Showing posts with label evolution. Show all posts
Showing posts with label evolution. Show all posts

Thursday, January 30, 2020

Pre-empting Pandora's Box - Update on Blastocystis Subtypes and Reference Data

Back in 2006, when we came up with the subtype terminology for Blastocystis, the spectrum of and boundaries between Blastocystis subtypes were quite clear and distinct. Since then, the genetic make-up of Blastocystis has appeared to be an even bigger universe than we (or at least I) expected, and we may be far from having explored the entire 'galaxy' yet.

New technologies make it easier to sequence DNA, and sequences attributed to Blastocystis are accumulating in the publicly available databases with great speed. While this situation is one of the things that stimulate research (genetic diversity, co-evolution, host specificity, parasite-host-microbiome interaction, etc.), issues have emerged when it comes quality-controlling DNA sequences and putting taxonomic identifiers on these sequences.

For Blastocystis, the main taxonomic identifier is the 'subtype'. In 2013, 17 subtypes of Blastocystis had been acknowledged based on SSU rDNA analysis, and since then, quite a few more have been suggested by independent researchers all around the world. While it's great to see the field advance and more and more researchers 'checking in' on Blastocystis, care should be taken to ensure that Blastocystis terminology remains a useful one. And this... is not an easy task!

Some things are relatively straightforward though. For instance, sequence quality control. A simple BLAST query in GenBank (NCBI Database) should tell you whether your sequence is Blastocystis or something else. Like banana. Or asparagus. DNA sequence chimeras are sequences where one piece of DNA is combined with a piece of DNA from another strain/species/genus/etc., which can happen during PCR-based amplification of DNA. Suppose you have a sequence that is 75% Blastocystis and 25% banana. If you BLAST such a sequence, you might get Blastocystis as the top hit, but with a modest amount of sequence identity - maybe 85%. If you're not cautious, you might jump to the conclusion that this might be a new subtype, since 85% similarity is a lot less than the 95-97% similarity that is used pragmatically to delimit the boundary between subtypes. But if you look carefully at the alignment of the query sequence and the reference sequence, you'll probably note that a large part of the sequence aligns very well to the most similar reference sequence, while a minor part of it has great dissimilarity. This should be a warning sign, and you should try and BLAST only the bit of the sequence not aligning up well... and when you do this, you might end up with... banana! In which case you would have to discard this part of the sequence. Please also see one of my recent posts for more on this. If you do not check for chimeras, you might end up including chimeric DNA sequences in your phylogenetic analyses that will distort and confuse the interpretation and - in the worst case - lead to erroneous calling of new subtypes.

What is less easy is to set a 'one-fits-all' threshold for sequence similarity... how similar can Blastocystis DNA sequences be to be considered the same subtype? When do you have evidence of a 'new' subtype? It's difficult to know, as long as the data available in public databases is so limited as it is. Moreover, researchers do not always use the same genetic markers. It's still common practice to amplify and sequence only about 1/3 of the SSU rRNA gene and use that as a taxonomic identifier. But if it's not the same 1/3 then it gets tricky to compare data. Moreover, we actually need near-complete SSU rDNA sequences (at least 1600 bp or so) to be able to infer robust phylogenetic relationships between reference sequences and sequences potentially reflecting new subtypes. Obviously, this is because variation can exist across the entire SSU rRNA gene.

One subtype that has proven particularly challenging is ST14, a subtype which is common in larger herbivourous mammals, is very difficult to delimit. It may easily be confused with other subtypes, if sufficiently long sequences are not used for investigation. To this end, we try to keep a pragmatic approach to Blastocystis subtype terminology, and it may turn out that it would be more practical and relevant to refer to ST24 and ST25 as ST14 (see figure below). For now, we suggest keeping them as separate subtypes. Near-complete Blastocystis SSU rDNA sequences from a lot of larger herbivorous mammals will help us resolve the taxonomy in the top part of the tree shown in the figure above.

In terms of acquiring near-complete SSU rDNA sequences, I would personally recommend MinION sequencing of PCR products obtained by the universal eukaryotic primers RD5 + RD3. And if DNA from cultures isused (yes, it IS possible to culture Blastocystis not only from human hosts, but also from a variety of animals), then then MinION sequencing and analysis of the data output should be a straight-forward and relative cost-effective task.

Figure. As of January 2020, 'real' Blastocystis subtypes are most likely subtypes 1–17, 21, 23–26. This simplified phylogeny gives and indication of the relatedness of the subtypes and the relative host specificity. Humans can host subtypes 1–9 and also 12; when subtypes other than 1–4 are encountered in human samples, this may reflect cases of zoonotic transmission.


Graham Clark and I just published an article in Trends in Parasitology on this, and we concluded that some of the newly proposed subtypes are in fact invalid. Invalid subtypes (subtypes 18, 19, 20, 22) typically reflected DNA sequence chimeras.

In the figure above, you can see the subtypes identified to date that we consider valid.

We also provided updated guidelines on Blastocystis subtyping. One very important thing to include here is reference sequence data. It would be very useful if our wonderful Blasto colleagues could all try and use the same reference sequences when they develop multiple sequence alignments for phylogenetic analyses. We have already done all the work for you, so all there is to it, is to download the sequences from London School of Hygiene and Tropical Medicine's server available here and align them with your own DNA sequences. It would make life easier for all of us!

๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž๐ŸŒž

Corrected proofs of the article can be downloaded here.

Thanks for reading!

Wednesday, May 22, 2019

Parasite Course: Concepts in Parasitology (Australia)

For those interested:


There will be a two-week specialist course for PhD students and earlycareer researchers (ECRs), 
run by the Australian Society for Parasitology (ASP) called Concepts in Parasitology.
  
The course is running again this year from the 25th of November to the 8th of December 2019 in New South Wales (Kioloa Coastal Campus), and the deadline for applications is the 13th of July 2019. 

Details of the course are here.


Monday, April 29, 2013

Transcriptomic Analysis of Blastocystis ST1!

BLASTing Breaking News!

Probably to support their genomic data, researchers in Andrew Roger's group in Canada have performed transcriptomic analysis of the Nand II strain, which belongs to Blastocystis sp. ST1.

Running from April 29 to May 2 is the SMBE (Society for Molecular Biology and Evolution) satellite meeting on Eukaryotic-Omics; the abstract booklet can be downloaded here. And fellow tweeps, don't let yourselves down by not following #SMBEeuks!

Until now, we've only known of one complete Blastocystis nuclear genome, namely that of ST7. Now, the release of the ST1 genome may be imminent! In any case, Roger's group have used their transcriptomic data to compare the protein content in ST1 with that in ST7, and it appears from their conference abstract that "the genes encoded by the Nand II strain (ST1) are surprisingly distantly related to ST7 orthologues, sharing on average ~50% identity at the protein level." And more: "Preliminary analyses allowed us to detect ~1000 genes in ST1 that have no homologue in Blastocystis sp. ST7". This means that the extreme genetic diversity that we see across the SSU ribosomal RNA genes is reflected and may be even more pronounced at nuclear genome level.

The group also studied genes acquired by lateral gene transfer (LGT; see previous post for more on LGT, also known as horizontal gene transfer), and what they basically found was that ST1 appears to have acquired bacterial genes related mainly to metabolism, while genes acquired from eukaryotes code for proteins related to cellular processes and signaling mechanisms.

Finally, they have discovered genes obtained by LGT that has had importance for Blastocystis' adaptation to parasitism; among these genes that enable resistance to host immune responses.

Roger's group is based at the Dept. of Biochemistry and Molecular Biology, Dalhouise University, Halifax, Nova Scotia in Canada.




Thursday, March 14, 2013

Extremophilic Eukaryotes

My recent post Blastocystis aux Enfers was my "literary take" on biological adaptation of intestinal parasitic protists, using Blastocystis as an example. As a parasitologist you'd come across many peculiar and shrewd biological adaptations and life cycles, and I hope to be able to give some examples in a future post. Actually, there is a parasite which is quite common in humans, maybe even just as common as Blastocystis, which is also single-celled, but which may have a much more complicated life cycle than Blastocystis, namely Dientamoeba fragilis; a colleague of mine is currently doing his PhD on Dientamoeba and he has collected multiple sources of evidence to confirm the hypothesis that this parasite is transmitted by a vector, namely pinworm, probably along the same way that Histomonas meleagridis – the cause of blackhead disease in especially turkeys – is transmitted by heterakids (which again are transmitted by parathenic hosts such as earthworms, which get eaten by turkeys, chickens, etc.). Anyway, I’ll probably get back to Dientamoeba, once his data are out.

Meanwhile, Blastocystis comes out of a very heterogeneous group of organisms called Stramenopiles, many of which are algae. Algae are photosynthetic organisms found in habitats as diverse as glacial ice and hot springs.One of these algae is named Galdieria sulphuraria, which is a remarkable unicellular eukaryote inhabiting hostile environments such as volcanic hot sulfur springs where it is responsible for about 90% of the biomass; indeed this certainly qualifies as "Galdieria aux enfers"!

Thursday, April 26, 2012

What is Blastocystis?

Intestinal parasites of humans can be divided into mainly helminths ('worms' including cestodes, nematodes and trematodes), and single-celled eukaryotic organisms. Most single-celled intestinal parasites belong to one of four main groups:
  • Archamoebae or Amoeboids (e.g. Entamoba, Iodamoeba, Endolimax)
  • Ciliates (e.g. Balantidium)
  • Sporozoa (e.g. Cryptosporidium, Cyclospora, Cystoisospora; even microsporidia)
  • Flagellates (e.g. Giardia, Chilomastix, Enteromonas, Pentatrichomonas, Retortamonas, Dientamoeba (unflagellated flagellate!))
Traditionally, these four groups have been referred to as protozoa.

However, the most common, single-celled intestinal parasitic eukaryote, Blastocystis, does not belong in any of these four categories. Taxonomically, Blastocystis belongs to the heterogeneous group of Stramenopiles, which includes slime nets, diatoms, water moulds and brown algae. Most stramenopiles are free-living organisms. Blastocystis is an atypical stramenopile not only as this group is named for the straw-like tubular hairs on the flagella and sometimes the cell body - Blastocystis has no flagella and lacks any tubular hairs - but also due to its parasitic nature.

Often, Blastocystis is referred to as a 'protozoon', although 'protist' is more appropriate. Protists can be defined basically as any eukaryote that is not a plant, an animal or a fungus.

One of the closest relatives of Blastocystis identified to date is Proteromonas lacertae, a parasite of reptiles.

Interestingly, Proteromonas does have flagella and hairs on the cell body. For comparison, the image below shows Blastocystis (culture) - appearing almost amoeboid, only with very limited morphological hallmarks (note examples of binary fission and the eccentrically located nuclei and mitochondrion-like organelles).

Blastocystis is one of two Stramenopiles known to infect humans, the other being Pythium insidiosum, which has been associated with keratitis and dermatological lesions mainly in SE Asia.

Other organisms with close relation to Blastocystis include Karotomorpha, Cepedea, Protoopalina and Opalina.

For further information, please visit

Silberman, J., Sogin, M., Leipe, D., & Clark, C. (1996). Human parasite finds taxonomic home Nature, 380 (6573), 398-398 DOI: 10.1038/380398a0  

HOEVERS, J., & SNOWDEN, K. (2005). Analysis of the ITS region and partial ssu and lsu rRNA genes of Blastocystis and Proteromonas lacertae Parasitology, 131 (2), 187-196 DOI: 10.1017/S0031182005007596  

Kostka, M., Cepicka, I., Hampl, V., & Flegr, J. (2007). Phylogenetic position of Karotomorpha and paraphyly of Proteromonadidae Molecular Phylogenetics and Evolution, 43 (3), 1167-1170 DOI: 10.1016/j.ympev.2006.11.002

Friday, April 6, 2012

Why "Blastocystis sp." and not "Blastocystis hominis"?

Blastocystis identified in humans used to be referred to as "Blastocystis hominis". However, after the advanced use of nucleic acid-based tools in the 90s and 00s it became clear that

1) morphologically identical Blastocystis can be genetically extremely diverse
2) Blastocystis in humans comprises at least 9 species (or, perhaps more correctly, ribosomal lineages), 8 of which can be found in other animals as well.

This means that host origin is not a reliable indicator of organism identity.

Blastocystis appears to exhibit only moderate host specificity - at least at subtype level - , and until a more substantial sampling from various hosts has been carried out, we will have to go with "Blastocystis sp." followed by an appropriate subtype (ST) number (according to species/ribosomal lineage), e.g. "Blastocystis sp. ST3", which is one of the 4 subtypes commonly found in humans.

In order to make subtype analysis very easy, we have created a site (together with Keith Jolley, Oxford University), where a bulk of sequences can be assigned to subtype in few seconds. Single sequence entries are also possible.

To sum up: Blastocystis hominis is a misleading and currently an invalid taxon.

(Read more about this in our Blastocystis consensus paper from 2007 in Trends in Parasitology)