Cryptosporidium gp60 subtyping.

Cryptosporidium spp. are coccidian protozoan parasites that infect various vertebrate and invertebrate hosts. At least seven Cryptosporidium species have been associated with gastro-intestinal disease in humans: C. hominis, C. parvum, C. meleagridis, C felis, C. canis, C. suis and C. muris. Of these, C. hominis and C. parvum are the two species found most often in humans. Cryptosporidium-detection methods based on PCR has been an important instrument for studying the taxonomy and transmission of the parasite. For the differentiation of Cryptosporidium species/genotypes, different types of molecular tools have been used. In many studies the subtyping is performed using the Small SubUnit ribosomal RNA (SSU rRNA). Another genetic target is an oocyst wall protein (COWP) gene. However, the COWP gene is less useful because of its narrow specificity (Xiao, 2010). One gene that is becoming popular for subtyping Cryptosporidium is the 60 kDa glycoprotein (gp60). A Cryptosporidium subtype will end up with a name such as “IaA23R4” or “IIdA18G1”. In this blog I will explain the methodology behind this classification of Cryptosporidum subtypes based on gp60 analysis.

The gp60 gene has tandem repeats of the serine-coding trinucleotide: TCA, TCG or TCT. These tandem repeats have varying lengths. The DNA sequence outside these repeats also shows considerable differences; they are used to categorize C. parvum and C. hominis each into several subtype families. The name of gp60 subtypes start by designating “I” for C. hominis and “II” for C. parvum. Subsequently, each new subtype family is given a letter. Ia, Ib, Id, etc. for C. hominis and IIa, IIb, IIc, etc. for C. parvum. What follows then is the letter and number of the trinucleotide repeats. TCA is represented by the letter A, TCG is represented by the letter G and TCT by the letter T. The letter R is designated for any other repeat.

So lets look at some examples. In this text-file you may find some Cryptosporidium subtype DNA sequences that I used: three C. hominis subtypes and three C. parvum subtypes. 

cryp alignment Screen Shot 2013-08-13 at 22.27.46

By the way, here are two useful online tools for DNA sequence analyses, which I often use. For DNA aligment: Multalin …and to draw phylogenetic trees: ClustalW2

For the first example I took the DNA sequence with GenBank Accession number AF164502.

Cryp subtype1.001

The dominant trinucleotide repeat is TCA (in red). The serine trinucleotide is repeated 23 times. Another repeat is with AAGACGGTGGTAAGG (in green). The sequence is repeated three times. However, the repeat is followed by a DNA sequence similar to the preceding one: AAACGGTGAAGG. In this case the researcher has decided that it should be part of the tandem repeat. So here we end up with a ‘Cryptosporidium hominis’ of the subtype family ‘Ia’ followed by ‘A23R4’.

Another one. Let’s take Accession number AY262034, C. parvum subtype family IIa.

Cryp subtype2.002

The dominant trinucleotide is TCA with 15 repeats (purple). In between are two TCG’s (green). Furthermore, this subtype also has one ‘R’ (other) repeat: ACATCA (orange). Huh? One repeat? Well, apparently there are similar subtypes with two copies of ACATCA. Hence, R=1 in this one, which makes IIaA15G2R1.

Ok, last one. Genbank number AF164491. A C. parvum beloning to subtype family IIc.

Cryp subtype3.003

Five times TCA (blue) and three times TCG (pink). In the IIc subtype family, all strains have A5G3 repeats. However, downstream in the 3’ region some strains are different. The diverged strains have subsequently an extension. In this case it’s IIcA5G3a.

Subtyping microbiological strains always had a slice of guesswork. How many nucleotide substitutions does it need to make the strain to be a new genotype? When would you denote a sequence to be a repeat? Molecular genotyping has provided many advantages over conventional typing methods. Sometimes it might seem that the resolution for differentiating strains with molecular methods are either to narrow or to broad. Still, as we see with the typing for Cryptosporidium strains, it takes a lot of effort, thought slowly a consensus is appearing.

Want to know more about sub typing Cryptosporidum strain? A good start is the article ‘Molecular epidemiology of cryptosporidiosis: An update‘ by Lihua Xiao in Experimental Parasitology (124 (2010) 80-90). Or if you have any specific questions, feel free to contact me.

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