This Month in Blastocystis Research (AUG 2014)

Some August highlights in Blastocystis research:

1) The PRE-IOPCA Molecular Parasitology Workshop took place from the 7-10 August at CINVESTAV, Mexico City. Top-motivated students from some 10-15 countries worked hard from 7 am to 7 pm in dry+wet lab sessions, and we all had a really great time, thanks to both participants and fantastic organisers. There was a 4 h session on Blastocystis molecular epidemiology, and I was pleased to learn that some of the participants currently work with (or plan to work with) Blastocystis. I look forward to doing something similar in Ankara, Turkey on the 27th of May next year (www.blastomeeting2015.com - did you bookmark it?).

Most of the task force of the Molecular Parasitology Workshop (ICOPA 2014).

2) At the actual ICOPA conference, I chaired a session on Blastocystis in the context of IBS, with talks delivered by Ken Boorom, Pablo Maravilla, Pauline Scanlan and myself. In the audience I was honoured to see and meet Dr Hisao Yoshikawa, who has been a main contributor to Blastocystis research over the past 25 years or so (you can look up the publications by Dr Yoshikawa here). Considering the focus of this post, I guess that Pauline's talk was of particular interest, since she presented the data that we just published in FEMS Microbiology and Ecology:

3) The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. That's the title of the paper appearing in FEMS Microbiology and Ecology. The study, led by Pauline, showed that Blastocystis was present in 56% of 105 healthy adults, which is much higher than previously reported from an industrialised county (Ireland). Moreover, a diversity of different subtypes (species) were detected and Blastocystis was present in a subset of individuals sampled over a period of time between six and ten years, indicating that it is capable of long-term host colonisation. These observations show that Blastocystis is a common and diverse member of the healthy gut microbiota, thereby extending our knowledge of the microbial ecology of the healthy human intestine. And one of the interesting things here is: Why do we see this great divide? Why does half of the population appear colonised while the other half not? What are the factors driving successful Blastocystis colonisation? Would some people be refractory to colonisation or does it really boil down to some sort of enterotype-driven phenomenon as previously indicated?

4) I would like to reiterate the paper by Klimes et al. published a study in Genome Biology and Evolution (GBE) on a striking finding in the Blastocystis nuclear genome. Stop codons created by mRNA polyadenylation have been seen so far in mitochondrial genomes only and not in nuclear genomes; however, the authors observered this feature in Blastocystis's nuclear genome. Partly due to limitations of currently available annotation software, this finding ostensibly calls for reannotation of the genome currently available in GenBank (ST7). The paper was highlighted in a separate article in GBE that can be accessed from here.

5) Speaking of Blastocystis genomes: The genome of Blastocystis ST4 (WR1 strain) is now available on-line and can be accessed here.

6) Wang and colleagues studied the location and pathogenic potential of Blastocystis in the porcine intestine. They studied a total of 28 pigs from a commercial piggery, a small animal farm, and a research facility, and all pigs were positive (for ST5, and mixed subtypes were also seen in some). Post-mortem analyses showed that all pigs were consistently found to harbour Blastocystis in the colon, and approximately 90% of the caeca and rectums examined were positive. Some of the pigs were immunosuppresed (Dexamethasone), and interestingly, Blastocystis was occasionally detected in the small intestine, notably in immunosuppressed pigs, suggesting that immunosuprression may alter host-agent relations and predispose to small intestinal colonisation. Histopathological analysis saw the presence of vacuolar and granular forms of Blastocystis, but there was no evidence of attachment or invasion of the intestinal epithelium. The lack of pathology, including inflammation, epithelial damage, mucosal sloughing, and lamina propria oedema, confirmed the trend from a previous study carried out by Ron Fayer's group (see reference below). The study adds to the string of papers finding no evidence in support for Blastocystis causing primary intestinal damage.

6) Lastly, I want to extend a cordial thank you to Shashiraja Padukone and Subhash Chandra Parija, Department of Microbiology, Jipmer, Puducherry, India, for writing up a review of my 'Thoughts on Blastocystis' available on Amazon for the price of only one US$ or so. The review was published recently in Tropical Parasitology and can be accessed here.

And, for those who are worried about researchers 'overselling' microbiome research, there is a small comment in Nature calling for sound scepticism to be applied to research dealing with the relationship between the microbiome and different types of diseases. There is much to be agreed upon, and what I find particularly important, is to take the reductionist approach where possible - in terms of Blastocystis there are lots of ways to study the impact of Blastocystis on bacteria in vitro, and also host microbiota, physiology and immunology in vivo; ways that can be controlled quite diligently. Also, I think that simple validation of methods applied to map e.g. intestinal microbiota is key. This is for some reason something that is generally being utterly and completely ignored. Why?

References 

Fayer R, Elsasser T, Gould R, Solano G, Urban J Jr, & Santin M (2014). Blastocystis tropism in the pig intestine. Parasitology Research, 113 (4), 1465-72 PMID: 24535732

Hanage, W. (2014). Microbiology: Microbiome science needs a healthy dose of scepticism Nature, 512 (7514), 247-248 DOI: 10.1038/512247a

Klimeš V, Gentekaki E, Roger AJ, & Eliáš M (2014). A large number of nuclear genes in the human parasite blastocystis require mRNA polyadenylation to create functional termination codons. Genome Biology and Evolution, 6 (8), 1956-61 PMID: 25015079

Scanlan PD, Stensvold CR, Rajilić-Stojanović M, Heilig HG, De Vos WM, O'Toole PW, & Cotter PD (2014). The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. FEMS Microbiology Ecology PMID: 25077936 

Venton, D. (2014). Highlight: Not Like a Textbook--Nuclear Genes in Blastocystis Use mRNA Polyadenylation for Stop Codons Genome Biology and Evolution, 6 (8), 1962-1963 DOI: 10.1093/gbe/evu167

Wang W, Bielefeldt-Ohmann H, Traub RJ, Cuttell L, & Owen H (2014). Location and pathogenic potential of Blastocystis in the porcine intestine. PloS One, 9 (8) PMID: 25093578 

This Month In Blastocystis Research (FEB 2014) - The Protease Edition

A few interesting papers on Blastocystis appeared this month on PubMed. I would like to give a great salute to Ron Fayer's group in Maryland who took to investigating faecal samples and tissue sections from naturally infected pigs. Due to the protease theme of this blog post, I won't go into detail with this paper, but only highlight a few points. The researchers found Blastocystis ST5 in faecal samples from all 11 pigs investigated. By examination of tissue sections they found that Blastocystis existed in the lumen of the jejunum, caecum, proximal and distal colon, but not in the duodenum and ileum. Moreover:

"In tissue sections, Blastocystis was found primarily in the lumen usually associated with digested food debris, sometimes in close proximity or appearing to adhere to the epithelium, but no stages were found to penetrate the epithelium or the lamina propria."

So, the authors did a great job to describe Blastocystis tropism in the pig intestine. It is new to me that the parasite can be found in the jejunum; if anything, I would have thought that the ileum might be 'affected', and certainly the caecum and possibly the remainder of the colon. It is also important to note that in these naturally infected pigs (ST5 is probably the most common subtype in pigs), no signs of invasiveness was detected.

Now, moving on to the proteases, there is a paper out by Arutchelvan Rajamanikam and Suresh K Govind called 'Amoebic forms of Blastocystis spp. - evidence for a pathogenic role'. The study links protease activity to amoebic forms of Blastocystis, which the authors found in symptomatic carriers but not in asymptomatic carriers. Amoeboid forms of Blastocystis being associated with symptomatic infections were described already in 2006 by T C Tan and K G Suresh (whom I believe is identical to S K Govind). While the study is small, investigation of Blastocystis proteases has been going on for a while, and I thought it would be useful to go over some of the literature.

Proteases (or proteinases or peptidases) are enzymes that degrade proteins and therefore useful for instance for the mobilisation and storage of proteins (i.e. 'food'), and the general development and differentiation of cells and tissues, but these enzymes may also be vital for for instance pathogen survival and virulence in the human body (i.e. 'defence' and 'invasion'). Proteases exist in all organisms, i.e. in pro- and eukaryotes + viruses. Proteases are classified on the basis of catalytic mechanism, and five known distinct classes are described: metallo, aspartic, cysteine, serine, and threonine. Being enzymes, proteases digest substrates, can be inhibited, and their functions are dependent on pH and temperature. Hence, proteases can be identified by substrate digestion and by intended inhibition by selective inhibitors (for cystein protease such inhibitors include N-ethylmaleimide, iodoacetamide, and para-hydroxymercuribenzoate for instance).

Turning to the intestinal protozoon Entamoeba for a short while, cysteine proteases have been studied in detail and are among the most likely candidates responsible for the differential pathogenocitiy (virulence factors) of morphologically similar species of Entamoeba: Entamoeba histolytica expresses at least 5 types of cysteine proteases (ACP1, ACP2, ACP3, EhCP5, and EhCP112) and can invade host tissue (leading to amoebiasis), while Entamoeba dispar expresses at least three types of cysteine proteases (EdCP1, EdCP2, and EdCP3) without the ability to invade host tissue. Clinical isolates of E. histolytica release 10- to 1,000-fold more cysteine proteinase activity into the supernatant than E. dispar isolates, although  significant day-to-day variability may be seen. Extracellular cysteine proteases cleave immune secretory IgA (facilitating adhesion of the organism (pathogen) to mucosal surfaces), degrade the extracellular matrix, activate complement, and degrade IgG to circumvent the host immune response. The first evidence of amoebic pathology is local depletion of intestinal mucus and disruption of the epithelial barrier as a result of degradation of the extracellular matrix, which occurs in part from the action of cysteine proteases. More than 80% of patients with amoebiasis develop antibodies against cysteine proteases. Please note that E. histolytica is not consistently invasive; only 10% of E. histolytica infections are believed to be invasive.

Importantly, cysteine proteases are critical to host invasion in a number of parasites. Specific inhibitors block invasion in Trypanosoma cruzi, Plasmodium falciparum, Cryptosporidium parvum, and Toxoplasma gondii.

The main reservoir of Blastocystis ST7 appears to include birds.

Now what do we know about Blastocystis and cysteine proteases? In 2005, Manoj K Puthia from Dr Kevin S W Tan's group in Singapore identified mainly cysteine protease activity in the 'B. hominis B' strain (which is the ST7 strain used in the genome sequencing and annotation study by Denoeud et al. (2011)) and aspartic protease activity in 'B. ratti WR1 strain' (which is a ST4 strain). Lysates and conditioned medium (culture supernatant) from both axenic strain cultures were able to degrade human secretory IgA over 2 h at 37 C, suggesting that Blastocystis actively secrets proteases that - among other things - degrade IgA, thereby potentially evading host mucosal immunity, and enhancing survival opportunities. Along theses lines, in 2006 Sio and colleagues from Tan's group used enzyme digestion (azocasein spectrophotometric assay and gelatin SDS-PAGE analysis), and inhibition assays to characterise proteases from 'B. hominis B' strain. They showed the existence of cysteine proteases with highest activity at neutral pH (the pH of the colon is neutral if even slightly acidic).

Mirza and Tan confirmed that cysteine protease activity was higher in ST7 than in ST4, while inter- and intra-subtype variation in activity was seen over time. In a small study of ST3 positive individuals, Abdel-Hameed and Hassanin were able to detect protease activity in 17/18 symptomatic individuals but only in 2/8 asymptomatic individuals, suggesting intra-subtype differential protease activity. I don't think they tested for protease activity in the culture supernatant.

Cysteine proteases from Blastocystis were reported by Puthia et al. (2008) to enable activation of interleukin 8 (IL-8) gene expression in the human colonic epithelial T84 cell line. IL-8 is a cytokine that attracts PMN and activates monocytes (interestingly, recent results from Olivo-Diaz et al. (2012) suggest that some IL-8 and IL-10 SNPs could change individual susceptibility increasing the relative risk in the development of irritable bowel syndrome (IBS) in Blastocystis carriers).

Gastrointestinal disorders, such as bacterial enteritis, celiac disease, and inflammatory bowel disease, are reported to be associated with a breakdown of epithelial barrier function which is mainly regulated by 'tight junctions'. There is some experimental evidence that Blastocystis may be able to interfere with this regulation and that it may induce host cell apoptosis without attaching to the gut mucosa. Puthia et al. (2006) explain:

"Pathogen invasion and induction of apoptosis are discrete processes, and there are pathogens that can invade but do not induce apoptosis. It appears that induction of apoptosis of host intestinal cells would not be advantageous to a noninvasive parasite like Blastocystis, as it would result in the loss of colonization sites for the parasite. This unintended induction of host cell apoptosis might be a host response against some parasitic factors like proteases which are necessary for the parasite's own life cycle."

Back to the paper by Rajamanikam and Govind: I cannot remember ever seeing amoeboid stages in Blastocystis cultures myself. But then again, in cultures, Blastocystis can take so many forms (some actually resembling the outline of the head of, well, Mickey Mouse (!) and other cuddly creatures (looks like budding off of new cells), and I wouldn't be able to define strict criteria for stratification of organisms into groups. Since we use Jones' Medium also, I do not suspect that it's a 'medium thing'. What we usually see in well-maintained cultures are small, quite inconspicuous and completely spherical cells. Using the aforementioned digestion assays, Rajamanikam and Govind found elevated protease activity related to patient Blastocystis cultures that had a higher percentage of amoebic forms with intense bands representing higher molecular weight proteases (60-100 kDa); the proteases previously described have been of a size of maximum 75 kDa; however, no attempts were made to characterise the proteases in this study. The authors did not include analysis of conditioned medium, and so we do not know whether these proteases were actually secreted. The proteases identified here may be expressed by the amoebic forms only and so they may be responsible for this particular life cycle stage. Knowledge of substrate specificity might have been useful, and it is also possible to actually determine the protein's amino acid sequence and thereby predict it's structure and function using e.g. mass spectrometry (MS) or Edman degradation of peptides.

Just like Ivan Wawrzyniak and colleagues who recently used SDS-PAGE and MS to characterise proteases secreted by the Blastocystis ST7 (B strain). They were able to match two cysteine proteases identified in the culture supernatant to 2 of 22 proteases predicted by in silico analysis of their ST7 B strain genome data, namely Cathepsin B cysteine protease (CBCP) and a Legumain cysteine protease, which the authors speculated to be potentially involved in pathological processes such as mucin degradation. Incidentally, silencing of CBCP has recently been shown to reduce gut penetration in the helminth Faciola hepatica.

Back in 2007, Jésus Serrano-Luna and colleagues studied proteases from pathogenic Naegleria fowleri (causing primary amoebic meningoencephalitis) and non-pathogenic Naegleria gruberi. They observed cysteine proteases in both species, but more proteases in the N. gruberi than in N. fowleri. Protease activity appeared to depend on pH and temp, and moreover, protease patterns for crude extracts and conditioned medium differed

It's probably fair to assume that the expression of potential virulence genes such as genes encoding cysteine proteases may depend on a multiple factors, most of which are yet to be identified, or at least, confirmed. For now, the marked differences in cysteine protease production/expression between and within Blastocystis STs together with experimental evidence highlighting a variation in pathophysiological effects and immunological responses to Blastocystis subtypes isolated from symptomatic and asymptomatic carriers, could be seen as supporting the hypothesis that cysteine proteases may be essential virulence factors responsible for variation in disease symptoms observed across carriers. For more on this, why not look up this paper (free in PubMed Central). However, it is also tempting to think that differential protease expression is merely reflecting various stages in the parasite's life cycle. Things would have been so much easier if we had access to a strain in culture capable of invasion or isolated from an outbreak of Blastocystis infection. But, contrary to parasites of 'acknowledged clinical significance', we do not have such a strain, and neither invasion nor outbreaks of Blastocystis have been reported of, at least not convincingly, I think; please correct me, if I'm wrong. I think it's time for a coffee...

Literature:

Abdel-Hameed DM, & Hassanin OM (2011). Proteaese activity of Blastocystis hominis subtype 3 in symptomatic and asymptomatic patients. Parasitology Research, 109 (2), 321-7 PMID: 21279383

Denoeud F, Roussel M, Noel B, Wawrzyniak I, Da Silva C, Diogon M, Viscogliosi E, Brochier-Armanet C, Couloux A, Poulain J, Segurens B, Anthouard V, Texier C, Blot N, Poirier P, Ng GC, Tan KS, Artiguenave F, Jaillon O, Aury JM, Delbac F, Wincker P, Vivarès CP, & El Alaoui H (2011). Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite. Genome Biology, 12 (3) PMID: 21439036 

Fayer R, Elsasser T, Gould R, Solano G, Urban J Jr, & Santin M (2014). Blastocystis tropism in the pig intestine. Parasitology Research PMID: 24535732

McGonigle L, Mousley A, Marks NJ, Brennan GP, Dalton JP, Spithill TW, Day TA, & Maule AG (2008). The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration. International Journal for Parasitology, 38 (2), 149-55 PMID: 18048044

Mirza H, & Tan KS (2009). Blastocystis exhibits inter- and intra-subtype variation in cysteine protease activity. Parasitology Research, 104 (2), 355-61 PMID: 18846388

Olivo-Diaz A, Romero-Valdovinos M, Gudiño-Ramirez A, Reyes-Gordillo J, Jimenez-Gonzalez DE, Ramirez-Miranda ME, Martinez-Flores WA, Martinez-Hernandez F, Flisser A, & Maravilla P (2012). Findings related to IL-8 and IL-10 gene polymorphisms in a Mexican patient population with irritable bowel syndrome infected with Blastocystis. Parasitology Research, 111 (1), 487-91 PMID: 22287022

Poirier P, Wawrzyniak I, Vivarès CP, Delbac F, & El Alaoui H (2012). New insights into Blastocystis spp.: a potential link with irritable bowel syndrome. PLoS Pathogens, 8 (3) PMID: 22438803

Puthia MK, Vaithilingam A, Lu J, & Tan KS (2005). Degradation of human secretory immunoglobulin A by Blastocystis. Parasitology Research, 97 (5), 386-9 PMID: 16151742

Puthia MK, Sio SW, Lu J, & Tan KS (2006). Blastocystis ratti induces contact-independent apoptosis, F-actin rearrangement, and barrier function disruption in IEC-6 cells. Infection and Immunity, 74 (7), 4114-23 PMID: 16790785

Que X, & Reed S L (2000). Cysteine Proteinases and the Pathogenesis of Amebiasis. Clinical Microbiology Reviews, 13 (2), 196-206 DOI: 10.1128/CMR.13.2.196-206.2000

Rajamanikam A, & Govind SK (2013). Amoebic forms of Blastocystis spp. - evidence for a pathogenic role. Parasites & Vectors, 6 (1) PMID: 24499467

Serrano-Luna J, Cervantes-Sandoval I, Tsutsumi V, & Shibayama M (2007). A biochemical comparison of proteases from pathogenic Naegleria fowleri and non-pathogenic Naegleria gruberi. The Journal of Eukaryotic Microbiology, 54 (5), 411-7 PMID: 17910685

Sio SW, Puthia MK, Lee AS, Lu J, & Tan KS (2006). Protease activity of Blastocystis hominis. Parasitology Research, 99 (2), 126-30 PMID: 16518611 

Wawrzyniak I, Texier C, Poirier P, Viscogliosi E, Tan KS, Delbac F, & El Alaoui H (2012). Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite. Parasitology International, 61 (3), 437-42 PMID: 22402106