

-0.030

-0.027

C

-0.012

-0.024

-0.016

-0.030

-0.029

-0.029

-0.024

-0.030

-0.028

-0.031

-0.035

-0.032

D

-0.033

-0.035

-0.028

-0.032

-0.031

-0.029

-0.025

-0.029

-0.029

-0.031

-0.035

-0.032

E

0.257

0.213

0.213

0.225

0.172

0.172

0.123

0.092

0.052

-0.030

-0.035

-0.031

F

0.269

0.245

0.280

0.195

0.111

0.046

0.111

-0.026

-0.025

-0.031

-0.033

-0.031

 

Table 1 shows the wavelengths (shown above in nm) read on a spectrometer set at A540nm of the lysed red blood cell concentration. The different well letters contained different substances, corresponding below. The different well numbers were the number of wells that contained each substance. The values were read in nm. The values with – proceeding were values that read negative on the spectrometer, meaning that no lysed products were detected. The first three Blank wells were wells that were read on the spectrometer and averaged to obtain the spectrometer “key” to read all of the wells set to this value. The “Blanks” were first read as following:  A1 = 0.119nm, A2 = 0.109nm, A3 = 0.116nm. The average of the three first wells was 0.114 nm. The well rows were filled with the following solutions:

            A: PBS solution without DTT added

            B: Wild Type AT without DTT added

            C: S220C/S269C mutant without DTT added

            D: PBS solution with DTT added

            E: Wild Type AT with DTT added

            F: S220C/S269C mutant with DTT added.

The A540 well containing 100% Hemolysis was found at well B1 (AT without DTT), and read on the spectrometer at 0.263 nm. The A540 value of 50% Hemolysis was 0.132, and this value was obtained by dividing the well with 100% Hemolysis by 2. This value was used to find the well containing 50% Hemolysis for all of the toxins.

LEAD Technologies Inc. V1.01

Figure 1

Figure 1 shows the graph of the hemolysis of red blood cells with the S220C/S269C mutant containing no DTT compared to the S220C/S269C mutant with the addition of DTT. Figure 1 shows that the mutant S220C/S269C without DTT (line marked with the ‘squares’) did not show any lysing from the spectrometer readings. The S220C/S269C mutant with the addition of DTT (the line marked with the ‘Xs’) gained back its lysing abilities, which was proven by the hemolytic assay. In addition, Figure 1 also compared the wild type alpha toxin without DTT (line marked with ‘circles’) to the wild type alpha toxin with DTT (line marked with ‘diamonds’).

 

Figure 2

 

            Figure 2 shows the results of a two tailed t-test that was calculated when the amount of lysed red blood cells without 5 mM DTT was compared to the amount of lysed red blood cells with 5 mM DTT. The value of –2.843 was calculated for the S220C/S269C mutant, the value of –0.569 was calculated for the wild type AT, and the value of zero was calculated for the PBS solution. Since the only value falling into the critical region was the S220C/S269C mutant, the working hypothesis, which stated that the movement of the loop region of Domain two in C.S. alpha toxin was required for pore formation, was accepted with a 95% level of confidence.

Discussion

            A statistical difference was found to exist between the percent of lytic activities of the S220C/S269C mutant containing no DTT and the S220C/S269C mutant containing DTT. A possible explanation for this difference in lysation could be that Aeromonas hydrophilia aerolysin is a toxin that has been proven in studies to be 72% similar to alpha toxin in the primary sequences, and possesses the same lysing abilities (10-12). The loop region of aerolysin has been proven in studies to be critical and important for pore formation and lytic, and supports the working hypothesis where the loop region in alpha toxin was required for pore formation (6).

 Another possible explanation for this difference in lytic percents could be that DTT is a reducing agent commonly used to reduce disulfide bonds containing two cystines, and in these results, DTT was proven to be a reducing agent for S220C/S269C (12).

            In conclusion, a statistical difference was found to exist in the lysing abilities between the mutant S220C/S269C and the mutant S220C/S269C containing DTT. The TMD was crucial in the alpha toxin lysing abilities, and a disulfide bond was able to halt these abilities.

Acknowledgements

 

            I would like to thank the following people for making this possible: First of all, Jody Melton for allowing me to spend time researching with her and allowing me to move in for a week; University of Oklahoma Health Sciences Center and Dr. Rodney K. Tweten for graciously allowing me to use and experiment with their fine equipment; My dad and Mark Smithey for teaching me how to do the advanced graphs and such on the computer; Mrs. Angle for her brilliant revising ideas; and last but not least, Sushi NeKo restaurant for the mind fuel during my research time.

 

References

 

1.   Hanne, Larry “Biology Lecture 29” 4 Nov. 2002: n.pag. On-Line. Internet 4 Sep. 2003 Available WWW. http:llwww.csucbico.edu/biollpersonnellHanne/MedBactNoteslLect29Clostridium .html

 

2.   Songer, Dr. Glenn “Pathogenic Bacteriology Lecture Notes” No Date n.pag. On-Line. Internet. 4 Sep. 2003 Available WWW:

http://www.microvet.arizona.edu/CoursesIMIC42O/lecture notes/clostridia/cl ostridia histotoxic/c septicum.html

 

3.   Wichroski, Michael J.; Melton, Jody A.; Donahue, Carolyn G.; Tweten, Rodney K.; Ward, Gary E. “Clostridium Septicum alpha-toxin is active against Toxoplasma tachyzoites and binds to SAG proteins” MPM 2001 Abstract #238B n.pag. On-Line Internet 20 Sep. 2003 Available: http://e2kroos.cis.unenn.edu/mpm-2001/abstacts/abstract.php3?id=385

 

4.   Wichroski, Michael J.; Melton, Jody A.; Donahue, Carolyn G.; Tweten, Rodney K.; Ward, Gary E. “Clostridium septicum Alpha-Toxin is active against the parasitic protozoan Toxoplasma gondii and targets members of the SAG family of Glycosylphosphophatidylinositol-Anchored surface proteins” Infection and Immunity Aug. 2002; Vol. 70, No. 8; 4353-4361 On-Line Internet 3 Sep. 2003 Available WWW. http://ncbi.n1m.nih.2ov/entrez/puery.fc2i?cmd=Retrieve&dp=PubMed&list uids=12117945&dopi=Abstract

 

5.   Amimoto, Katsuhiko; Ohgitani, Toshiaki; Sasaki, Osamu; Oishi, Eiji; Katayama, Shigeji; Isogai, Makoto; Ota, Sotoyuki “Protective Effect of Clostridium septicum alpha-toxin vaccine against challenge with spores in Guinea Pigs” 2002; Vol. 64, No. 1; pp.67-69 On-Line Internet 22 Sep. 2003 Available WWW: http://jvms.jstage.j st.go.jp/cgi-­bin/rs.c2i?SID=f5fefd39efdl7b2949e749d6ede4f0a5

 

6.   Gordon, V. M.; Benz, R.; Fujii, K.; Leppla, S. H.; Tweten, R. K. “Clostridium septicum alpha-toxin is proteolytically activated by furin” Infection and Immunity Oct. 1997; Vol. 65, No. 10; 4130-4134 On-line Internet 5 Sep. 2003 Available WWW: http://iai.asm.or2/c2i/content/abstract/65/1O/4130

 

7.    Selleman, B. R.; Kagan B. L.; Tweten, R. K. “Generation of a membrane-bound, oligomerized pre-pore complex is necessary for pore formation by Clostridium Septicum alpha toxin” Mol. Microbiol Feb. 1997; Vol. 23, No. 3, 551-8 On­Line Internet 22 Sep. 2003 Available WWW: http://ncbi.nlm.nih.2ov/entrez/uuerv.fc2i?cmd=Retrieve&db=pubMed&list.uids9044288&doDt=Abstract&itool=iconabstr

 

8.   Tweten, R. K. “Clostridium perfringens beta toxin and Clostridium septicum alpha toxin: their mechanisms and possible role in pathogenesis” Vet Microbiol 3 Sep. 2001 Vol. 82, No. 1, 1-9 On-Line Internet 5 Sep. 2003 Available WWW: http://chaos.swmed.edu/c2i-bin/etblast/abstract?pmid=1 1423190&user id= 1023227436&iteration id=10 23227436&user name=kgoble

 

9. *  Gordon, Valery M.; Nelson, Kim L.; Buckley, J. Thomas; Stevens, Victoria L.; Tweten, Rodney K.; Elwood, Patrick C.; Leppla, Stephen H. “Clostridium septicum alpha toxin uses Glycosylphosphophatidylinositol-anchored Protein Receptors” The Journal of Biological Chemistry Sept. 17 1999 Vol. 274 No. 38 pp.27274-27280 13 Nov. 2003 Available: The Journal of Biological Chemistry

 

10. *  Hong, Yeongjin; Ohishi, Kazuhito; Norimitsu, Inoune; Kang, Ji Young; Shime, Hiroak; Horiguchi, Yasuhiko; Goot, F. Gisou van der; Sugimoto, Nakaba; Kinosita, Taroh “Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum alpha toxin” The EMBO Journal 2002 Vol. 21 No. 19 5047-5056 13 Nov. 2003 Available: The EMBO Journal

 

11. * Ballard, Jimmy; Crabtree, Judy; Roe, Bruce A.; Tweten, Rodney K. “The Primary Structure of Clostridium septicum Alpha Toxin Exhibits Similarity with that of Aeromonas hydrophilia Aerolysin” Infection and Immunity Jan. 1995: 340-344 13 Nov. 2003 Available: American Society for Microbiology

 

12. *  Singh, R. Rajesh; Chang, Jui-Yoa “Structural stability of human alpha-thrombin studied by disulfide reduction and scrambling” Biochimica et Biophysica Acta (BBA)-Proteins & Proteomics 23 Sept. 2003 Vol. 1651 Issues 1-2 pages 85-92 30 Nov. 2003 Available: Biochimica et Biophysica Acta (BBA)