Introduction

A poisonous substance which produced by microorganisms is known as Toxins. Toxins can be classified into two groups such as Exotoxins ( A protein substance that are released by organisms) and Endotoxins ( A lipopolysaccharide substance that are present in outer membrane of Gram Negative bacteria). Based on the site of damage, the toxins are classified into various categories namely

• Neurotoxins (it affects nervous system)

• Enterotoxin ( it affects intestinal tract)

• Cytotoxin ( it affects several different kind of cells like RBC,Leukocytes.)

One of the most commonly occurring food poisoning in the world is Staphylococcal food intoxication. It is due to the ingestion of food having enterotoxin which produced by Staphylococcus aureus. Staphylococcus aureus is Gram positive, facultatively anaerobic bacterium which appears in grape like clusters when observed in microscope and most of enterotoxin producing strain is Coagulase positive. Staphylococcus aureus produce six different type of enterotoxin like A,B,C1,C2,D and E. Staphylococcal Enterotoxin B (SEB) is one of the toxin responsible for staphylococcal food intoxication and some countries produce SEB as a Biological Weapon (Institute for International Cooperation in Animal Biologics, Iowa, USA).

During prolonged refrigeration, the organism deposit their toxin on food. It will consumed into intestinal tract if the food is eaten without sufficient heating. After ingestion, the incubation period is about 3 to 10 hours. When the toxin enter into body, it absorbed in to mucosa and cause tissue damage then induce abdominal pain, nausea, vomiting and diarrhea.

Materials And Methods

MATERIALS:

Dry Lab:

A System with Internet Access.

OS Name: Microsoft Windows XP Professional.

Programs: Internet Explorer or Mozilla Firefox (2.0.0.18)

Biological Software: Rasmol

Input file: Protein Sequence ( PDB file format, FASTA Format, Raw sequence )

Wet Lab:

No wet lab experiments.

Methods

Sequence Retrieval:

The sequence details of Staphylococcal Enterotoxin B were retrieved from Protein Data Bank (Brookhaven National Laboratory).

Protein Structure Analysis:

Primary Structure:

ProtParam (Gasteiger E et al, Protein Identification and Analysis Tools on the ExPASy Server) was used to predicate amino acid composition, atomic composition, molecular weight, Theoretical pI of the protein (toxin). The raw sequence of SEB was used as input file.

Secondary structure:

SOPMA (Self-Optimized Prediction Method with Alignment) (Geourjon and Deléage, 1995) was used to predicate the secondary structure ( Alpha Helix, Beta Sheet, Turn and Coil) of SEB protein.

Tertiary structure:

FUGUE v2.s.07 (J. Shi, T. L. Blundell, and K. Mizuguchi (2001) was used to recognize the distant homologues by sequence-structure comparison.

Molecular visualization:

Rasmol (Herbert J. Bernstein 2000 -- 2006.) was used to visualize the protein molecule. Here PDB format of SEB protein sequence was used as input file. The following command was used to label the three letter code of amino acid on structure.

Syntax: label <%n>

Transcription and Translation Analysis:

Protein to DNA:

Reverse Translate: Reverse Translate (Stothard P (2000), (Sequence Manipulation Suite) was used to get DNA sequence from protein sequence. Here SEB protein sequence (one letter amino acid code ) in Raw format was used as input to get DNA sequence. Input limit of Reverse Translate tool is 20000 characters.

DNA to RNA to Protein:

Transcription and Translation Tools (1998,1999 Attotron Biosensor Corporation) was used to determine the RNA sequence for translation of SEB protein. Here DNA sequence which got from reverse translate tool, was used as input file.

Enzymatic cleavage of SEB:

ExPASy PeptideCutter tool (Gasteiger E., et al) was used to predicate the potential cleavage site of SEB by selective Enzymes and Chemicals. Here the enzymes namely Proteinase K

Pepsin (pH1.3), Trypsin and a chemical (Formic Acid) was used.

Peptidemass tool (Wilkins, M.R. et al, 1997) was used to calculate the peptidemass which generate from above enzyme cleavage.

Results And Discussion

Sequence Retrieval:

The sequence details of Staphylococcus Enterotoxin B were retrieved from Protein Data Bank (Brookhaven National Laboratory). The PDB Code is 3SEB. The sequence details in FASTA format are as follws…

>3SEB:A|PDBID|CHAIN|SEQUENCE ESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFLYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDK YVDVFGANYYYQCYFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQEL DYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKK

Primary Structure:

The primary structure or amino acid sequence is unique for each protein. The ProtParam tool predicted the molecular weight of SEB toxin (protein) as 28.23 kDa and theoretical pI as 7.84. Amino acid composition, Atomic composition , half life, Instability index and Aliphatic index of SEB protein were also predicated by using ProtParm. Number of amino acids in Staphylococcus Enterotoxin B is 238. The amino acid composition of SEB is in Table : 1

Figure 1

Atomic composition of SEB is in Table: 2

Figure 2

Formula: C1269H1932N324O387S10

Estimated half-life:

The N-terminal of the sequence considered is E (Glu).

The estimated half-life is: 1 hours (mammalian reticulocytes, in vitro).

30 min (yeast, in vivo).

>10 hours (Escherichia coli, in vivo).

Instability index:

The instability index (II) is computed to be 32.84

Aliphatic index: 62.56

Grand average of hydropathicity (GRAVY): -0.915

SEB protein having high amount of lysine (13.4%) and aspartate ( 10.1%). The total number of negatively charged residues (aspartate + glutamine) is 36 and positively charged residues ( arginine+ lysine) is 37. The total number of atoms in SEB is 3922. The instability index (II) is computed to be 32.84. This classifies the protein as stable.

Secondary Structure:

The most common secondary structure of a protein is alpha helix and beta sheets. SOPMA (Self-Optimized Prediction Method with Alignment) predicated 22.27 % of Alpha helix, 29.83 % of Extended strand, 8.82 % of Beta turn and 39.08 % of Random coil in Staphylococcal Enterotoxin B where as Staphylococcal enterotoxin type C2 ( PDB code: 1 STE) contains 23.85 % of Alpha helix and 28.03% of Extended strand.

Tertiary Structure:

Fugue predicated following results for input amino acid sequence

Figure 3

The profile Stap_Strap toxin having high ZSCORE and ZORI for input amino acid sequence and the Stap_Strap toxin profile having 6 types of structure and alignment with our SEB toxin protein sequence.

Figure 4

The alignment of SEB protein sequence with above proteins are as follows…

Figure 5

Figure 6

Staphylococcus enterotoxin type c-2 (PDB Code: 1ste), streptococcal superantigen ( PDB Code: 1bxt) are having slightly similar protein composition with SEB.

Molecular visualization:

The structure of SEB protein was viewed by using Rasmol.

Figure 7

Fig.1: SEB in Strands

Figure 8

Fig.2: SEB in Strands, labeled with three letter code of amino acids

Transcription and Translation Analysis:

The transcription and translation analysis was done to predicate the DNA and RNA sequence which code for SEB protein.

Protein to DNA:

Reverse Translate tool predicated the following DNA sequence from input

>reverse translation of 3SEB:A|PDBID|CHAIN|SEQUENCE to a 714 base sequence of most likely codons.

Gaaagccagccggatccgaaaccggatgaactgcataaaagcagcaaatttaccggcctgatggaaaacatgaaagtgctgtatgatgataaccatgtgagcgcgattaacgtgaaaagcattgatcagtttctgtattttgatctgatttatagcattaaagataccaaactgggcaactatgataacgtgcgcgtggaatttaaaaacaaagatctggcggataaatataaagataaatatgtggatgtgtttggcgcgaactattattatcagtgctattttagcaaaaaaaccaacgatattaacagccatcagaccgataaacgcaaaacctgcatgtatggcggcgtgaccgaacataacggcaaccagctggataaatatcgcagcattaccgtgcgcgtgtttgaagatggcaaaaacctgctgagctttgatgtgcagaccaacaaaaaaaaagtgaccgcgcaggaactggattatctgacccgccattatctggtgaaaaacaaaaaactgtatgaatttaacaacagcccgtatgaaaccggctatattaaatttattgaaaacgaaaacagcttttggtatgatatgatgccggcgccgggcgataaatttgatcagagcaaatatctgatgatgtataacgataacaaaatggtggatagcaaagatgtgaaaattgaagtgtatctgaccaccaaaaaa

DNA to RNA to Protein:

Transcription and Translation Tool predicated following RNA and then protein sequence from input DNA sequence.

DNA Sequence (input file)

gaaagccagccggatccgaaaccggatgaactgcataaaagcagcaaatttaccggcctgatggaaaacatgaaagtgctgtatgatgataaccatgtgagcgcgattaacgtgaaaagcattgatcagtttctgtattttgatctgatttatagcattaaagataccaaactgggcaactatgataacgtgcgcgtggaatttaaaaacaaagatctggcggataaatataaagataaatatgtggatgtgtttggcgcgaactattattatcagtgctattttagcaaaaaaaccaacgatattaacagccatcagaccgataaacgcaaaacctgcatgtatggcggcgtgaccgaacataacggcaaccagctggataaatatcgcagcattaccgtgcgcgtgtttgaagatggcaaaaacctgctgagctttgatgtgcagaccaacaaaaaaaaagtgaccgcgcaggaactggattatctgacccgccattatctggtgaaaaacaaaaaactgtatgaatttaacaacagcccgtatgaaaccggctatattaaatttattgaaaacgaaaacagcttttggtatgatatgatgccggcgccgggcgataaatttgatcagagcaaatatctgatgatgtataacgataacaaaatggtggatagcaaagatgtgaaaattgaagtgtatctgaccaccaaaaaa

Figure 9

GAAAGCCAGCCGGAUCCGAAACCGGAUGAACUGCAUAAAAGCAGCAAAUUUACCGGCCUGAUGGAAAACAUGAAAGUGCUGUAUGAUGAUAACCAUGUGAGCGCGAUUAACGUGAAAAGCAUUGAUCAGUUUCUGUAUUUUGAUCUGAUUUAUAGCAUUAAAGAUACCAAACUGGGCAACUAUGAUAACGUGCGCGUGGAAUUUAAAAACAAAGAUCUGGCGGAUAAAUAUAAAGAUAAAUAUGUGGAUGUGUUUGGCGCGAACUAUUAUUAUCAGUGCUAUUUUAGCAAAAAAACCAACGAUAUUAACAGCCAUCAGACCGAUAAACGCAAAACCUGCAUGUAUGGCGGCGUGACCGAACAUAACGGCAACCAGCUGGAUAAAUAUCGCAGCAUUACCGUGCGCGUGUUUGAAGAUGGCAAAAACCUGCUGAGCUUUGAUGUGCAGACCAACAAAAAAAAAGUGACCGCGCAGGAACUGGAUUAUCUGACCCGCCAUUAUCUGGUGAAAAACAAAAAACUGUAUGAAUUUAACAACAGCCCGUAUGAAACCGGCUAUAUUAAAUUUAUUGAAAACGAAAACAGCUUUUGGUAUGAUAUGAUGCCGGCGCCGGGCGAUAAAUUUGAUCAGAGCAAAUAUCUGAUGAUGUAUAACGAUAACAAAAUGGUGGAUAGCAAAGAUGUGAAAAUUGAAGUGUAUCUGACCACCAAAAAA

Figure 10

ESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFLYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANYYYQCYFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKK

Enzymatic cleavage of SEB:

ExPASy PeptideCutter tool predicated potential cleavage sites of SEB by using 3 enzymes (Proteinase K , Pepsin (pH1.3),Trypsin ) and a chemical i.e Formic Acid.

Proteinase K produced 93 cleavage on Staphylococcus Enterotoxin B protein. The cleavage site are as follows..

11 17 18 20 26 27 28 33 35 36 38 41 44 45 46 47 49 50 51 53 56 58 61 64 66 68 73 74 77 81 82 84 85 87 89 90 91 94 95 99 102 107 112 115 118 119 126 129 132 133 134 136 137 143 144 146 148 150 155 156 157 160 162 163 164 167 168 169 174 175 177 182 184 186 187 189 190 196 197 198 203 208 213 214 217 223 228 230 232 233 234 235 236

/// : The symbol indicating where cleavage done.

ESQPDPKPDEL///HKSSKF///T///GL///MENMKV///L///Y///DDNHV///SA///I///NV///KSI///DQF///L///Y///F///DL///I///Y///SI///KDT///KL///GNY///DNV///RV///EF///KNKDL///A///DKY///KDKY///V///DV///F///GA///NY///Y///Y///QCY///F///SKKT///NDI///NSHQT///DKRKT///CMY///GGV///T///EHNGNQL///DKY///RSI///T///V///RV///F///EDGKNL///L///SF///DV///QT///NKKKV///T///A////QEL///DY///L///T///RHY///L///V///KNKKL///Y///EF///NNSPY///ET///GY///I///KF///I///ENENSF///W///Y///DMMPA///PGDKF///DQSKY///L///MMY///NDNKMV///DSKDV///KI///EV///Y///L///T///T///KK

Pepsin ( pH 1.3) ( preferentially cleaves at Phe, Tyr, Trp and Leu in position P1 or P1'(Keil, 1992) produce 84 cleavage and cleavage site are as follows…

10 11 16 17 19 20 26 28 43 44 44 45 45 46 46 47 48 49 50 51 57 58 60 61 68 72 76 77 81 84 85 88 89 89 90 90 91 93 94 94 95 114 115 125 126 128 129 136 142 144 145 146 159 160 161 162 162 163 166 173 176 177 181 185 186 188 189 195 196 196 197 197 198 207 208 212 213 213 216 217 232 233 233 234

ESQPDPKPDE///L///HKSSK///F///TG///L///MENMKV///LY///DDNHVSAINVKSIDQ///F///L///Y///F///D///L///I///Y///SIKDTK///L///GN///Y///DNVRVEF///KNKD///LADK///Y///KDKY///VDV///F///GAN///Y///Y///Y///QC///Y///F///SKKTNDINSHQTDKRKTCM///Y///GGVTEHNGNQ///L////DK///Y///RSITVRV///FEDGKN///LL///S///F///DVQTNKKKVTAQE///L///D///Y///L///TRH///YLVKNKK///LYE///F///NNSP///YETG///Y///IK///F///IENENS///F///W///Y///DMMPAPGDK///F///DQSK///Y///LMM///Y///NDNKMVDSKDVKIEV///Y///L///TTKK

Trypsin produced 36 cleavage and cleavage site are as follows…

13 16 25 39 54 57 65 69 71 76 78 80 97 98 109 110 111 128 130 135 141 152 153 154 165 170 172 173 188 207 212 221 226 229 237 238

ESQPDPKPDELHK///SSK///FTGLMENMK///VLYDDNHVSAINVK///SIDQFLYFDLIYSIK///DTK///LGNYDNVR///VEFK///NK///DLADK///YK///DK///YVDVFGANYYYQCYFSK///K///TNDINSHQTDK///R///K////TCMYGGVTEHNGNQLDK///YR///SITVR///VFEDGK///NLLSFDVQTNK///K///K///VTAQELDYLTR///HYLVK///NK///K///LYEFNNSPYETGYIK///FIENENSFWYDMMPAPGDK///FDQSK///YLMMYNDNK///MVDSK///DVK///IEVYLTTK///K///

Formic acid (Cleaves at Asp in position P1 (Li et al., 2001).) produce 24 cleavages and the sites are as follows..

5 9 29 30 42 48 55 62 72 75 79 83 101 108 127 139 147 161 199 206 209 219 224 227

ESQPD///PKPD///ELHKSSKFTGLMENMKVLYD///D///NHVSAINVKSID///QFLYFD///LIYSIKD///TKLGNYD///NVRVEFKNKD///LAD///KYKD///KYVD///VFGANYYYQCYFSKKTND///INSHQTDK///RKTCMYGGVTEHNGNQLDK///YRSITVRVFEDG///KNLLSFDV///QTNKKKVTAQELDY///LTRHYLVKNKKLYEFNNSPYETGYIKFIENENSFWYDM///MPAPGDK///FDQ///SKYLMMYNDN///KMVDS///KDV///KIEVYLTTKK

Proline-endopeptidase, Hydroxylamine are produce only one cleavage on site 8 and 122 respectively. Caspase1,Caspase2,Caspase3,Caspase 4,Caspase 5,Caspase 6 ,Caspase 7, Caspase 8 ,Caspase 9,Caspase10,Enterokinase, Factor Xa, GranzymeB, Thrombin,Tobacco etch virus protease are not produce any cleavage on SEB protein.

PeptideMass:

PeptideMass tool calculated masses of generated peptide during enzymatic cleavages of Staphylococcus Enterotoxin B by enzymes ( Proteinase K, Pepsin pH 1.3 and Trypsin)

Proteinase K:

The Position of cleavages, Sequence and Masses of Peptide generated during cleavage by Proteinase K are in Table : 3

Figure 11

Table 3

Pepsin (pH 1.3):

The Position of cleavages, Sequence and Masses of Peptide generated during cleavage by Pepsin ( PH 1.3) are in Table : 4

Figure 12

Table 4

Trypsin:

The Position of cleavages, Sequence and Masses of Peptide generated during cleavage by Trypsin are in Table: 5

Figure 13

Table 5

Conclusion

Bioinformatics is the application of Information Technology to the Biological Science. Bioinformatics tools like ProtParam, SOPMA, FUGUE v2.s.07,Rasmol etc were used to predicate the Primary, Secondary and Tertiary structure of SEB protein. SEB contains 238 amino acid residues and having slightly similar amino acid composition with Staphylococcal Enterotoxin Type C 2 and Streptococcal superantigen. The enzymatic cleavage of SEB protein by various enzymes were also predicated by using bioinformatics tool namely ExPASy PeptideCutter tool. Proteinase K and Pepsin produce 93 and 84 cleavages respectively on SEB protein. Caspase 1 to 10 are not produce cleavage on SEB.