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.
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| 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
