Comparison of alpha toxin gene among different types of Clostridium perfringens

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1 Comparison of alpha toxin gene among different types of Clostridium perfringens (Received: ; Accepted: ) M.M. Effat* and Y.A. Abdallah** * Microbiology and Immunology Department National Research Centre, Giza, Egypt ** Veterinary Serum and Vaccine Research Institute, Abbassia, Egypt, ABSTRACT Alpha toxin is found to be produced by all types of Clostridium perfringens. The genes encoding alpha toxin from the available five types of Clostridium perfringens [A I chicken strain), A (rabbit strain), B, C and D were PCR amplified using specific primers and the PCR products were examined on 1.5% (w/v) agarose gel and demonstrated the same bands comparable to the published alpha toxin gene. The PCR products of a toxin gene from the five types were separately sequenced and aligned with all published a toxin genes of Clostridium perfringens. Identities among all studied alpha toxin gene sequences and with the published ones were nearly 96-98%. There are no any significant differences among these nucleotide sequences. It is concluded that a toxin gem; sequences among different types of Clostridium perfringens are similar and highly conserved. Key words: Clostridium perfringens, Alpha toxin, phospholipase C, DNA sequencing. INTRODUCTION C lostridium perfringens, an anaerobic spore former rod is widely distributed in the environment and in the intestines of humans, animals and some wildlife (Nillo, 1993). Clostridium perfringens type A (Alpha toxin producer) is common in the intestinal tract of chicks, soil, dust contaminated feed and liter (EL-Seedy, 1990). The Alpha toxin is the principal lethal toxin of Clostridium perfringens is a multifunctional phospholipase produced by nearly all isolates. The toxin is haemolytic, necrotizing and potently lethal. The hydrolytic action of the toxin on membrane phospholipids found in the erythrocytes, platelets, leukocytes, endothelial cells and muscle cells results in lysis or other forms of cytotoxicity (Songer 1996). The alpha toxin gene (cpa) has been cloned and sequenced and homologous genes have been found in other clostridia. (Leslie et. al., 1989). Phospholipases are enzymes that degrade phospholipids and their classification is based on the site of cleavage. Phospholipase C cleaves between glycerol and the phosphate moieties. Phospholipases are found in all types of cells and various subcellular locations within eukaryotic cells (Zubay, 1988). Phospholipases may exert a direct biological effect on an animal since they can destroy the cell membrane phospholipids resulting in both cytotoxic and haemolytic effect. Moreover, subhaemolytic doses of phospholipases are capable of degenerating mast cells leading to local changes in vascular permeability and elevation in blood kinins and development of anaphlactoid syndrome (Tizard et. al., 1978). Phospholipases, exotoxins are virulence factors that damage the host, they hydrolyse

2 254 M.M. Effat and Y.A. Abdallah phospholipids in the host cell membrane leading to its disruption and killing the host cells by lysing them and aiding the phagocytosed bacteria to escape the phagocytic vesicle and enter the host cell cytoplasm (Salyers and Whitt, 1994). The five types of Clostridium perfringens could not be differentiated reliably on the basis of cellular or colonial morphology, biochemical reactions or gas liquid chromatographic analyses of fatty and organic acids and products of metabolism. Alpha toxin of Clostridium perfringens a key virulence determinant was suggested to be a cause of necrotic enteritis in chickens.analysis of alpha toxin of 25 chicken derived Clostridium perfringens strains demonstrated high homology to mammal derived strains rather than to the only avian Clostridium perfringens us alpha toxin sequence reported (Scot, et. al., 2004). Alpha toxin may be produced more by isolates from birds with necrotic enteritis than by isolates from normal birds (Hofshagen and Stenwig, 1992). Molecular structure of Clostridium perfringens alpha toxin revealed two domain protein; amino terminal domain containing phospholipase C active site and non-toxic carboxyterminal domain a paralogue of lipid binding domains (Titball, et al., 1999). Alpha toxin is produced by all types of Clostridium perfringens (Yoo et. al., 1997; Effat et. al., 2007). Genetic analysis of alpha toxin gene among different types of Clostridium perfringens is performed to check if it is conserved or not There is no any published article dealt with the study of DNA sequence among different types of this organism especially rabbit and chicken isolates of Clostridium perfringens type A. This study looked for the presence of any difference of the alpha toxin gene among different types of Clostridium perfringens. MATERIALS AND METHODS Five reference strains of Clostridium perfringens type A (Rabbit strain, Chicken strain); type B; type C; and type D used in this study, were provided by the Serum and Vaccine Research Institute, Abbassia, Egypt. Amplification of Alpha toxin gene among these 5 strains was performed using the following: The primer nucleotide sequences for Alpha toxin gene and the melting temperature (Tm) for each primer are as follows: Forward primer: 5' GTTGATAGCGC AGGACATGTTAAG 3' (Tm 61.0) Reverse primer: 5'CATGTAGTCATC TGTTCCAGCATC3' (Tm 61.0) Primers used in this study were designed according to (Yoo et. al., 1997) and obtained from Metabion International AG, Germany. Qiagen master mix was used to amplify the gene and the PCR solutions are: 25µl master mix, 2.0 µl forward primer (10 pmol each µl), 2.0 µl reverse primer (10 pmol each µl), 11 µl distilled water and 10 µl template (heat blocked supernatant of each type of Clostridium perfringens. PCR protocol The PCR thermal cycler was programmed for Clostridium perfringens as follows: Initial dentauration for 5 minutes at 94 C then 30 cycles each consists of dentauration at 94 o C for one minute, annealing at 55 C for one minute and extension at 72 C for one minute. The cycles were followed by final extension for 10 minutes at 72 C. The Program was adjusted at 4 C (as pause for keeping PCR product refrigerated) after ending the cycles and the final elongation (Yoo et. al., 1997), 10 µl from each PCR product were mixed with 2 µl loading buffer and analyzed on 1.5 % (w/v) agarose gel in IX TBE buffer and run along with 6 µl of 100 bp

3 Alpha toxin gene in C. perfringens 255 DNA ladder. Ethidium bromide was added to a final concentration 1:20,000 from a stock solution of 10 mg/ml (Maniatis et. al., 1982) the electric current volt was adjusted at 50. The gel was examined under UV transilluminator and the pictures were taken using normal digital Kodak camera. RESULTS AND DISCUSSION PCR amplification of alpha toxin gene Alpha toxin gene PCR amplification for the available 5 types of Clostridium perfringens was performed using a specific set of primers. Bands of PCR products for Clostridium perfringens types demonstrated the same as that published at nearly 402 bp (Yoo et. al., 1997). No nucleotide sequence differences inside the alpha toxin gene were detected and the gene seems conserved among the 5 types of Clostridium perfringens studied. It is concluded that all studied alpha toxin genes obtained by PCR amplification from the five types of Clostridium perfringens under study are the same. no significant difference in nucleotide sequence was seen and thus alpha toxin genes for the 5 studied types of that pathogens are highly conserved. bp Fig.( 1): exhibits PCR product of alpha toxin gene of different types of C. perfringens, lane l:100 bp ladder, lane 2: C. perfringens type A Rabbit strain, lane 3: C. perfringens type A; chicken strain, lane 4: C. perfringens type B, lane5: C. perfringens type C, and lane 6: C. perfringens type D. TTTAGCTTTGCGACGAAGAAACACAGTATAAAATAAACAGCAGGTTGCAAAACTAATGAGGCTTTTT ATACTGATATCTTAAAAAACAAAGATTTTAATGCATGGTCAAAAGAATATGCAAGAGGTTTTGCTAA AACAGGAAAATCAATATACTATAGTCATGCTAGCATGAGTCATAGTTGGGATGATTGGGATTATGCA GCAAAGGTAACTTTAGCTAACTCTCAAAAAGGAACAGCGGGATATATTTATAGATTCTTACACGATG TATCAGAGGGTAATGATCCATCAGTTGGAAAGAATGTAAAAGAACTAGTAGCTTACATATCAACTAG TGGTGAGAAAGATGCTGGAACAGATGACTACATGTATTTTGG Fig. (2): Nucleotide sequence of alpha toxin gene of C. perfringens type A chicken strain.

4 256 M.M. Effat and Y.A. Abdallah Query 361 GACTACATGTATTTTGG 377 Sbjct GACTACATGTATTTTGG Fig. (3): Alignment of the a toxin gene of C. perfringens type A chicken strain with all published a toxin gene of C. perfringens especially C. perfringens ATCC Identities = 370/377 (98%) TTTRGACTTTGCAGAGCAAGAAAGAACAGTATAAAATAAACACAGCAGGTTGCAAAACTAATGACGA TTTTTATGCTGATATCTTAAAAAACAAAGATTTTAATCGCATGGTCAAAAGAATATGCAAGAGGTTTT GCTAAAACAGGGAAATCAATATACTATAGTCATGCTAGCATGAGTCATAGTTGGGATGATFGGGATT ATGCAGCAAAGGTAACTCTAGCTAACTCTCAAAAAGGAACAGCGGGATATATTTATAGATTCTTACA CGATGTATCAGAGGGTAATGATCCATCAGTTGGAAAGAATGTAAAAGAACTAGTAGCTTACATATCA ACTAGTGGTGAAAAAGATGCTGGAACAGATGACTACATGATTTTA Fig.(4): Nucleotide sequence of Alpha toxin gene of C. perfringens type A rabbit strain.

5 Alpha toxin gene in C. perfringens 257 Fig. (5): Alignment of a toxin gene of C. perfringens type A Rabbit strain with all published a toxin gene of C perfringens especially Clostridium perfringens ATCC Identities = (97%).

6 258 M.M. Effat and Y.A. Abdallah TTTGGCTTITGCGAGGAAGAAAGACCOTATAAAATAAACACAGCAGGTTGCAAAACTAATGAGGCTTTTT ATACTGATATCTTAAAAAACAAAGATTTTAATGCATGGTCAAAAGAATATGCAAGAGGTTTTGCTAAAAC AGGAAAATCAATATACTATAGTCATGCTAGCATGAGTCATAGTTGGGATGATTGGGATTATGCAGCAAAG GTAACTTTAGCTAACTCTCAAAAAGGAACAGCGGGATATATTTATAGATTCTTACACGATGTATCAGAGGG TAATGATCCATCAGTTGGAAAGAATGTAAAAGAACTAGTAGCTTACATATCAACTAGTGGTGAGAAAGAT GCTGGAACAGATGACTACATGT Fig. (6): Nucleotide sequence of Alpha toxin gene of C. perfringens type B. Fig. (7): Alignment of a toxin gene of C. perfringens type B with all published a toxin gene of C. perfringens especially C, perfringens ATCC Identities = 367 / 372 (98 %).

7 Alpha toxin gene in C. perfringens 259 TTGACTTTGCGAGGAAGAAGACCAGTATAAAATAAACACAGCAGGTTGCAAAACTAATGAGGATTTTTAT GCTGATATCTTAAAAAACAAAGATTTTAATGCATGGTCAAAAGAATATGCAAGAGGTTTTGCTAAAACAG GGAAATCAATATACTATAGTCATGCTAGCATGAGTCATAGTTGGGATGATTGGGATTATGCAGCAAAGGT AACTCTAGCTAACTCTCAAAAAGGAACAGCGGGATATATTTATAGATTCTTACACGATGTATCAGAGGGTA ATGATCCATCAGTTGGAAAGAATGTAAAAGAACTAGTAGCTTACATATCAACTAGTGGTGAAAAAGATGC TGGAACAGATGACTACATGCCCCC Fig. (8): Nucleotide sequence of Alpha toxin gene of C. perfringens type C. Fig. (9): Alignment of a toxin gene of C. perfringens type C strain with all published a toxin gene of C. perfringens especially C. perfringens ATCC Identities = 362/373 (97%).

8 260 M.M. Effat and Y.A. Abdallah TGGACTTTTGCAGAGGAAAGAAAAGACAGTATAAAATAAACACAGCAGGTTGCAAAACTAATGAGGATTT TTATGCTGATATCTTAAAAAACAAGGATTTTAATGCATGGTCAAAAGAATATGCAAGAGGTTTTGCTAAAA CAGGAAAATCAATATACTATAGTCATGCTAGCATGAGTCATAGTTGGGATGATTGGGACTATGCAGCAAA GGTAACTCTAGCTAACTCTCGAAAAGGGAACAGCAGGATATATTTATAGATTCTTACACGATGTATCAGAG GGTAATGATCCATCAGTTGGAAAGAATGTAAAAGAACTAGTAGCTTACATATCAACTAGTGGTGAAAAAG ATGCTGGAACAGATGACTACATGATATTTT Fig.(10): Nucleotide sequence of Alpha toxin gene of C. perfringens type D. Fig. (11): Alignment of a toxin gene of C. perfringens type D with all published a toxin gene of C. perfringens especially C. perfringens ATCC Identities = 370 / 381 (97 %). Nucleotide sequence and alignment Alpha toxin, a necrotizing toxin commonly produced by all five strains of C perfringens, is believed to be a major factor responsible for the organism tissue pathology and has been suggested to be a key virulence determinant and predominant product of C. perfringens type A (Yoo et al., 1997; Scott et.

9 Alpha toxin gene in C. perfringens 261 al., 2004). Previous studies revealed that alpha toxin gene is found in all types of Clostridium perfringens (Effat et. al, 2007). However, no articles are found dealt with studying the nucleotide sequences among different types of Clostridium perfringens. The present studies demonstrated the nucleotide sequence among different types and different strains in the same type (A rabbit and chicken strains). Similar to the present results (Scott et. al, 2004) found that on applying sequencing for alpha toxin of 25 chickens derived C. perfringens, demonstrated high homology to mammal derived strains rather than to the only avian derived strain (Figs). REFERENCES Effat, M. M.; Abdallah, Y. A.; Soheir, M. F. and Rady, M. M. (2007).Characterization of Clostridium perfringens field isolates, implicated in necrotic enteritis outbreaks on broiler farms in Cairo, by multiplex PCR. African Journal of Microbiology Research (3): El-Seedy, F. (1990). Studies on necrotic enteritis in chickens Vet. Med. J. Giza, 38: Holshagen, M. and H. Stenwig (1992). Toxin production by Clostridium perfringens isolated from broiler chickens and capercallies (Tetrao urogallus) with and without necrotizing enteritis. Avian Diseases, 36: Leslie, D.; Fairweather, N; Pickard, D; Dougan, G and Kehoe, M. (1989). Phospholipase C and haemolytic activities of C. perfringens alpha toxin cloned in Escherichia coli: sequence and homology with Bacillus cereus phospholipase C. Mol. Microbiol. (3): Maniatis, T.; Fritsch, E.F., and Sambrook, J. (1982). Molecular cloning; a laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Nillo, L.(1993): Enterotoxemic Clostridium perfringens in pp in Gyles CL, Thoen CO, (Ed.) pathogenesis of bacterial infections in animals. Iowa State University, Ames. Salyers, A. A., and Whitt, D. D. (1994):Virulence factors that damage the host. In pp in Chapter 4;Bacterial pathogenesis, A molecular approach. ASM press Washington D.C., USA Scott, A.S.; Aaron, B.I.; Julian, I.R., and Robert, J.M. (2004):Highly conserved alpha toxin sequences of avian isolates of Clostridium perfringens Journal of Clinical Microbiology, 42(3): Songer, J.G. (1996). Clostridial enteric diseases of domestic animals. Clinical Microbiology Reviews 9(2): Titball, R.W.; Naylor, C.E., and Basak, A.K. (1999):The Clostridium perfringens Alpha toxin. Anaerobe, 5: Tizard, I; Neilesen, K.H.; Seed, J.R. and Hall, J.E. (1978). Biologically active production from African Trypanosomes. Microbiol. Re. 42 (4): YOO, H. S.; Sang, U.; Kyoung, Y.P., and Yonk, H. P. (1997). Molecular typing and epidemiological survey of prevalence of Clostridium perfringens types by multiplex PCR. J. Clinical Microbiology, 35(1): Zubay, G. (1988). Phospholipases Biochemistry (3 rd ed) W.M.C. Brown publishers, Columbia University.

10 262 M.M. Effat and Y.A. Abdallah א ص א و ن ن א א و د وמ ز א محمد محمد عفت* و ياسر ا حمد عبد االله** *قسم الميكرو بيولوجي والمناعة المركز القومي للبحوث الدقي الجيزة ** معهد بحوث الا مصال واللقاحات البيطرية العباسية القاهرة ينتج الا لفاتوآسين من جميع عترات الكلوستريديوم برفرينجينز وفي هذه الدراسة تم استخدام خم س عت رات للكلوس تريديوم برفرينجينز وهي نوع ) أ ( المعزول من الا رانب ونوع ) أ ( المعزول من الدواجن ونوع ) ب ( ون وع ) ج ( ون وع ) د ( المعزول ة ايضا م ن ال دواجن والج ين الم سي ول ع ن الا لفاتوآ سين ت م ف صله عل ى الاج اروز ج ل % 1.5 ولجمي ع العت رات و ت م عم ل اختب ار البلمرة المتسلسل باستخدام البادي ات النوعية المتخص صة. ث م ت م عم ل تحلي ل ترتي ب تسل سلى لقواع د الجين ات المف صولة م ن ن واتج اختبار البلمرة المتسلسل و تم مقارنتها بالتسلسل المرجعى للجينات المفصولة من العترات المرجعية ووجد نسبة ت شابه تت راوح م ا ب ين % ٩٦-٩٨ % ولا يوج د اخ تلاف ج وهري ب ين الترتي ب التسل سلي لقواع د الجين ات المف صولة م ن العت رات الم ستخدمة ف ي الدراسة وجينات العترات المرجعية.