Lab 1

Vibrio cholerae

Even nowadays, cholera is still a prevalent epidemic that is responsible for millions of deaths, especially due to the lack of a vaccine that can particularly target this bacterium ( Reyburn et al., 2011). The Cholera Toxin B found in Vibrio cholerae might be a strong “immunological adjuvant” or agent that that could be used as a potential vaccine against cholera. Cholera toxin B, often referred as CT, is an antigen and holotoxin (made up of many subunits) that has an active A subunit connected to B subunits (made up of pentamers) which binds to ganglioside GM1(Negri et al., 2009). This leads to the intake of toxin B by intestinal epithelial cells through endocytosis and results in diarrhea (Jayasekera et al., 2008).

It is often difficult to find a vaccine that targets a pathogenic bacterium without affecting human cells as well as the other bacteria that are present in the human body and that are actually not harmful. If there are no other or a very small number of proteins that are identical or homologous to Toxin B then this would be a perfect candidate for a vaccine because it would be very specific to the bacterium. Cholera toxin B is made up of 124 amino acids with alanine, isoleucine and lysine being the most abundant and tryptophan being the least (Figure 1).

1 miklkfgvff tvllssayah gtpqnitdlc aeyhntqiyt lndkifsyte slagkremai
61 itfkngaifq vevpgsqhid sqkkaiermk dtlriaylte akveklcvwn nktphaiaai
121 sman
Figure 1: Amino Acid sequence of Cholera Toxin B.

The sequence of Toxin B illustrates many repeats of adenine and thymine (Figure 2). It is a fairly large protein and there doesn’t seem that be another protein that could be revolutionary related to Toxin B in Vibrio cholerae.

1 atgattaaat taaaatttgg tgtttttttt acagttttac tatcttcagc atatgcacat
61 ggaacacctc aaaatattac tgatttgtgt gcagaatacc acaacacaca aatatatacg
121 ctaaatgata agatattttc gtatacagaa tctctagctg gaaaaagaga gatggctatc
181 attactttta agaatggtgc aatttttcaa gtagaagtac caggtagtca acatatagat
241 tcacaaaaaa aagcgattga aaggatgaag gataccctga ggattgcata tcttactgaa
301 gctaaagtcg aaaagttatg tgtatggaat aataaaacgc ctcatgcgat tgccgcaatt
361 agtatggcaa atttg
Figure 2: Nucleotide sequence of Cholera Toxin B.

From looking through the BLAST results, only one protein (CTxB) appeared to have an association with Toxin B, however they were only 33% identical (Figure 3). This is pretty low a comparison and we cannot conclude that they are evolutionary related, thus toxin B might the only one of its kind in Vibrio cholerae.

gb|AAQ84036.1| CtxB cholerae//
Length=41
Score = 85.5 bits (210), Expect = 2e-15, Method: Compositional matrix adjust.
Identities = 40/41 (98%), Positives = 40/41 (98%), Gaps = 0/41 (0%)
Query 84 KAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN 124
KAIERMKDTLRIAY TEAKVEKLCVWNNKTPHAIAAISMAN
Sbjct 1 KAIERMKDTLRIAYFTEAKVEKLCVWNNKTPHAIAAISMAN 41
Figure 3: Protein that is very unlikely related to toxin B.

If we are to look across species, there appears to be another protein that is homologous to toxin B. The LTB (heat-labile enterotoxin b subunit) protein of E.coli which is 100% identical to toxin B of Vibrio cholerae (Figure 4). The LTB protein also has 124 amino acids, though in this case there are a couple of tryptophan amino acids that are present in this protein.

gb|ABV01333.1| LTB [Escherichia coli]
Length=124
Score = 216 bits (551), Expect = 5e-55, Method: Compositional matrix adjust.
Identities = 101/124 (82%), Positives = 112/124 (91%), Gaps = 0/124 (0%)
Query 1 MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAI 60
M K+KF V FTVLLSS AHG PQ+IT+LC+EY NTQIYT+NDKI SYTES+AGKREM I
Sbjct 1 MNKVKFYVLFTVLLSSLCAHGAPQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVI 60
Query 61 ITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAI 120
ITFK+GA FQVEVPGSQHIDSQKKAIERMKDTLRIAYLTE K++KLCVWNNKTP++IAAI
Sbjct 61 ITFKSGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTETKIDKLCVWNNKTPNSIAAI 120
Query 121 SMAN 124
Figure 4: LTB protein of E.coli that is significantly identical to Toxin B.

The LTB protein not only appears to have a similar structure to Toxin B, but also a close and similar function (Rosales-Mendoza et al., 2007). Since there might very few homologs of Toxin B, this could be a great candidate for a vaccine target because it can be very specific to this particular bacterium. The development of this vaccine would help stop the spread of the epidemic, especially in third world countries where there is a strong need for vaccines against this pathogen.

REFERENCES

Jayasekera et al. (2008). Cholera Toxin–Specific Memory B Cell Responses Are Induced in Patients with Dehydrating Diarrhea Caused by Vibrio cholerae O1. J Infect Dis. 198(7) 1055-1061.
Negri et al. (2009). Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin, but Not Their Nontoxic Counterparts, Improve the Antigen-Presenting Cell Function of Human B Lymphocytes. American Society for Microbiology. 77(5): 1924–1935.
Reyburn et al. (2005). The Case for Reactive Mass Oral Cholera Vaccinations. 5 (1): 1-10.
Rosales-Mendoza et al. 2007. Expression of Escherichia coli heat-labile enterotoxin b subunit (LTB) in carrot (Daucus carota L.) Plant Cell Rep. 26:969–976
Tinker et al. (2003). Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B5 Interface: Mutational Analysis and Development of an In Vitro Assembly System. Infection and Immunity. 71(7) 4093-4101